U.S. patent application number 09/359325 was filed with the patent office on 2002-08-08 for assays and reagents for identifying anti-fungal agnets, amd uses related thereto.
Invention is credited to BERLIN, VIVIAN, LEVIN, DAVID E., OHYA, YOSHIKAZU.
Application Number | 20020107226 09/359325 |
Document ID | / |
Family ID | 24530697 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020107226 |
Kind Code |
A1 |
BERLIN, VIVIAN ; et
al. |
August 8, 2002 |
ASSAYS AND REAGENTS FOR IDENTIFYING ANTI-FUNGAL AGNETS, AMD USES
RELATED THERETO
Abstract
The present invention relates to rapid, reliable and effective
assays for screening and identifying pharmaceutically effective
compounds that specifically inhibit the biological activity of
fungal GTPase proteins, particularly GTPases involved in cell wall
integrity, hyphael formation, and/or other cellular functions
critical to pathogenesis.
Inventors: |
BERLIN, VIVIAN; (DUNSTABLE,
MA) ; LEVIN, DAVID E.; (OWINGS MILLS, MD) ;
OHYA, YOSHIKAZU; (TOKYO, JP) |
Correspondence
Address: |
ROPES & GRAY
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
24530697 |
Appl. No.: |
09/359325 |
Filed: |
July 22, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09359325 |
Jul 22, 1999 |
|
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08631319 |
Apr 11, 1996 |
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Current U.S.
Class: |
514/100 |
Current CPC
Class: |
C12Q 1/6895 20130101;
G01N 2333/40 20130101; C12Q 1/6897 20130101; G01N 2333/91091
20130101; A61K 38/00 20130101; G01N 2333/91165 20130101; G01N
33/573 20130101; C12Q 1/18 20130101; C07K 16/14 20130101; C07K
14/40 20130101; C12Q 1/48 20130101 |
Class at
Publication: |
514/100 |
International
Class: |
A61K 031/665; A01N
057/00 |
Goverment Interests
[0001] Work described herein was supported in part by funding from
the National Institute of Health. The United States Government has
certain rights in the invention.
Claims
We claim:
1. An assay for identifying compounds having potential anti-fungal
activity, comprising: (a) forming a reaction mixture including: (i)
a fungal geranylgeranyl transferase (GGPTase), (ii) a GGPTase
substrate, and (iii) a test compound; and (b) detecting interaction
of the GGPTase substrate with the GGPTase, wherein a statistically
significant decrease in the interaction of the GGPTase substrate
and GGPTase in the presence of the test compound, relative to the
level of interaction in the absence of the test compound, indicates
a potential anti-fungal activity for the test compound.
2. The assay of claim 1, wherein GGPTase substrate comprises target
polypeptide comprising a fungal Rho-like GTPase, or a polypeptide
portion thereof including at least one of (a) a prenylation site
which can be enzymatically prenylated by the GGPTase, or (b) a
GGPTase binding sequence which specifically binds the GGPTase.
3. The assay of claim 1, wherein the reaction mixture is a
prenylation system including an activated geranylgeranyl group, and
the step of detecting the interaction of the GGPTase substrate with
the GGPTase comprises detecting conjugation of the geranylgeranyl
group to the GGPTase substrate.
4. The assay of any of claims 3 or 23, wherein at least one of the
geranylgeranyl group and the GGPTase substrate comprises a
detectable label, and the level of geranylgeranyl group-conjugated
to the GGPTase substrate is quantified by detecting the label in at
least one of the GGPTase substrate, the geranylgeranyl group, and
geranylgeranyl-conjugate- d GGPTase substrate.
5. The assay of claim 1, wherein the step of detecting the
interaction of the GGPTase substrate with the GGPTase comprises
detecting the formation of complexes including the GGPTase
substrate with the GGPTase.
6. The assay of claim 5, wherein at least one of the GGPTase and
the GGPTase substrate comprises a detectable label, and the level
of GGPTase/GGPTase substrate complexes formed in the reaction
mixture is quantified by detecting the label in at least one of the
GGPTase substrate, the GGPTase, and GGPTase/GGPTase substrate
complexes.
7. The method of any of claims 4 or 6, wherein the label group is
selected from a group consisting of radioisotopes, fluorescent
compounds, enzymes, and enzyme co-factors.
8. The assay of claim 4, wherein the substrate target comprises a
fluorescent label, the fluorescent characterization of which is
altered by the level of prenylation of the substrate target.
9. The assay of claim 8, wherein the substrate target comprises a
dansylated peptide substrate of the fungal GGPTase.
10. The assay of any of claims 3 or 23, wherein conjugation of the
geranylgeranyl group to the GGPTase substrate is detected by an
immunoassay.
11. The assay of claim 5, wherein the formation of protein-protein
complexes including the GGPTase substrate with the GGPTase is
detected by an immunoassay.
12. The assay of any of claims 1 or 23, wherein the reaction mixure
is reconstituted protein mixture.
13. The assay of any of claims 1 or 23, wherein the reaction mixure
comprises a cell lysate.
14. The assay of any of claims 2 or 23, wherein the fungal Rho-like
GPTase is selected from the group consisting of Rho1, Rho2,
Rsr1/Bud1 and Cdc42, and homologs thereof.
15. The assay of claim 1, wherein the reaction mixture is a whole
cell comprising heterologous nucleic acid recombinantly expressing
one or more of the fungal GGPTase subunits and GGPTase
substrate.
16. The assay of claim 1, wherein the reaction mixture is a whole
cell comprising a heterologous reporter gene construct comprising a
reporter gene in operable linkage with a transcriptional regulatory
sequence sensitive to intracellular signals transduced by
interaction of the GGPTase substrate and GGPTase.
17. The assay of any of claims 1, 23, 24, 25, or 26 wherein the
assay is repeated for a variegated library of at least 100
different test compounds.
18. The assay of any of claims 1, 23, 24, 25, or 26 wherein the
test compound is selected from the group consisting of small
organic molecules, and natural product extracts.
19. The assay of any of claims 2 or 23, wherein one or more of the
GGPTase and target polypeptide are derived from a human pathogen
which is implicated in mycotic infection.
20. The assay of claim 19, wherein the mycotic infection is a
mycosis selected from a group consisting of candidiasis,
aspergillosis, mucormycosis, blastomycosis, geotrichosis,
cryptococcosis, chromoblastomycosis, penicilliosis,
conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis,
maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and
sporotrichosis.
21. The assay of claim 19, wherein the human pathogen is selected
from a group consisting of Candida albicans, Candida stellatoidea,
Candida tropicalis, Candida parapsilosis, Candida krusei, Candida
pseudotropicalis, Candida quillermondii, Candida rugosa,
Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,
Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus,
Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor
pusillus.
22. The assay of claim 19, wherein the human pathogen is
Pneumocystis carinii.
23. An assay for identifying compounds having potential anti-fungal
activity, comprising: (a) forming a cell-free reaction mixture
including: (i) a fungal geranylgeranyl transferase (GGPTase), (ii)
a target polypeptide comprising a fungal Rho-like GTPase, or a
polypeptide portion thereof including a prenylation site (iii) an
activated geranylgeranyl group, (iv) a divalent cation, and (v) a
test compound; (b) detecting conjugation of the gernaylgernayl
group of the target polypeptide in the reaction mixture, wherein a
statistically significant decrease in the prenylation of the target
polypeptide and GGPTase in the presence of the test compound,
relative to the level of prenylation in the absence of the test
compound, indicates a potential anti-fungal activity for the test
compound.
24. A method of identifying an agent which disrupts the ability of
an geranylgeranyl protein transferase (GGPTase) to bind to a fungal
GTPase, comprising: i. providing an interaction trap system
including (a) a first fusion protein comprising at least a portion
of a fungal GGPTase subunit, (b) second fusion protein comprising
at least a portion of a fungal GTPase, and (c) a reporter gene,
including a transcriptional regulatory sequence sensitive to
interactions between the GGPTase portion of the first fusion
protein and the GTPase portion of the second polypeptide; ii.
contacting the interaction trap system with a candidate agent; iii.
measuring the level of expression of a reporter gene in the
presence of the candidate agent; and iv. comparing the level of
expression of the reporter gene in the presence of the candidate
agent to a level of expression in the absence of the candidate
agent, wherein a decrease in the level of expression of the
reporter gen in the presence of the candidate agent is indicative
of an agent that inhibits interaction of the GGPTase and
GTPase.
25. An assay for identifying compounds having potential anti-fungal
activity, comprising: (i) providing a first recombinant cell
including a first prenylation substrate derived from a Rho-like
GTPase which is a substrate for a geranylgeranyl transferase
expressed by the cell; (ii) providing a second recombinant cell
including a second prenylation substrate identical to the first
prenylation substrate except that it is mutated to be a substrate
for a farnesyl transferase expressed by the recombinant cell; (iii)
contacting the first and second cells with a candidate agent; and
(iv) comparing the level of prenylation of the Rho-like GTPases in
first and second cells, wherein a statistically significant
decrease in the prenylation of the first prenylation substrate,
relative to the level of prenylation of the second prenylation
substrate, is indicative of an agent that inhibits interaction of a
GGPTase and GTPase.
26. An assay for screening test compounds to identify agents which
modulate the interaction of a fungal geranylgeranyl transferase
(GGPTase) with a fungal Rho0-like GTPase, comprising: i. providing
a cell expressing a recombinant form of one or more of a fungal
GGPTase and a fungal Rho-like GTPase; ii. contacting the cell with
a test compound; and iii. detecting the level of interaction of the
GGPTase and Rho-like GTPase, wherein a statistically significant
change in the level of interaction of the GGPTase and Rho-like
GTPase is indicative of an agent that modulates the interaction of
those two proteins.
27. The method of claim 23, wherein one or both of a GGPTase
subunit or the Rho-like GTPase are fusion proteins.
28. The method of claim 23, wherein the level of interaction of the
GGPTase and Rho-like GTPase is detected by detecting prenylation of
the Rho-like GTPase.
29. The method of claim 25, wherein the Rho-like GTPase is a fusion
protein further comprising a transcriptional regulatory protein,
and level of prenylation of the Rho-like GTPase is detected by
measuring the level of expression of a reporter gene construct
which is sensitive to the transcriptional regulatory protein
portion of the fusion protein, wherein inhibition of prenylation of
the fusion protein results in loss of membrane partitioning of the
fusion protein and increases expression of the reporter gene
construct.
30. The assay of any of claims 1, 20, 21, 22, or 23, which
comprises a further step of preparing a pharmaceutical preparation
of one or more compounds identified as having potential antifungal
activity.
31. An assay for identifying compounds having potential antifungal
activity, comprising: i. forming a reaction mixture including a
fungal Rho-like GTPase, a fungal protein kinase C (PKC), and a test
compound; and ii. detecting interaction of the Rho-like GTPase and
PKC, wherein a statistically significant decrease in the
interaction of the Rho-like GTPase and PKC in the presence of the
test compound, relative to the level of interaction in the absence
of the test compound, indicates a potential antifungal activity for
the test compound.
32. The assay of claim 31, wherein the reaction mixture is a kinase
system including ATP and a PKC substrate, and the step of detecting
interaction of the GTPase and PKC comprises detecting
phosphorylation of the PKC substrate by a PKC/GTPase complex.
33. The assay of claim 32, wherein at least one of the PKC
substrate and ATP comprises a detectable label, and the level of
phosphorylation of the PKC substrate is quantified by detecting the
label in at least one of the PKC substrate or ATP.
34. The assay of claim 31, wherein the step of detecting the
interaction of the GTPase with the PKC comprises detecting the
formation of protein-protein complexes including the GTPase and
PKC.
35. The assay of claim 34, wherein at least one of the PKC and
GTPase comprises a detectable label, and the level of PKC/GTPase
complexes formed in the reaction mixture is quantified by detecting
the label in at least one of the GTPase, the PKC, and PKC/GTPase
complexes.
36. The method of any of claims 33 or 35, wherein the label group
is selected from a group consisting of radioisotopes, fluorescent
compounds, enzymes, and enzyme co-factors.
37. The assay of claim 36, wherein the detectable label is a
protein having a measurable activity, and one of the PKC or GTPase
is fusion protein including the detectable label.
38. The assay of claim 33, wherein the PKC substrate comprises a
fluorescent label, the fluorescent characterization of which is
altered by the level of phosphorylation of the PKC substrate.
39. The assay of claim 33, wherein phosphorylation of the PKC
substrate is detected by immunoassay.
40. The assay of claim 34, wherein the formation of protein-protein
complexes including the GTPase and PKC is detected by an
immunoassay.
41. The assay of claim 31, wherein the reaction mixure is
reconstituted protein mixture.
42. The assay of claim 31, wherein the reaction mixure comprises a
cell lysate.
43. The assay of claim 31, wherein the GPTase is selected from the
group consisting of Rho1, Rho2, Rsr1/Bud1 and Cdc42, and fungal
homologs thereof.
44. The assay of claim 31, wherein the reaction mixture is a whole
cell comprising heterologous nucleic acid recombinantly expressing
one or more of the PKC and GTPase.
45. The assay of claim 31, wherein the reaction mixture is a whole
cell comprising a heterologous reporter gene construct comprising a
reporter gene in operable linkage with a transcriptional regulatory
sequence sensitive to intracellular signals transduced by
interaction of the GTPase and PKC.
46. The assay of claim 31, wherein the assay is repeated for a
variegated library of at least 100 different test compounds.
47. The assay of claim 31, wherein the test compound is selected
from the group consisting of small organic molecules, and natural
product extracts.
48. The assay of claim 31, wherein one or more of the PKC and
GTPase are derived from a human pathogen which is implicated in
mycotic infection.
49. The assay of claim 48, wherein the mycotic infection is a
mycosis selected from a group consisting of candidiasis,
aspergillosis, mucormycosis, blastomycosis, geotrichosis,
cryptococcosis, chromoblastomycosis, penicilliosis,
conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis,
maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and
sporotrichosis.
50. The assay of claim 48, wherein the human pathogen is selected
from a group consisting of Candida albicans, Candida stellatoidea,
Candida tropicalis, Candida parapsilosis, Candida krusei, Candida
pseudotropicalis, Candida quillermondii, Candida rugosa,
Aspergillusfumigatus, Aspergillusflavus, Aspergillus niger,
Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus,
Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor
pusillus.
51. The assay of claim 48, wherein the human pathogen is
Pneumocystis carinii.
52. The assay of claim 31, which comprises a further step of
preparing a pharmaceutical preparation of one or more compounds
identified as having potential antifungal activity.
53. An assay for identifying compounds having potential antifungal
activity, comprising: i. forming a reaction mixture including a
fungal Rho-like GTPase, a fungal glucan synthase complex or subunit
thereof (GS protein), and a test compound; and ii. detecting
interaction of the Rho-like GTPase and GS protein, wherein a
statistically significant decrease in the interaction of the
Rho-like GTPase and GS protein in the presence of the test
compound, relative to the level of interaction in the absence of
the test compound, indicates a potential antifungal activity for
the test compound.
54. The assay of claim 53, wherein the reaction mixture is a glucan
synthesis system including a GTP and a UDP-glucose, and the step of
detecting interaction of the GTPase and GS protein comprises
detecting formation of glucan polymers in the reaction mixture.
55. The assay of claim 54, wherein the UDP-glucose comprises a
detectable label, and the level of glucan polymer formation is
quantified by detecting the labelled glucan polymers.
56. The assay of claim 53, wherein the step of detecting the
interaction of the GTPase with the GS protein comprises detecting
the formation of protein-protein complexes including the GTPase and
GS protein.
57. The assay of claim 56, wherein at least one of the GS protein
and GTPase comprises a detectable label, and the level of GS
protein/GTPase complexes formed in the reaction mixture is
quantified by detecting the label in at least one of the GTPase,
the GS protein, and GS protein/GTPase complexes.
58. The method of any of claims 53 or 57, wherein the label group
is selected from a group consisting of radioisotopes, fluorescent
compounds, enzymes, and enzyme co-factors.
59. The assay of claim 56, wherein the formation of protein-protein
complexes including the GTPase and GS protein is detected by an
immunoassay.
60. The assay of claim 53, wherein the reaction mixure is
reconstituted protein mixture.
61. The assay of claim 53, wherein the reaction mixure comprises a
cell lysate.
62. The assay of claim 53, wherein the GPTase is selected from the
group consisting of Rho1, Rho2, Rsr1/Bud1 and Cdc42, and fungal
homologs thereof.
63. The assay of claim 53, wherein the reaction mixture is a whole
cell comprising heterologous nucleic acid recombinantly expressing
one or more of the GS protein and GTPase.
64. The assay of claim 53, wherein the assay is repeated for a
variegated library of at least 100 different test compounds.
65. The assay of claim 53, wherein the test compound is selected
from the group consisting of small organic molecules, and natural
product extracts.
66. The assay of claim 53, wherein one or more of the GS protein
and GTPase are derived from a human pathogen which is implicated in
mycotic infection.
67. The assay of claim 66, wherein the mycotic infection is a
mycosis selected from a group consisting of candidiasis,
aspergillosis, mucormycosis, blastomycosis, geotrichosis,
cryptococcosis, chromoblastomycosis, penicilliosis,
conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis,
maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and
sporotrichosis.
68. The assay of claim 66, wherein the human pathogen is selected
from a group consisting of Candida albicans, Candida stellatoidea,
Candida tropicalis, Candida parapsilosis, Candida krusei, Candida
pseudotropicalis, Candida quillermondii, Candida rugosa,
Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,
Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus,
Rhizopus oryzae, Absidia corymbifera, Absidia ramrosa, and Mucor
pusillus.
69. The assay of claim 66, wherein the human pathogen is
Pneumocystis carinii.
70. The assay of claim 53, which comprises a further step of
preparing a pharmaceutical preparation of one or more compounds
identified as having potential antifungal activity.
71. A recombinant cell comprising (i) exogenous nucleic acid
encoding one or more subunits of a fungal geranylgeranyl protein
transferase (GGPTase), and (ii) exogenous nucleic acid encoding a
fungal Rho-like GTPase or a fragment thereof including at least one
of (a) a prenylation site which can be enzymatically prenylated by
the GGPTase, or (b) a GGPTase binding sequence which specifically
binds the GGPTase.
72. The cell of claim 71, wherein one or more of the nucleic acids
encoding the GGPTase and GTPase are derived from a human pathogen
which is implicated in mycotic infection.
73. The cell of claim 72, wherein the mycotic infection is a
mycosis selected from a group consisting of candidiasis,
aspergillosis, mucormycosis, blastomycosis, geotrichosis,
cryptococcosis, chromoblastomycosis, penicilliosis,
conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis,
maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and
sporotrichosis.
74. The cell of claim 72, wherein the human pathogen is selected
from a group consisting of Candida albicans, Candida stellatoidea,
Candida tropicalis, Candida parapsilosis, Candida krusei, Candida
pseudotropicalis, Candida quillermondii, Candida rugosa,
Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,
Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus,
Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor
pusillus.
75. The cell of claim 72, wherein the human pathogen is
Pneumocystis carinii.
76. The cell of claim 71, which cell is a recombinantly manipulate
yeast cell selected from the group consisting of Kluyverei spp,
Schizosaccharomyces spp, Ustilaqo spp and Saccharomyces spp.
77. The cell of claim 71, which cell is a recombinantly manipulate
Schizosaccharomyces cerivisae cell.
78. The cell of claim 71, which cell is constitutively or inducibly
defective for an endogenous activity corresponding to one or more
of the GGPTase and GTPase encoded by the exogenous nucleic
acids.
79. A reconstituted protein mixture or a cell lysate mixture
comprising (i) a recombinant fungal geranylgeranyl protein
transferase (GGPTase), and (ii) a recombinant fungal Rho-like
GTPase or a fragment thereof including at least one of (a) a
prenylation site which can be enzymatically prenylated by the
GGPTase, or (b) a GGPTase binding sequence which specifically binds
the GGPTase.
80. The mixture of claim 79, wherein one or more of the recombinant
GGPTase and GTPase are derived from a human pathogen which is
implicated in mycotic infection.
81. The mixture of claim 80, wherein the mycotic infection is a
mycosis selected from a group consisting of candidiasis,
aspergillosis, mucormycosis, blastomycosis, geotrichosis,
cryptococcosis, chromoblastomycosis, penicilliosis,
conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis,
maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and
sporotrichosis.
82. The mixture of claim 80, wherein the human pathogen is selected
from a group consisting of Candida albicans, Candida stellatoidea,
Candida tropicalis, Candida parapsilosis, Candida krusei, Candida
pseudotropicalis, Candida quillermondii, Candida rugosa,
Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,
Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus,
Rhizopus oryzae, Absidia corymbifera, Absidia ramrosa, and Mucor
pusillus.
83. The mixture of claim 80, wherein the human pathogen is
Pneumocystis carinii.
84. A recombinant cell comprising (i) exogenous nucleic acid
encoding a fungal Rho-like GTPase, and (ii) exogenous nucleic acid
encoding a fungal protein selected from the group consisting of a
fungal protein kinase C (PKC) or one or more subunits of a fungal
glucan synthase.
85. A reconstituted protein mixture or a cell lysate mixture
comprising (i) a recombinant fungal Rho-like GTPase, and (ii) a
recombinant fungal protein selected from the group consisting of a
fungal protein kinase C (PKC) or a fungal glucan synthase.
86. A recombinant cell comprising exogenous nucleic acid encoding
one or more subunits of a geranylgeranyl protein transferase
(GGPTase) cloned from a human fungal pathogen.
87. A recombinant cell comprising exogenous nucleic acid encoding a
Rho-like GTPase cloned from a human fungal pathogen.
88. A recombinant cell comprising exogenous nucleic acid encoding
one or more subunits of a glucan synthase cloned from a human
fungal pathogen.
89. A recombinant cell comprising exogenous nucleic acid encoding a
protein kinase C cloned from a human fungal pathogen.
90. A reconstituted protein mixture or a cell lysate mixture
comprising one or more subunits of a recombinant geranylgeranyl
protein transferase (GGPTase) cloned from a human fungal
pathogen.
91. A reconstituted protein mixture or a cell lysate mixture
comprising a recombinant Rho-like GTPase cloned from a human fungal
pathogen.
92. Areconstituted protein mixture or a cell lysate mixture
comprising one or more recombinantn subunits of a glucan synthase
cloned from a human fungal pathogen.
93. A reconstituted protein mixture or a cell lysate mixture
comprising a recombinant protein kinase C cloned from a human
fungal pathogen.
94. The cell of any of claims 86-93 wherein the human pathogen is
selected from a group consisting of Candida albicans, Candida
stellatoidea, Candida tropicalis, Candida parapsilosis, Candida
krusei, Candida pseudotropicalis, Candida quillermondii, Candida
rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus
niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus
arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and
Mucor pusillus.
Description
BACKGROUND OF THE INVENTION
[0002] Fungal infections of humans range from superficial
conditions, usually caused by dermatophytes or Candida species,
that affect the skin (such as dermatophytoses) to deeply invasive
and often lethal infections (such as candidiasis and
cryptococcosis). Pathogenic fungi occur worldwide, although
particular species may predominate in certain geographic areas.
[0003] In the past 20 years, fungal infections have increased
dramatically--along with the numbers of potentially invasive
species. Indeed, fungal infections, once dismissed as a nuisance,
have begun to spread so widely that they are becoming a major
concern in hospitals and health departments. Fungal infections
occur more frequently in people whose immune system is suppressed
(because of organ transplantation, cancer chemotherapy, or the
human immunodeficiency virus), who have been treated with
broad-spectrum antibacterial agents, or who have been subject to
invasive procedures (catheters and prosthetic devices, for
example). Fungal infections are now important causes of morbidity
and mortality of hospitalized patients: the frequency of invasive
candidiasis has increased tenfold to become the fourth most common
blood culture isolate (Pannuti et al. (1992) Cancer 69:2653).
Invasive pulmonary aspergillosis is a leading cause of mortality in
bone-marrow transplant recipients (Pannuti et al., supra), while
Pneumocystis carinii pneumonia is the cause of death in many
patients with acquired immunodeficiency syndrome in North America
and Europe (Hughes (1991) Pediatr Infect. Dis J. 10:391). Many
opportunistic fungal infections cannot be diagnosed by usual blood
culture and must be treated empirically in severely
immunocompromised patients (Walsh et al. (1991) Rev. Infect. Dis.
13:496).
[0004] The fungi responsible for life-threatening infections
include Candida species (mainly Candida albicans, followed by
Candida tropicalis), Aspergillus species, Cryptococcus neoforms,
Histoplasma capsulatum, Coccidioides immitis, Pneumocystis carinii
and some zygomycetes. Treatment of deeply invasive fungal
infections has lagged behind bacterial chemotherapy.
[0005] There are numerous commentators who have speculated on this
apparent neglect. See, for example, Georgopapadakou et al. (1994)
Science 264:371. First, like mammalian cells, fungi are eukaryotes
and thus agents that inhibit fungal protein, RNA, or DNA
biosynthesis may do the same in the patient's own cells, producing
toxic side effects. Second, life-threatening fungal infections were
thought, until recently, to be too infrequent to warrant aggressive
research by the pharmaceutical industry. Other factors have
included:
[0006] (i) Lack of drugs. A drug known as Amphotericin B has become
the mainstay of therapy for fungal infection despite side effects
so severe that the drug is known as "amphoterrible" by patients.
Only a few second-tier drugs exist.
[0007] (ii) Increasing resistance. Long-term treatment of oral
candidiasis in AIDS patients has begun to breed species resistant
to older anti-fungal drugs. Several other species of fungi have
also begun to exhibit resistance.
[0008] (iii) A growing list of pathogens. Species of fungi that
once posed no threat to humans are now being detected as a cause of
disease in immune-deficient people. Even low-virulence baker's
yeast, found in the human mouth, has been found to cause infection
in susceptible burn patients.
[0009] (iv) Lagging research. Because pathogenic fungi are
difficult to culture, and because many of them do not reproduce
sexually, microbiological and genetic research into the
disease-causing organisms has lagged far behind research into other
organisms.
[0010] In the past decade, however, more antifungal drugs have
become available. Nevertheless, there are still major weaknesses in
their spectra, potency, safety, and pharmacokinetic properties, and
accordingly it is desirable to improve the the panel of anti-fungal
agents available to the practioner.
[0011] I. The Fungal Cell
[0012] The fungal cell wall is a structure that is both essential
for the fungus and absent from mammalian cells, and consequently
may be an ideal target for antifungal agents. Inhibitors of the
biosynthesis of two important cell wall components, glucan and
chitin, already exist. Polyoxins and the structurally related
nikkomycins (both consist of a pyrimidine nucleoside linked to a
peptide moiety) inhibit chitin synthase competitively, presumably
acting as analogs of the substrate uridine diphosphate
(UDP)-N-acetylglucosamine (chitin is an N-acetylglucosamine
homopolymer), causing inhibition of septation and osmotic lysis.
Unfortunately, the target of polyoxins and nikkomycins is in the
inner leaflet of the plasma membrane; they are taken up by a
dipeptide permease, and thus peptides in body fluids antagonize
their transport.
[0013] In most fungi, glucans are the major components that
strengthen the cell wall. The glucosyl units within these glucans
are arranged as long coiling chains of .beta.-(1,3)-linked
residues, with occasional sidechains that involve .beta.-(1,6)
linages. Three .beta.-(1,3) chains running in parallel can
associate to form a triple helix, and the aggregation of helicies
produces a network of water-insoluble fibrils. Even in the
chitin-rich filamentous aspergilli, .beta.-(1,3)-glucan is required
to maintain the integrity and form of the cell wall (Kurtz et al.
(1994) Antimicrob Agents Chemother 38:1408-1489), and, in P.
carinii, it is important during the life cycle as a constituent of
the cyst (ascus) wall (Nollstadt et al. (1994) Antimicrob Agents
Chemother 38:2258-2265.
[0014] In a wide variety of fungi, .beta.-(1,3)-glucan is produced
by a synthase composed of at least two subunits (Tkacz, J. S.
(1992) in Emerging Targets in Antibacterial and Antifungal
Chemotherapy (Sutcliffe and Georgopapadakou, eds), pp495-523,
Chapman & Hall; and Kang et al. (1986) PNAS 83:5808-5812). One
subunit is localized to the plasma membrane and is thought to be
the catalytic subunit, while the second subunit binds GTP and
associates with and activates the catalytic subunit (Mol et al.
(1994) J Biol Chem 269:31267-31274).
[0015] Two groups of anticandidal antibiotics known in the art
interfere with the formation of .beta.-(1,3)-glucan: the
papulacandins and the echinocandins (Hector et al. (1993) Clin
Microbiol Rev 6:1-21). However, many of the papulacandins are not
active against a variety of Candida species, or other pathogenic
fungi including aspergillus. The echinocandins, in addition to
suffering from narrow activity spectrum, are not in wide use
because of lack of bioavilability and toxicity.
[0016] II. Protein Prenylation
[0017] Covalent modification by isoprenoid lipids (prenylation)
contributes to membrane interactions and biological activities of a
rapidly expnanding group of proteins (see, for example, Maltese
(1990) FASEB J 4:3319; and Glomset et al. (1990) Trends Biochem Sci
15:139). Either farnesyl (15-carbon) or geranylgeranyl (20-carbon)
isoprenoids can be attached to specific proteins, with
geranylgeranyl being the predominant isoprenoid found on proteins
(Fransworth et al. (1990) Science 247:320).
[0018] Three enzymes have been described that catalyze protein
prenylation: farnesyl-protein transferase (FPTase),
geranylgeranyl-protein transferase type I (GGPTase-I), and
geranylgeranyl-protein transferase type-II (GGPTase-II, also called
Rab GGPTase). These enzymes are found in both yeast and mammalian
cells (Schafer et al. (1992) Annu. Rev. Genet. 30:209-237). FPTase
and GGPTase-I are .alpha./.beta. heterodimeric enzymes that share a
common .alpha. subunit; the .beta. subunits are distinct but share
approximately 30% amino acid similarity (Brown et al. (1993).
Nature 366:14-15; Zhang et al. (1994). J. Biol. Chem.
269:3175-3180). GGPTase II has different .alpha. and .beta.
subunits and complexes with a third component (REP, Rab Escort
Protein) that presents the protein substrate to the .alpha./.beta.
catalytic subunits. Each of these enzymes selectively uses farnesyl
diphosphate or geranylgeranyl diphosphate as the isoprenoid donor
and selectively recognizes the protein substrate. FPTase
famesylates CaaX-containing proteins that end with Ser, Met, Cys,
Gln or Ala. GGPTase-I geranylgeranylates CaaX-containing proteins
that end with Leu or Phe. For FPTase and GGPTase-I, CaaX
tetrapeptides comprise the minimum region required for interaction
of the protein substrate with the enzyme. GGPTase-II modifies XXCC
and XCXC proteins; the interaction between GGPTase-II and its
protein substrates is more complex, requiting protein sequences in
addition to the C-terminal amino acids for recognition. The
enzymological characterization of these three enzymes has
demonstrated that it is possible to selectively inhibit one with
little inhibitory effect on the others (Moores et al. (1991) J.
Biol. Chem. 266:17438).
[0019] GGPTase I transfers the prenyl group from geranylgeranyl
diphosphate to the sulphur atom in the Cys residue within the CAAX
sequence. S Cerevisiae proteins such as the Ras superfamily
proteins Rho1, Rho2, Rsr1/Bud1 and Cdc42 appear to be GGPTase
substrates (Madaule et al. (1987) PNAS 84:779-783; Bender et al.
(1989) PNAS 86:9976-9980; and Johnson et al. (1990) J Cell Biol
111:143-152).
[0020] III. Protein Kinase C
[0021] Members of the family of phospholipid-dependent,
serine/threonine-specific protein kinases known collectively as
protein kinase C (PKC) respond to extracellular signals that act
through receptor-mediated hydrolysis of
phosphatidylinositol-4,5-bisphosphate to diacyl-glycerol (DAG) and
inositol-1,4,5-trisphosphate (IP.sub.3) (Hokin (1985) Annu. Rev.
Biochem. 54, 205-235.). DAG serves as a second messenger to
activate PKC (Takai et al. (1979) Biochem. Biophys. Res. Commun.
91, 1218-1224; Kishimoto et al. (1980) J. Biol. Chem. 255,
2273-2276; Nishizuka 1986) Science 233, 305-312; and Nishizuka
(1988) Nature 334, 661-665), and IP.sub.3 functions to mobilize
Ca.sup.2+ from intracellular stores (Berridge et al. (1984) Nature
312, 215-321). Twelve distinct subtypes of mammalian PKC have been
reported to date (Nishizuka, Y. (1992) Science 258, 607-614; Decker
et al. (1994) TIBS 19:73-77). The four initially identified
isozymes, .alpha., .beta.I, .beta.II, and .gamma., are structurally
closely related to each other and display similar catalytic
properties.
[0022] Mammalian PKC is thought to play a pivotal role in the
regulation of a host of cellular functions through its activation
by growth factors and other agonists. These functions include cell
growth and proliferation, release of various hormones, and control
of ion conductance channels. Indirect evidence suggests that PKC
induces the transcription of a wide array of genes, including the
proto-oncogenes c-myc, c-fos, and c-sis, human collagenase,
metallothionein II.sub.A, and the SV40 early genes.
[0023] The PKC1 gene of budding yeast encodes a homolog of the
.alpha., .beta., and .gamma. isoforms of mammalian Protein Kinase C
that regulates a MAPK-activation pathway. Loss of PKC1 function
results in a cell lysis defect that is due to a deficiency in cell
wall construction.
SUMMARY OF THE INVENTION
[0024] The present invention provides drug screening assays for
identifying pharmaceutically effective compounds that specifically
inhibit the biological activity of fungal GTPase proteins,
particularly GTPases involved in cell wall integrity, hyphael
formation and other cell functions critical to pathogenesis.
Briefly, as described in greater detail below, Applicants have
discovered the critical involvment of Rho-like GTPase activities in
cell wall integrity. For instance, the fungal Rho1 GTPase is
required for glucan synthase activity, copurifies with
1,3-.beta.-glucan synthase, and is found to associate with the
Gsc1/Fks1 subunit of this complex in vivo. Rho1 is an regulatory
subunit of 1,3-.beta.-glucan synthase, and accordingly this
interaction, and the resulting enzyme complex, are potential
therapeutic targets for development of antifungal agents. Moreover,
Rho1 is required for protein kinase C (PKC1) mediated MAPK
activation, amd confers upon PKC1 the ability to be stimulated by
phosphatidylserine (PS), indicating that Rho1 controls signal
transmission through PKC1. Loss of PCK1 activity results in cell
lysis. Also, we demonstrate that prenylation of Rho1 by a
geranylgeranyl transferase is a critical step to maintenance of
cell wall integrity in yeast. As described in the appended
examples, prenylation of Rho1 is required for sufficient glucan
synthase activity. Loss of Rho1 prenylation results in cell lysis.
In general, a salient feature of the subject assays is that the
each is generated to detect agent which are potentially cytotoxic
to a fungal cell, rather than merely cytostatic. Moreover, given
the uniqueness of the therapeutic fungal targets of the present
assays, e.g., relative to homolgous proteins in mammalian cells,
the therapeutic targeting of Rho-like GTPase(s) involvement in such
interactions and complexes in yeast presents an opportunity to
define antifungal agents which are highly selective for yeast cells
relative to mammalian cells.
[0025] In one aspect, the present invention provides an assay for
identifying potential anti-fungal agents by targeting the
GGPTase/GTPase interaction. For instance, the assay can be run by
forming a reaction mixture including (i) a fungal geranylgeranyl
transferase (GGPTase), (ii) a substrate for the GGPTase, such as a
target polypeptide comprising a fungal Rho-like GTPase such as
Rho1, Rho2, Rsr1/Bud1 and Cdc42, or a polypeptide portion thereof
including at least one of (a) a prenylation site which can be
enzymatically prenylated by the GGPTase, or (b) a GGPTase binding
sequence which specifically binds the GGPTase, and (iii) a test
compound. The interaction of the target polypeptide with the
GGPTase can be detected. A statistically significant decrease in
the interaction of the target polypeptide and GGPTase in the
presence of the test compound, relative to the level of interaction
in the absence of the test compound (or other control), indicates a
potential anti-fungal activity for the test compound.
[0026] The reaction mixture can be a reconstituted protein mixture,
a cell lysate or a whole cell. For instance, the reaction mixture
can be a prenylation system including an activated geranylgeranyl
group, and the step of detecting the interaction of the target
polypeptide with the GGPTase includes detecting conjugation of the
geranylgeranyl group to the target polypeptide. In preferred
embodiments of such prenylation systems at least one of the
geranylgeranyl group and the target polypeptide has a detectable
label, and the level of geranylgeranyl group conjugated to the
target polypeptide is quantified by detecting the label in at least
one of the target polypeptide, free geranylgeranyl groups, and
geranylgeranyl-conjugated target polypeptide. As illustrated below,
the substrate target can incorporate a fluorescent (or other)
label, the fluorescent characterization of which is altered by the
level of prenylation of the substrate target, e.g., the substrate
target can be a dansylated peptide substrate of the fungal
GGPTase.
[0027] In other embodiments, the step of detecting the interaction
of the target polypeptide with the GGPTase includes detecting the
formation of protein-protein complexes including the target
polypeptide with the GGPTase. For example, at least one of the
GGPTase and the target polypeptide can include a detectable label,
and the level of GGPTase/target polypeptide complexes formed in the
reaction mixture is quantified by detecting the label in at least
one of the target polypeptide, the GGPTase, and GGPTase/target
polypeptide complexes. Exemplary labels for such embodiments, and
for the prenylation assays above, include radioisotopes,
fluorescent compounds, enzymes, and enzyme co-factors. For
instance, the detectable label can be a protein having a measurable
activity, and one of the PKC or GTPase is fusion protein including
the detectable label. In other exemplary embodiments, conjugation
of the geranylgeranyl group to the target polypeptide is detected
by an immunoassay.
[0028] Where the reaction mixture is a whole cell, the cell will
preferably include heterologous nucleic acid recombinantly
expressing one or more of the fungal GGPTase subunits and target
polypeptide. In certain preferred embodiments, the cell will also
include a heterologous reporter gene construct having a reporter
gene in operable linkage with a transcriptional regulatory sequence
sensitive to intracellular signals transduced by interaction of the
target polypeptide and GGPTase.
[0029] In one preferred embodiment, the assay includes forming a
cell-free reaction mixture including: (i) a fungal GGPTase, (ii) a
GGPTase substrate, e.g., a target polypeptide comprising a fungal
Rho-like GTPase, or a polypeptide portion thereof including a
prenylation site, (iii) an activated geranylgeranyl group, (iv) a
divalent cation, and (v) a test compound. The assay is derived to
detect conjugation of the gernaylgernayl group of the target
polypeptide in the reaction mixture, and a statistically
significant decrease in the prenylation of the target polypeptide
and GGPTase in the presence of the test compound, relative to an
appropriate control, indicates a potential anti-fungal activity for
the test compound.
[0030] In another preferred embodiment, the method utilizes an
interaction trap system including (a) a first fusion protein
comprising at least a portion of a fungal GGPTase subunit, (b) a
second fusion protein comprising at least a portion of a fungal
GTPase, and (c) a reporter gene, including a transcriptional
regulatory sequence sensitive to interactions between the GGPTase
portion of the first fusion protein and the GTPase portion of the
second polypeptide. After contacting the interaction trap system
with a candidate agentthe level of expression of a reporter gene is
measured and compared to the level of expression in the absence of
the candidate agent. A decrease in the level of expression of the
reporter gene in the presence of the candidate agent is indicative
of an agent that inhibits interaction of the GGPTase and
GTPase.
[0031] In still another embodiment, the assay is derived from a a
recombinant cell expressing a recombinant form of one or more of a
fungal GGPTase and a fungal Rho-like GTPase. The cell is contacted
with a test compound, and the level of interaction of the GGPTase
and Rho-like GTPase is detected. A statistically significant change
in the level of interaction of the GGPTase and Rho-like GTPase is
indicative of an agent that modulates the interaction of those two
proteins. In preferred embodiments, one or both of a GGPTase
subunit or the Rho-like GTPase are fusion proteins, e.g., the
fusion protein providing a detectable label and/or an affinity tag
for purification. In a preferred embodiment, the Rho-like GTPase is
a fusion protein further comprising a transcriptional regulatory
protein, and level of prenylation of the Rho-like GTPase is
detected by measuring the level of expression of a reporter gene
construct which is sensitive to the transcriptional regulatory
protein portion of the fusion protein, wherein inhibition of
prenylation of the fusion protein results in loss of membrane
partitioning of the fusion protein and increases expression of the
reporter gene construct.
[0032] In other preferred embodiments, the level of interaction of
the GGPTase and Rho-like GTPase is detected by detecting
prenylation of the Rho-like GTPase.
[0033] In yet another preferred embodiment, the assay is generated
from a set of cells in which prenylation of endogenous Rho-like
GTPases by GGPTase I is made dispensible. According to this
embodiment, the assay provides a first test cell in which one or
more Rho-like GPTases are mutated to be a substrate for a farnesyl
transferase expressed by the cell such that GGPTase I is
dispensible for cell growth; and a second test cell identical to
the first cell except that the Rho-like GTPases are substrates for
GGPTase I and are indispensible for cell growth. The first and
second cells are contacted with a candidate agent, and the level of
prenylation of the Rho-like GTPases in first and second test cells
are compared. A statistically significant decrease in the
prenylation of the GTPases in the second test cell, relative to the
level of prenylation of the GTPase in the first cell, is indicative
of an agent that inhibits interaction of a GGPTase and GTPase.
[0034] Yet another aspect of the present invention, the subject
assays are derived for detecting agents which disrupt the formation
of, or function of fungal protein complexes including Rho-like
GTPases and PKC proteins. In one embodiment, the assay provides a
reaction mixture including a fungal Rho-like GTPase, a fungal
protein kinase C (PKC), and a test compound. Interaction of the
Rho-like GTPase and PKC is detected in the reaction mixture,
wherein a statistically significant decrease in the interaction of
the Rho-like GTPase and PKC in the presence of the test compound,
relative to the level of interaction in the absence of the test
compound, indicates a potential antifungal activity for the test
compound.
[0035] The reaction mixture can be a reconstituted protein mixture,
a cell lysate or a whole cell. In preferred embodiments, the
reaction mixture is a kinase system including ATP and a PKC
substrate, and the step of detecting interaction of the GTPase and
PKC includes detecting phosphorylation of the PKC substrate by a
PKC/GTPase complex. Preferably, at least one of the PKC substrate
and ATP includes a detectable label, and the level of
phosphorylation of the PKC substrate is quantified by detecting the
label in at least one of the phosphorylated PKC substrate or ATP.
For instance, the PKC substrate may include a fluorescent (or
other) label, the fluorescent characterization of which is altered
by the level of phosphorylation of the PKC substrate.
[0036] In other preferred embodiments, the step of detecting the
interaction of the GTPase with the PKC includes detecting the
formation of protein-protein complexes including the GTPase and
PKC. For instance, at least one of the PKC and GTPase includes a
detectable label, and the level of PKC/GTPase complexes formed in
the reaction mixture is quantified by detecting the label in at
least one of the GTPase, the PKC, and PKC/GTPase complexes. For
instance, phosphorylation of the PKC substrate is detected by
immunoassay.
[0037] Cell-based assays are also provided, including cells
comprising reporter gene constructs sensitive to PKC/GTPase
complexes. In one embodiment, PKC/GTPases interaction trap assays
are used for drug screening according to the present invention.
[0038] In still another aspect of the present invention, the
subject assays are derived for detecting agents which disrupt the
formation of, or function of fungal protein complexes including
Rho-like GTPases and glucan synthase complexes or subunits thereof.
In a preferred embodiment, the assay includes forming a reaction
mixture including a fungal Rho-like GTPase, a fungal glucan
synthase complex or subunit thereof (collectively "GS protein"),
and a test compound. The interaction of the Rho-like GTPase and GS
protein can be detected in the reaction mixture. Similar to the
assay embodiments set out above, a statistically significant
decrease in the interaction of the Rho-like GTPase and GS protein
in the presence of the test compound, relative to the level of
interaction in the absence of the test compound, indicates a
potential antifungal activity for the test compound.
[0039] The reaction mixture can be a reconstituted protein mixture,
a cell lysate or a whole cell. In preferred embodiments, the
reaction mixture is a glucan synthesis system including a GTP and a
UDP-glucose, and the step of detecting interaction of the GTPase
and GS protein includes detecting formation of glucan polymers in
the reaction mixture, e.g., the UDP-glucose can include a
detectable label, and the level of glucan polymer formation is
quantified by detecting the labeled glucan polymers.
[0040] In other embodiments, the step of detecting the interaction
of the GTPase with the GS protein includes detecting the formation
of protein-protein complexes including the GTPase and GS protein.
As above, at least one of the GS protein and GTPase can include a
detectable label, and the level of GS protein/GTPase complexes
formed in the reaction mixture is quantified by detecting the label
in at least one of the GTPase, the GS protein, and GS
protein/GTPase complexes. Alternatively, the formation of
protein-protein complexes including the GTPase and GS protein is
detected by an immunoassay.
[0041] As above, cell-based assays are also provided, including
cells comprising reporter gene constructs sensitive to GS/GTPase
complexes. Permeabilization of cells due to disruption of GS
activity by the test compound can also be detected by loss of
cytoplasmic localization or cytoplasmic exclusion (depending on the
embodiment) of a detectable label.
[0042] For each of the assay embodiments set out above, the assay
is preferably repeated for a variegated library of at least 100
different test compounds, though preferably libraries of at least
10.sup.3, 10.sup.5, 10.sup.7, and 10.sup.9 compunds are tested. The
test compound can be, for example, small organic molecules, and/or
natural product extracts.
[0043] Also, in preferred embodiments of the subject assay, one or
more of the GTPase of other proteins which interacting with the
GTPase (e.g., GGPTase subunits, PKC and glucan synthase subunits)
are derived from a human pathogen which is implicated in mycotic
infection.
[0044] The subject assay also preferably includes a further step of
preparing a pharmaceutical preparation of one or more compounds
identified as having potential antifungal activity.
[0045] Still another aspect of the invention concerns various
compositions and reagents for performing the subject drug screening
assays. For instance, the present invention provides a variety of
recombinant cells expressing one or more different fungal proteins
implicated as targets in the subject screening assays. In a
preferred embodiment, the recombinant cell includes exogenous
nucleic acid (e.g., expression vectors) encoding a fungal Rho-like
GTPase. In a more preferred embodiment, the recombinant cell
includes (i) exogenous nucleic acid(s) encoding one or more
subunits of a fungal geranylgeranyl protein transferase (GGPTase),
and (ii) exogenous nucleic acid encoding a fungal Rho-like GTPase
or a fragment thereof including at least one of (a) a prenylation
site which can be enzymatically prenylated by the GGPTase, or (b) a
GGPTase binding sequence which specifically binds the GGPTase. In
still other preferred embodiments, the cell inlcudes (i) exogenous
nucleic acid encoding a fungal Rho-like GTPase, and (ii) exogenous
nucleic acid encoding a fungal protein selected from the group
consisting of a fungal protein kinase C (PKC) or one or more
subunits of a fungal glucan synthase.
[0046] The nucleic acids encoding the GGPTase, GTPase, PKC and/or
glucan synthase are preferably derived from a human pathogen which
is implicated in mycotic infection. For instance, the recombinant
genes can be derived from fungus involved in such mycotic
infections as selected from a group consisting of candidiasis,
aspergillosis, mucormycosis, blastomycosis, geotrichosis,
cryptococcosis, chromoblastomycosis, penicilliosis,
conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis,
maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and
sporotrichosis. To further illustrate, the expression vectors can
be generated from genes cloned from human pathogen selected from a
group consisting of Candida albicans, Candida stellatoidea, Candida
tropicalis, Candida parapsilosis, Candida krusei, Candida
pseudotropicalis, Candida quillermondii, Candida rugosa,
Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,
Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus,
Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor
pusillus. Another source for recombinant genes is the human
pathogen is Pneumocystis carinii.
[0047] In preferred embodiments, the cell is a recombinantly
manipulated yeast cell selected from the group consisting of such
genuses as Kluyverei, Schizosaccharomyces, Ustilaqo and
Saccharomyces, though a prefered host cell is the
Schizosaccharomyces cerivisae cell. Moreover, the host cell can be
constitutively or inducibly defective for an endogenous activity
corresponding to one or more of the GGPTase and GTPase encoded by
the exogenous nucleic acids.
[0048] In similar fashion, another aspect of the present invention
concerns reconstituted protein mixtures or cell lysate mixtures
including a recombinant fungal Rho-like GTPase, .e.g, or a fragment
thereof including at least one of (a) a prenylation site which can
be enzymatically prenylated by the GGPTase, or (b) a GGPTase
binding sequence which specifically binds the GGPTase, along with
one or more of a recombinant fungal glucan synthase, a recombinant
fungal GGPTase, and/or a recombinant fungal PKC. As above, the
fungal target proteins are preferably derived from a human pathogen
which is implicated in mycotic infection.
[0049] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1. Overexpression of PKC1 suppresses the cell lysis
defect of a rho1.sup.ts mutant. (A) The rho1-5 allele lyses at
restrictive temperature. Yeast strains patched on a YPD plate were
incubated at 23.degree. C. for 3 days, then shifted overnight to
37.degree. C. The patches were assayed in situ for release of
alkaline phosphatase as an indication of cell lysis. 1, wild-type;
2, rho1-3; 3, rho1-5; 4, pkc1.sup.ts (stt1-1; SYT11-12A). (B) An
episomal plasmid (YEp352) with or without PKC1 was transformed into
the rho1.sup.ts mutants (rho1-3 and rho1-5). Transformants were
streaked onto a YPD plate and incubated at 37.degree. C. for 3
days.
[0051] FIG. 2. RHO1 is required for Mpk1 activation in response to
heat shock. (Top panel) Phosphorylation of myelin basic protein
(MBP) by Mpk1.sup.HA immunoprecipitated from extracts of cells
shifted from growth at 23.degree. C. to 39.degree. C. for 30 min.
This treatment did not affect the viability of the mutant strains
(data not shown). Mpk1 activity in rho1-5 (lanes 4 and 5) and
rho1-3 (lanes 6 and 7) relative to wild-type (RHO1; lane 1-3)
maintained at 23.degree. C. (lane 1) is indicated. (Bottom panel)
Immunoblot of immunoprecipitated Mpk1.sup.HA.
[0052] FIG. 3. PKC1 associates with Rho1 in vivo and in vitro. (A)
.sup.HARho1 was immunoprecipitated from extracts of cells growing
at 23.degree. C. (lane 4), or shifted from 23.degree. C. to
39.degree. C. for 30 min (lane 6). .sup.HARho1 immunoprecipitates
(left) and whole-cell extracts (100 .mu.g protein; right) were
analyzed by immunoblot with anti-PKC1 antibodies (top panels), or
with anti-HA (to detect .sup.HARho1; bottom panels). Untagged Rho1
was used as a negative control (lanes 1, 2, and 7). Band indicated
by * is derived from immunoprecipitating antibodies. (B)
Recombinant GST-Rho1 (1 .mu.g), purified from Sf9 insect cells and
bound to glutathione agarose beads, was preloaded with the
indicated guanine nucleotide (lanes 2-5). Soluble yeast cell
extract (400 .mu.g protein) containing PKC1.sup.HA was incubated
with the beads (lanes 1, 3, and 5), and bound PKC1.sup.HA was
detected by immunoblot analysis. A control in which naked
glutathione agarose beads were used (lane 1) demonstrates
dependence of PCK1.sup.HA binding on GST-Rho1.
[0053] FIG. 4. Rho1 allows cofactors to activate PKC1. (A)
Phosphorylation of synthetic Bck1 peptide by PKC1.sup.HA
immunoprecipitated from 50 .mu.g of soluble yeast cell extract
protein. Recombinant GST-Rho1 or GST-Cdc42 (1 .mu.g) was preloaded
with the indicated guanine nucleotide. Cofactors (80 .mu.g/ml PS, 8
.mu.g/ml DAG, and 100 .mu.M CaCl.sub.2) were added to the reaction
where indicated. Lanes 1 and 2 are control reactions with no
GTPase. Mean and standard error for three experiments is shown. (B)
PS alone is sufficient to stimulate PKC1 fully in the presence of
Rho1. Phosphorylation of Bck1 peptide by PKC1.sup.HA in the
presence of GTP.gamma.S-bound GST-Rho1 and the indicated cofactors.
Conditions were as in A, except for PMA (16 ng/ml). Concentrations
of PS as low as 8 .mu.g/ml fully activated PKC1 (data not
shown).
[0054] FIG. 5. Model for the dual role of Rho1 in the maintenance
of cell integrity.
[0055] FIG. 6. GS activity from rho1 mutants (See reference of
Example 3). (A) GS activity is thermolabile in rho1.sup.ts mutants.
Crude extracts were made from cells growing at room temperature,
and assayed for GS activity at the indicated temperatures in the
presence of 50 .mu.M GTP.gamma.S. (B) Reconstitution of GS activity
in rho1-3 membranes with recombinant Rho1. GS activity in rho1-3
membrane fractions was measured at 37.degree. C. in the presence of
1 .mu.g of the indicated recombinant GTPase and 50 .mu.M
GTP.gamma.S (19). (C) Reconstituted GS activity requires GTP. GS
activity in wild-type membranes or rho1-3 membranes complemented
with 1 .mu.g of GST-Rho1 was measured at 37.degree. C. in the
presence of the indicated guanine nucleotide (20 .mu.M). Results
are expressed as percent activity relative to GTP.gamma.S.
[0056] FIG. 7. GS activity in a constitutively active RHO1 mutant
is GTP independent. Cultures of rho1-3 cells harboring plasmids
with either RHO1 or RHO1-Q68H (M. S. Boguski et al. (1992) New
Biol. 4:408) under the control of the inducible GAL1 promoter were
grown at room temperature in medium containing 2% raffinose
(repressing conditions). Galactose was added (to 2%) to half of
each culture, and cells were cultured for an additional 4 h to
induce expression of RHO1. GS activity in membrane fractions was
assayed at 37.degree. C. in the presence or absence of
GTP.gamma.S.
[0057] FIG. 8. Rho1 and Gsc1/Fks1 are enriched during purification
of GS. GS was purified from a wild-type strain (A451; 3). (A)
Immunoblot analysis of Rho1 (upper) and Gsc1/Fks1 (lower) through
purification (See reference 20 of Example 2). (B) GS specific
activity through purification. Purification steps were: lane 1,
membrane fraction; lane 2, detergent extract; lane 3, first product
entrapment; lane 4, second product entrapment.
[0058] FIG. 9. (A) Coimmunoprecipitation of Rho1 with Gsc1/Fks1
(21). Partially purified GS was incubated with anti-Gsc1/Fks1
monoclonal antibodies, 1A6 (lane 1) and 1F4 (lane 2), and
anti-human endothelin B type receptor (lane 3) (3).
Immunoprecipitates were analyzed by SDS-PAGE followed by
immunoblotting. (B) Colocalization of Gsc1/Fks1 and Rho1 at sites
of cell wall remodeling (See reference 22 of Example 2). Indirect
immunofluorescence microscopy was used to visualize Gsc1/Fks1 and
.sup.HARho1 in double-stained cells.
[0059] FIG. 10. Alignments of the .beta.-subunits of GGPTase-Is
showing cal1/cdc43 mutations. Positions of the cal1/cdc43 mutations
are shown under the box representing the CAL1/CDC43 coding region.
The closed box represents the homologous region among the
.beta.-subunits of the protein isoprenyltransferase. Cluster was
used to align Cal1p, and the .beta.-subunits of the S. pombe, rat
and human GGPTase-Is near the cal1/cdc43 mutation points.
[0060] FIG. 11. Overproduction of CDC42 is toxic in cal1-1 cells.
cal1-1 (1), cdc43-2 (2), cdc43-3 (3) cdc43-4 (4) cdc43-5 (5),
cdc43-6 (6), cdc43-7 (7) and wild-type strain (M. S. Boguski et al.
(1992) New Biol. 4:408) harboring pGAL-CDC42 were streaked on the
plate containing glucose (A) or galactose (B), and incubated at
23.degree. C. for 1 week.
[0061] FIG. 12. Fractionation of Rho1p and Cdc42p in wild-type and
mutant strains. Yeast strains were grown to midlog phase at the
permissive temperature (23.degree. C.), shifted to the restrictive
temperature, collected after 2 hr (37.degree. C.), and the cell
lysates were prepared. Rho1p was detected by Western blotting
analysis with guinea pig polyclonal antibody against Rho1p. In
order to express HA-tagged version of Cdc42p, yeast strains
transformed with pYO920 were incubated at 23.degree. C. in 2%
galactose-containing medium for 6 hr before the temperature shift.
HA-tagged version of Cdc42p was detected by Western blotting
analysis with 12CA5. WT, YPH500; cal1-1, YOT159-3C; cdc43-5,
YOT435-1A.
[0062] FIG. 13. Reduced GS activity in the membrane fractions of
GGPTase I-deficient cells. Cultures of wild-type (YPH500), cal1-1
(YOT159-3C), cdc43-5 (YOT435-1A) cells were grown at room
temperature in YPD medium. GS activity in membrane fractions was
assayed at 30.degree. C. according to Inoue et al. (1995) Eur. J.
Biochem. 231: 845. Reconstitution of GS activity in cal1-1 membrane
was performed with recombinant mutant Rho1 (G19V) which is
constitutively active for its activity.
[0063] FIG. 14. Thin section electron micrograph of Pkc1-depleted
cells demonstrating cell lysis.
DETAILED DESCRIPTION OF THE INVENTION
[0064] The use of, and need for anti-fungal agents is widespread
and ranges from the treatment of mycotic infections in animals; to
additives in feed for livestock to promote weight gain; to
disinfectant formulations. In general, a salient feature of
effective anti-fungal agents is that the agent is cytotoxic to a
fungal cell rather than only cytostatic. The mere knowledge that a
particular protein is critical to cell growth is accordingly not
sufficient to render that protein a suitable target for generation
of anti-fungal agents. Rather, assays which are useful for
identifying potential anti-fungal agents should target a fungal
bioactivity which, when altered in a particular manner, results in
cell death rather than quiescence or sporulation. For example, as
is illustrated in FIG. 14, cell lysis is a preferred outcome to
treatment with the potential antifungal agent in order to ensure
destruction of the pathogen. Moreover, at least for anti-fungal
agents which are to be administered to humans and other animals,
the therapeutic index is preferably such that toxicity to the host
is several orders of magnitude less than it is for the targeted
fungus.
[0065] The present invention relates to rapid, reliable and
effective assays for screening and identifying pharmaceutically
effective compounds that specifically inhibit the biological
activity of fungal GTPase proteins, particularly GTPases involved
in cell wall integrity, hyphael formation, and other cellular
functions critical to pathogenesis.
[0066] The cell wall of many fungus, as set out above, is required
to maintain cell shape and integrity. The main structural component
responsible for the rigidity of the yeast cell wall is
1,3-.beta.-linked glucan polymers with some branches through
1,6-.beta.-linkages. The biochemistry of the yeast enzyme
catalyzing the synthesis of 1,3-.beta.-glucan chains has been
studied extensively, but little was previously known at the
molecular level about the genes encoding subunits of this enzyme.
Only a pair of closely related proteins (Gsc1/Fks1 and Gsc2/Fks2)
had previously been described as subunits of the 1,3-.beta.-glucan
synthase (GS) (Inoue et al., (1995) Eur. J. Biochem. 231:845; and
Douglas et al., (1994) PNAS 91:12907). GS activity in many fungal
species, including S. cerevisiae, requires GTP or a
non-hydrolyzable analog (e.g. GTP.gamma.S) as a cofactor,
suggesting that a GTP-binding protein stimulates this enzyme (Mol
et al. (1994) J. Biol. Chem. 269:31267).
[0067] As described in the appended examples, we demonstrate that
the Rho1 GTPase activity is required for glucan synthase activity,
copurifies with 1,3-.beta.-glucan synthase, and is found to
associate with the Gsc1/Fks1 subunit of this complex in vivo. Both
proteins were also found to reside predominantly at sites of cell
wall remodeling. Therefore, Rho1 is an regulatory subunit of
1,3-.beta.-glucan synthase, and accordingly this interaction, and
the resulting enzyme complex, are potential therapeutic targets for
development of antifungal agents. Moreover, given the uniqueness of
the yeast glucan cell wall relative to mammalian cells, the
therapeutic targeting of Rho-like GTPase(s) involvement in glucan
synthase complexes in yeast presents an opportunity to define
antifungal agents which are highly selective for yeast cells
relative to mammalian cells.
[0068] We have also discovered other interactions with Rho1-like
GTPase which are consequential to cell integrity in yeast. As
described in the appended examples, we find that Rho1 is required
for protein kinase C (PKC1) mediated MAPK activation. Moreover,
PKC1 co-immunoprecipitates with Rho1 in yeast extracts, and
recombinant Rho1 associates with PKC1 in vitro in a GTP-dependent
manner. Moreover, the data provided herein demonstrates that
recombinant Rho1 confers upon PKC1 the ability to be stimulated by
phosphatidylserine (PS), indicating that Rho1 controls signal
transmission through PKC1. This applications provides the first
example of a PKC isoform whose stimulation by cofactors is
dependent on a GTPase, and provides the basis for yet other drug
screening assays that target the interaction of a PKC and GTPase,
or the catalytic activity of the resulting complex. Furthermore, no
mammalian PKC activities have been reported to require a G-protein
co-factor, suggesting that the fungal Rho/PKC complex represents a
specific target for developing antiproliferative agents selective
for yeast cells.
[0069] Finally, we have demonstrated that prenylation of Rho1 by a
geranylgeranyl transferase is a critical step to maintenance of
cell wall integrity in yeast. As described in the appended
examples, prenylation of Rho1 is required for sufficient glucan
synthase activity. Taken together with the results respecting
Rho1's participation as a GS subunit, we demonstrate that not only
is the prenylatin of Rho1 by GGPTase I critical to cell growth, but
inhibition of the prenylation reaction is a potential target for
developing a cytotoxic agent for killing various fungi. Moreover,
the relatively high divergence between fungal and human GGPTase
subunits suggests that selectivity for the fungal GGPTase activity
may be obtained to provide antifungal agents having desirable
therapeutic indices.
[0070] In one embodiment, the subject assay comprises a prenylation
reaction system that includes a fungal geranylgeranyl protein
transferase (GGPTase), a fungal GTPase protein, or a portion
thereof, which serves as a prenylation target substrate, and an
activated geranylgeranyl moiety which can be covalent attached to
the prenylation substrate by the GGPTase. The level of prenylation
of the target substrate brought about by the system is measured in
the presence and absence of a candidate agent, and a statistically
significant decrease in the level prenylation is indicative of a
potential anti-fungal activity for the candidate agent.
[0071] As described below, the level of prenylation of the GTPase
target protein can be measured by determining the actual
concentration of substrate:geranylgeranyl conjugates formed; or
inferred by detecting some other quality of the target substrate
affected by prenylation, including membrane localization of the
target. In certain embodiments, the present assay comprises an in
vivo prenylation system, such as a cell able to conduct the target
substrate through at least a portion of a geranylgeranyl
conjugation pathway. In other embodiments, the present assay
comprises an in vitro prenylation system in which at least the
ability to transfer isoprenoids to the GTPase target protein is
constituted. Still other embodiments provide assay format which
detect protein-protein interaction between the GGPTase and a target
protein, rather than enzymatic activity per se.
[0072] With respect to the interaction of the fungal GTPase with
other cellular components, and the significance of those
interactions to cell wall integrity, another aspect of the present
invention relates to assays which seek to identify agents which
alter protein-protein interactions involving a fungal GTPase and
PKC or glucan synthase subunits, or which inhibit the catalytic
activity of a protein complex resulting from such interactions. For
instance, as described in more detail below, one therapeutic target
of interest are glucan synthase complexes which include a Rho1-like
GTPase. In another embodiment, the therapeutic target is a protein
kinase C complex including a GTPase. The particular assay format
selected will reflect the desire to identify compounds which
disrupt protein-protein interactions and thereby alter the enzyme
complex, or which disrupt the interaction with, and chemical
alteration of a given substrate by the enzyme complex. For
instance, the interaction with, and chemical alteration of a given
substrate by the enzyme complex. For instance, the interaction of
Rho1 with the glucan synthase subunit Gce1 can be the screening
target in some embodiments, while the synthase activity of the
resulting complex can be the screening target in other embodiments.
Likewise, screening assays targeting PKC1/Rho1 complex can provide
agents which disrupt the formation of the complex, or target the
complex's interaction with substrate proteins.
[0073] As described herein, inhibitors of a fungal GTPase
bioactivity refer generally to those agents which may act anywhere
along the prenylation pathway, e.g., from the reaction steps
leading up to and including conjugation of an isoprenoid to the
GTPase target, to the interaction of the GTPase protein with other
cellular proteins, such as glucan synthase subunits and/or PKC. A
subset of this class of inhibitors comprises the prenylation
inhibitors, which include those agents that act at the level of
preventing conjugation of geranylgeranyl moieties to the target
GTPase, rather than at the steps of protein-protein interactions
involving the prenylated GTPase, e.g., as part of enzymatic
complexes. Moreover, as will be clear from the following
description, particular embodiments of the present assay can be
chosen so as to discriminate between prenylation inhibitors and
inhibitors of prenylated-GTPase complexes.
[0074] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0075] As used herein, "recombinant cells" include any cells that
have been modified by the introduction of heterologous DNA. Control
cells include cells that are substantially identical to the
recombinant cells, but do not express one or more of the proteins
encoded by the heterologous DNA, e.g., do not include or express a
recombinant Rho1-like GTPase, a recombinant GGPTase, a recombinant
glucan synthase and/or a recombinant PKC1.
[0076] The terms "recombinant protein", "heterologous protein" and
"exogenous protein" are used interchangeably throughout the
specification and refer to a polypeptide which is produced by
recombinant DNA techniques, wherein generally, DNA encoding the
polypeptide is inserted into a suitable expression vector which is
in turn used to transform a host cell to produce the heterologous
protein. That is, the polypeptide is expressed from a heterologous
nucleic acid.
[0077] As used herein, "heterologous DNA" or "heterologous nucleic
acid" include DNA that does not occur naturally as part of the
genome in which it is present or which is found in a location or
locations in the genome that differs from that in which it occurs
in nature. Heterologous DNA is not endogenous to the cell into
which it is introduced, but has been obtained from another cell.
Generally, although not necessarily, such DNA encodes RNA and
proteins that are not normally produced by the cell in which it is
expressed. Heterologous DNA may also be referred to as foreign DNA.
Any DNA that one of skill in the art would recognize or consider as
heterologous or foreign to the cell in which is expressed is herein
encompassed by heterologous DNA. Examples of heterologous DNA
include, but are not limited to, isolated DNA that encodes a
Rho1-like GTPase, a GGPTase, a glucan synthase and/or a PKC1.
[0078] "Inactivation", with respect to genes of the host cell,
means that production of a functional gene product is prevented or
inhibited. Inactivation may be achieved by deletion of the gene,
mutation of the promoter so that expression does not occur, or
mutation of the coding sequence so that the gene product is
inactive (constitutively or inducibly). Inactivation may be partial
or total.
[0079] "Complementation", with respect to genes of the host cell,
means that at least partial function of inactivated gene of the
host cell is supplied by an exogenous nucleic acid. For instance,
yeast cells can be "mammalianized", and even "humanized", by
complementation of Rho1 with mammalian homologs such as RhoA.
[0080] As used herein, a "reporter gene construct" is a nucleic
acid that includes a "reporter gene" operatively linked to a
transcriptional regulatory sequences. Transcription of the reporter
gene is controlled by these sequences. The transcriptional
regulatory sequences include the promoter and other regulatory
regions, such as enhancer sequences, that modulate the activity of
the promoter, or regulatory sequences that modulate the activity or
efficiency of the RNA polymerase that recognizes the promoter, or
regulatory sequences are recognized by effector molecules.
[0081] The term "substantially homologous", when used in connection
with amino acid sequences, refers to sequences which are
substantially identical to or similar in sequence, giving rise to a
homology in conformation and thus to similar biological activity.
The term is not intended to imply a common evolution of the
sequences.
[0082] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein.
[0083] As used here, the terms "geranylgeranyl protein transferase"
and "GGPTase are art recognized and refer to the enzyme complexes
responsible for the covalent modification of proteins with
geranylgeranyl moieties. Particular reference to fungal GGPTases
sub-types such as GGPTase-I, or the subunits of a fungal GGPTase,
such as cdc43 and RAM2 (unless otherwise evident from the contest)
is intended to refer generically to the analogous GGPTase complex
and/or subunits in any fungal cell. Accordingly, reference to the
subunit cdc43 (also referred to as CAL1 and DPR1) refers to the S.
cerevisiae-i subunit as well as homologous proteins in that cell or
other fungi which form a GGPTase I enzyme complex.
[0084] Likewise, the terms "Rho-like GTPase" and "fungal GTPase"
will refer generally to GTPases related structurally to the yeast
GTPases Rho1, Rho2, cdc42, and/or Rsr1/Bud1, whether the enzyme is
isolated from S. cerevisiae or other fungi.
[0085] In similar fashion, the term "glucan synthase" refers
generically to fungal enzymes involved in synthesis of a
.beta.-(1,3)-glucan and comprised of subunits including Gsc1 (also
called Fks1) homologs and Rho-like GTPases. As above, reference to
a "Gsc1 subunit" refers to the S. cerevisiae-i protein as well as
structurally and functionally related homologs from other
fungi.
[0086] The terms "PKC" and "PKC1" are also used generically to
refer to protein kinase C homologs in fungi, and other fungal
homologs of the PKC1 protein of S. cerevisiae, respectively.
[0087] The terms "fungi" and "yeast" are used interchangeably
herein and refer to the art recognized group of eukaryotic protists
known as fungi. That is, unless clear from the context, "yeast" as
used herein can encompass the two basic morphologic forms of yeast
and mold and dimorphisms thereof.
[0088] The present invention provides a systematic and practical
approach for the identification of candidate agents able to inhibit
one or more of the cellular functions of fungal GTPase proteins. In
a general sense, the assays of the present invention evaluate the
ability of a compound to modulate binding between a GTPase protein
and another protein, whether the GTPase is acting as a subunit of a
multiprotein complex or as a substrate for modification. The assays
may be formatted to evaluate the ability of a compound to modulate
(i) protein complexes which include a GTPase protein; (ii) the
enzymatic activity of such multiprotein complexes; or (iii) the
enzymatic activity which produces a prenylated GTPase.
[0089] Exemplary compounds which can be screened for activity
against fungal GTPase activity include peptides, nucleic acids,
carbohydrates, small organic molecules, and natural product extract
libraries, such as isolated from animals, plants, fungus and/or
microbes.
[0090] Cell-free Assay Formats
[0091] In many drug screening programs which test libraries of
compounds and natural extracts, high throughput assays are
desirable in order to maximize the number of compounds surveyed in
a given period of time. Assays which are performed in cell-free
systems, such as may be derived with purified or semi-purified
proteins or cell-lysates, are often preferred as "primary" screens
in that they can be generated to permit rapid development and
relatively easy detection of an alteration in a molecular target
which is mediated by a test compound. Moreover, the effects of
cellular toxicity and/or bioavailability of the test compound can
be generally ignored in the in vitro system, the assay instead
being focused primarily on the effect of the drug on the molecular
target as may be manifest in an alteration of binding affinity with
upstream or downstream elements. Accordingly, in an exemplary
screening assay of the present invention, a reaction mixture is
generated to include a fungal GTPase polypeptide, compound(s) of
interest, and a "target polypeptide", e.g., a protein, which
interacts with the GTPase polypeptide, whether as a prenylating
activity, or by some other protein-protein interaction. Exemplary
target polypeptides include GGPTase activities such as GGPTase I,
PKC homologs such as PKC1, and glucan synthase subunits such as
Gsc1. Detection and quantification of the enzymatic conversion of
the fungal GTPase, or the formation of complexes containing the
fungal GTPase protein, provide a means for determining a compound's
efficacy at inhibiting (or potentiating) complex the bioactivity of
the GTPase. The efficacy of the compound can be assessed by
generating dose response curves from data obtained using various
concentrations of the test compound. Moreover, a control assay can
also be performed to provide a baseline for comparison.
[0092] In one embodiment, the subject drug screening assay
comprises a prenylation system, e.g. a reaction mixture which
enzymatically conjugates isoprenoids to a target protein, which is
arranged to detect inhibitors of the prenylation of a Rho-like
GTPase with a geranylgeranyl group. For instance, in one embodiment
of a cell-free prenylation system, one or more cell lysates
including a fungal GGPTase, a fungal Rho-like GTPase (or substrate
analog thereof), and an activated geranylgeranyl group are
incubated with the test compound and the level of prenylation of
the Rho-like GTPase substrate is detected. Lysates can be derived
from cells expressing one or more of the relevant proteins, and
mixed appropriately (or spilled) where no single lysate contains
all the components necessary for generating the prenylation system.
In preferred embodiments, one or more of the components, especially
the substrate target, are recombinantly produced in a cell used to
generate a lysate, or added by spiking a lysate mixture with a
purified or semi-purified preparation of the substrate. These
embodiments have several advantages including: the ability to use a
labeled substrate, e.g. a dansylated peptide, or fusion protein for
facilitating purification e.g. a Rho1-GST fusion protein; the
ability to carefully control reaction conditions with respect to
concentrations of reactants; and where targets are derived from
fungal pathogens, the ability to work in a non-pathogenic system by
recombinantly or synthetically producing by components from the
pathogen for constituting the prenylation system.
[0093] The prenylates can be derived from any number of cell types,
ranging from bacterial cells to yeast cells to cells from metazoan
organisms including insects and mammalian cells. To illustrate, a
fungal prenylation system can be reconstituted by mixing cell
lysates derived from insect cells expressing fungal GGPTase
subunits cloned into baculoviral expression vectors. For example,
the exemplary GGPTase-I expression vectors described below in the
section Reagents-i can be recloned into baculoviral vectors (e.g.
pVL vectors), and recombinant GGPTase-I produced in transfected
spodoptera-i fungiperda cells. The cells can than be lysed, and if
the RAM2 and CDC43 subunits are produced by different sets of
cells, cell lysates can be accordingly mixed to produce an active
fungal GGPTase. The level of activity can be assessed by enzymatic
activity, or by quantitating the level of expression by detecting,
e.g., an exogenous tag added to the recombinant protein. Substrate
and activated geranylgeranyl diphosphate can be added to the lysate
mixtures. As appropriate, the transfected cells can be cells which
lack an endogenous GGTase activity, or the substrate can be chosen
to be particularly sensitive to prenylation by the exogenous fungal
GGTPase relative to any endogenous activity of the cells. In other
cell-free embodiments of the present assay, the prenylation system
comprises a reconstituted protein mixture of at least semi-purified
proteins. By semi-purified, it is meant that the proteins utilized
in the reconstituted mixture have been previously separated from
other cellular proteins. For instance, in contrast to cell lysates,
the proteins involved in conjugation of geranylgeranyl moieties to
a target protein, together with the target protein, are present in
the mixture to at least 50% purity relative to all other proteins
in the mixture, and more preferably are present at 90-95% purity.
In certain embodiments of the subject method, the reconstituted
protein mixture is derived by mixing highly purified proteins such
that the reconstituted mixture substantially lacks other proteins
which might interfere with or otherwise alter the ability to
measure specific prenylation rates of the target GTPase
substrate.
[0094] Each of the protein components utilized to generate the
reconstituted prenylation system are preferably isolated from, or
otherwise substantially free of, other proteins normally associated
with the proteins in a cell or cell lysate. The term "substantially
free of other cellular proteins" (also referred to herein as
"contaminating proteins") is defined as encompassing individual
preparations of each of the component proteins comprising less than
20% (by dry weight) contaminating protein, and preferably comprises
less than 5% contaminating protein. Functional forms of each of the
component proteins can be prepared as purified preparations by
using a cloned gene as described below and known in the art. By
"purified", it is meant, when referring to the component protein
preparations used to generate the reconstituted protein mixture,
that the indicated molecule is present in the substantial absence
of other biological macromolecules, such as other proteins
(particularly other proteins which may substantially mask,
diminish, confuse or alter the characteristics of the component
proteins either as purified preparations or in their function in
the subject reconstituted mixture). The term "purified" as used
herein preferably means at least 80% by dry weight, more preferably
in the range of 95-99% by weight, and most preferably at least
99.8% by weight, of biological macromolecules of the same type
present (but water, buffers, and other small molecules, especially
molecules having a molecular weight of less than 5000, can be
present). The term "pure" as used herein preferably has the same
numerical limits as "purified" immediately above. "Isolated" and
"purified" do not encompass either protein in its native state
(e.g. as a part of a cell), or as part of a cell lysate, or that
have been separated into components (e.g., in an acrylamide gel)
but not obtained either as pure (e.g. lacking contaminating
proteins) substances or solutions. The term isolated as used herein
also refers to a component protein that is substantially free of
cellular material or culture medium when produced by recombinant
DNA techniques, or chemical precursors or other chemicals when
chemically synthesized.
[0095] In the subject method, prenylation systems derived from
purified proteins may have certain advantages over cell lysate
based assays. Unlike the reconstituted protein system, the
prenylation activity of a cell-lysate may not be readily
controlled. Measuring kinetic parameters is made tedious by the
fact that cell lysates may be inconsistent from batch to batch,
with potentially significant variation between preparations. In
vitro evidence indicates that prenyltransferases have the ability
to cross-prenylate CAAX-related sequences, so that famesyl
transferase present in a lysate may provide an unwanted kinetic
parameter. Moreover, cycling of prenylated proteins by guanine
nucleotide dissociation inhibitor (GDI)-like proteins in the lysate
could further complicate kinetics of the reaction mixture.
Evaluation of a potential inhibitor using a lysate system is also
complicated in those circumstances where the lysate is charged with
mRNA encoding the GTPase substrate polypeptide or GGPTase activity,
as such lysates may continue to synthesize proteins active in the
assay during the development period of the assay, and can do so at
unpredictable rates. Knowledge of the concentration of each
component of the prenylation system can be required for each lysate
batch, along with the overall kinetic data, in order to determine
the necessary time course and calculate the sensitivity of
experiments performed from one lysate preparation to the next. The
use of reconstituted protein mixtures can allow more careful
control of the reaction conditions in the prenylation reaction.
[0096] The purified protein mixture includes a purified preparation
of the substrate polypeptide and a geranylgeranyl isoprenoid (or
analog thereof) under conditions which drive the conjugation of the
two molecules. For instance, the mixture can include a fungal
GGPTase I complex including RAM2 and CDC43 subunits, a
geranylgeranyl diphosphate, a divalent cation, and a substrate
polypeptide, such as may be derived from Rho1.
[0097] Furthermore, the reconstituted mixture can also be generated
to include at least one auxiliary substrate recognition protein,
such as a Rab escort protein where GGPTase II is the prenylase
employed in the reaction mixture.
[0098] Prenylation of the target regulatory protein via an in vitro
prenylation system, in the presence and absence of a candidate
inhibitor, can be accomplished in any vessel suitable for
containing the reactants. Examples include microtitre plates, test
tubes, and micro-centrifuge tubes. In such embodiments, a wide
range of detection means can be practiced to score for the presence
of the prenylated protein.
[0099] In one embodiment of the present assay, the products of a
prenylation system are separated by gel electrophoresis, and the
level of prenylated substrate polypeptide assessed, using standard
electrophoresis protocols, by measuring an increase in molecular
weight of the target substrate that corresponds to the addition of
one or more geranylgeranyl moieties. For example, one or both of
the target substrate and geranylgeranyl group can be labeled with a
radioisotope such as .sup.35S, .sup.14C, or .sup.3H, and the
isotopically labeled protein bands quantified by autoradiographic
techniques. Standardization of the assay samples can be
accomplished, for instance, by adding known quantities of labeled
proteins which are not themselves subject to prenylation or
degradation under the conditions which the assay is performed.
Similarly, other means of detecting electrophoretically separated
proteins can be employed to quantify the level of prenylation of
the target substrate, including immunoblot analysis using
antibodies specific for either the target substrate or
geranylgeranyl epitopes.
[0100] As described below, the antibody can be replaced with
another molecule able to bind one of either the target substrate or
the isoprenoid. By way of illustration, one embodiment of the
present assay comprises the use of a biotinylated target substrate
in the conjugating system. Indeed, biotinylated GGPTase substrates
have been described in the art (c.f. Yokoyama et al. (1995)
Biochemistry 34:1344-1354). The biotin label is detected in a gel
during a subsequent detection step by contacting the
electrophoretic products (or a blot thereof) with a
streptavidin-conjugated label, such as a streptavidin linked
fluorochrome or enzyme, which can be readily detected by
conventional techniques. Moreover, where a reconstituted protein
mixture is used (rather than a lysate) as the conjugating system,
it may be possible to simply detect the target substrate and
geranylgeranyl conjugates in the gel by standard staining
protocols, including coomassie blue and silver staining.
[0101] In a similar fashion, prenylated and unprenylated substrate
can be separated by other chromatographic techniques, and the
relative quantities of each determined. For example, HPLC can be
used to quantitate prenylated and unprenylated substrate (Pickett
et al. (1995) Analytical Biochem 225:60-63), and the effect of a
test compound on that ratio determined.
[0102] In another embodiment, an immunoassay or similar binding
assay, is used to detect and quantify the level of prenylated
target substrate produced in the prenylation system. Many different
immunoassay techniques are amenable for such use and can be
employed to detect and quantitate the conjugates. For example, the
wells of a microtitre plate (or other suitable solid phase) can be
coated with an antibody which specifically binds one of either the
target substrate or geranylgeranyl groups. After incubation of the
prenylation system with and without the candidate agent, the
products are contacted with the matrix bound antibody, unbound
material removed by washing, and prenylated conjugates of the
target substrate specifically detected. To illustrate, if an
antibody which binds the target substrate is used to sequester the
protein on the matrix, then a detectable anti-geranylgeranyl
antibody can be used to score for the presence of prenylated target
substrate on the matrix.
[0103] Still a variety of other formats exist which are amenable to
high through put analysis on microtitre plates or the like. The
prenylation substrate can be immobilized throughout the reaction,
such as by cross-linking to activated polymer, or sequestered to
the well walls after the development of the prenylation reaction.
In one illustrative embodiment, a Rho-like GTPase, e.g. a fungal
Rho1, Rho2, Cdc42 or Rsr1/Bud1, is cross-linked to the polymeric
support of the well, the prenylation system set up in that well,
and after completion, the well washed and the amount of
geranylgeranyl sidechains attached to the immobilized GTPase
detected. In another illustrative embodiment, wells of a microtitre
plate are coated with streptavidin and contacted with the developed
prenylation system under conditions wherein a biotinylated
substrate binds to and is sequestered in the wells. Unbound
material is washed from the wells, and the level of prenylated
target substrate is detected in each well. There are, as evidenced
by this specification, a variety of techniques for detecting the
level of prenylation of the immobilized substrate. For example, by
the use of dansylated (described infra) or radiolabelled
geranylgeranyl diphosphaste in the reaction mixture, addition of
appropriate scintillant to the wells will permit detection of the
label directly in the microtitre wells. Alternatively, the
substrate can be released and detected, for example, by any of
those means described above, e.g. by radiolabel, gel
electrophoresis, etc. Reversibly bound substrate, such as the
biotin-conjugated substrate set out above, is particularly amenable
to the latter approach. In other embodiments, only the
geranylgeranyl moiety is released for detection. For instance, the
thioether linkage of the isoprenoid with the substrate peptide
sequence can be cleaved by treatment with methyl iodide. The
released geranylgeranyl products can be detected, e.g., by
radioactivity, HPLC, or other convenient format.
[0104] Other geranylgeranyl derivatives include detectable labels
which do not interfere greatly with the conjugation of that group
to the target substrate. For example, in an illustrative
embodiment, the assay format provides fluorescence assay which
relies on a change in fluorescent activity of a group associated
with a GGPTase substrate to assess test compounds against a fungal
GGPTase. To illustrate, GGPTase-I activity can be measured by a
modified version of the continuous fluorescence assay described for
farnesyl transferases (Cassidy et al., (1985) Methods Enzymol. 250:
30-43; Pickett et al. (1995) Analytical Biochem 225:60-63; and
Stirtan et al. (1995) Arch Biochem Biophys 321:182-190). In an
illustrative embodiment, dansyl-Gly-Cys-Ile-Ile-Leu (d-GCIIL) and
the geranylgeranyl diphosphate are added to assay buffer, along
with the test agent or control. This mixture is preincubated at
30.degree. C. for a few minutes before the reaction is initiated
with the addition of GGPTase enzyme. The sample is vigorously
mixed, and an aliquot of the reaction mixture immediately
transferred to a prewarmed cuvette, and the fluorescence intensity
measured for 5 minutes. Useful excitation and emission wavelengths
are 340 and 486 nm, respectively, with a bandpass of 5.1 nm for
both excitation and emission monochromators. Generally,
fluorescence data are collected with a selected time increment, and
the inhibitory activity of the test agent is determined by
detecting a decrease in the initial velocity of the reaction
relative to samples which lack a test agent.
[0105] In yet another embodiment, the geranylgeranyl transferase
activity against a particular substrate can be detected in the
subject assay by using a phosphocellulose paper absorption system
(Roskoski et al. (1994) Analytical Biochem 222:275-280), or the
like. To effect binding of a peptidyl substrate to phosphocellulose
at low pH, several basic residues can be added, preferably to the
amino-terminal side of the CAAX target sequence of the peptide, to
produce a peptide with a minimal minimum charge of +2 or +3 at pH
less than 2. This follows the strategy used for the
phosphocellulose absorption assay for protein kinases. In an
illustrative embodiment; the transfer of the [H.sup.3]
geranylgeranyl group from [H.sup.3]-geranylgeranyl pyrophosphate to
KLKCAIL or other acceptor peptides can be measured under conditions
similar to the famesyl transferase reactions described by Reiss et
al. (Reiss et al., (1990) Cell 62: 81-88) In an illustrative
embodiment, reaction mixtures can be generated to contain 50 mM
Tris-HCL (pH 7.5), 50 .mu.M ZnCl.sub.2, 20 mM KCl, 1 mM
dithiothreitol, 250 .mu.M KLKCAIL, 0.4 .mu.M [H.sup.3]
geranylgeranyl pyrophosphate, and 10-1000 .mu.g/ml of purified
fungal GGPTase protein. After incubation, e.g., for 30 minutes at
37.degree. C., samples are applied to Whatman P81 phosphocellulose
paper strips. After the liquid permeates the paper (a few seconds),
the strips are washed in ethanol/phosphoric acid (prepared by
mixing equal volumes of 95% ethanol and 75 mM phosphoric acid) to
remove unbound isoprenoids. The samples are air dried, and
radioactivity can be measured by liquid scintillation spectrometry.
Background values are obtained by using reaction mixture with
buffer in place of enzyme.
[0106] An added feature of this strategy is that it produces
hydrophilic peptides that are more readily dissolved in water.
Moreover, the procedure outlined above works equally well for
protein substrates (most proteins bind to phosphocellulose at
acidic pH), so should be useful where full length protein, e.g.,
Rho1 or Cdc42, are utilized as the GGPTase substrate.
[0107] Likewise, a variety of techniques are known in the art for
accessing the activity of a glucan synthase and can be adapted for
generating drug screening assays designed to detect inhibitors of a
fungal glucan synthase complex which includes a Rho-like GTPase. As
above, the cell-free glucan synthesis systems can be utilized in
the subject assay, and include reconstituted protein mixtures
and/or cell lysates/membrane preparations. Accordingly, in
preferred embodiments, the glucan synthesis system is derived from
purified protein preparations (preferably reconstituted in a lipid
formulation) or membrane preparations derived from a reagent cells,
e.g., a cell expressing a recombinant Rho1/Gsc1 complex. To
illustrate, membrane extracts are prepared from selected cells,
homogenized with glass beads, and unbroken cells and debris are
removed by centrifugation. The supernatant fluids are centrifuged
at high speed, and the resulting pellets are washed with buffer
containing 0.05M potassium phosphate (pH 7.5), 0.5 mM DTT, and 1.0
mM PMSF. The washed pellet is resuspended in the same buffer
containing 5% glycerol. This protein extract serves as the source
for .beta.(1-3)-glucan synthase in the enzymatic assays.
[0108] The .beta.(1-3)-glucan synthase reactions can be performed
similar to those described in the art (e.g., Cabib et al. (1987)
Methods Enzymol. 138:637-642) and the appended examples. Briefly, a
reaction mixture is generated containing Tris (or other suitable
buffer), dithiothretol, KF, glycerol, PMSF, UDP-glucose, guanosine
5'-(.gamma.-S)-triphosphate (GTP.gamma.S), UDP-[.sup.3H]glucose
(Amersham) plus a sample of membrane protein extract. Optionally,
.alpha.-amylase can be added to reaction mixtures to eliminate the
contribution of [3H]glucose incorporation into glycogen. The
reactions are performed in the presence or absence of the test
compound. Following incubation for a selected time, the
[.sup.3H]-glucose incorporated into trichloroacetic acid-insoluble
material is collected onto glass fiber filters and measured using a
liquid scintillation counter.
[0109] In still other embodiments of the subject assay, cell-free
mixtures can be utilized to identify agents which inhibit the
enzymatic activity of a fungal PKC/GTPase complex such as the
PKC1/Rho1 complex. In an exemplary embodiment, the kinase activity
of a PKC1/GTPase complex can assessed by such methods as described
in Watanabe et al. (1994) J Biol Chem 269:16829-16836. For
instance, phosphorylation reactions can be initiated by adding
reaction cocktail (40 mM MOPS pH 7.5, 10 mM MgCl.sub.2, 1 mM DTT,
50 .mu.M [.gamma.-.sup.32P]ATP [6 .mu.Ci/reaction], a substrate
peptide and the PKC/GTPase complex, and incubated for the reaction
to develop. Reactions can be terminated by adding 4.times.
Laemmli's sample buffer, and the samples boiled and subjected to
SDS/PAGE. After electrophoresis, gels are fixed in 12.5%
trichloroacetic acid for 10 min, washed in 10% methanol/10% acetic
acid to reduce background, dried, and subjected to autoradiography.
Likewise, capillary zone electrophoresis (CZE) techniques can be
used to separate and quantitate phosphorylated and unphosphorylated
PKC substrate, especially peptide substrates, following such
protocols as described by Dawson et al. (1994) Analytical Biochem
220: 340-345. Alternatively, reactions can be terminated by
spotting onto P81 paper (Whatman). The paper washed three times
with 75 mM H.sub.3PO.sub.4 and subjected to scintillation
counting.
[0110] In another embodiment, the assay is started with the
addition of enzyme and stopped after a set time by the addition of
25% trichloroacetic acid (TCA) and 1.0 mg/ml bovine serum albumin
(BSA). The radioactive product is retained and washed on glass
fiber filters that allow the unreacted .sup.32P-ATP to pass
through. As above, the amount of phosphorylation is determined by
the radioactivity measured in a scintillation counter.
[0111] In still another embodiment, the kinase substrate can be
separated by affinity tags. For instance, a biotinylated peptide
substrate of the PKC/Rho I complex can be provided in the kinase
reaction mixture with [.alpha..sup.32P]ATP, the .sup.32P label
incorporated into the peptide substrate can be detected by standard
scintillation methods. An advantage to the biotin-capture system is
that it tends to be more quantitative with respect to peptide
sequestration relative to, for example, phosocellulose paper.
[0112] The artificial substrate used can be a synthetic peptide
resembling the pseudosubstrate site of PKC1p. All known isoforms of
PKC possess a sequence within their regulatory domains that is
related to PKC phosphorylation sites, except for an alanine in
place of the target serine or threonine of a substrate. These
sequences, known as pseudosubstrate sites, have been proposed to
act as autoinhibitors of PKC activity. Autoinhibition is thought to
be relieved upon binding of activating cofactors to the regulatory
domain. Peptides resembling pseudosubstrate sites, except with a
serine or threonine in place of alanine, are known to be excellent
substrates for PKC (House et al. (1987) Science 238:1726-1728).
Therefore, one substrate that may be used to test fungal PKC1
complexes is the 15-amino acid peptide, GGLHRHGTIINRKEE,
corresponding to residues 394-408 of PKC1p of S. cerevisae (the
putative pseudosubstrate site), with a threonine in place of
alanine at position 401.
[0113] Yet another technique which can be used to follow the kinase
activity of a PKC/GTPase complex in the presence of a test agent
involves a spectrophotometric assay relying on an ADP produced by
the kinase-mediated phosphorylation reaction. Briefly, the
formation of ADP in the kinase reaction can be coupled to the
pyruvate kinase reaction to produce pyruvate which is, in turn,
coupled to the lactate dehydrogenase reaction with the concomitant
oxidation of DPNH to DPN+. The decrease in absorbance of 340 nm is
used to determine the reaction rate. See, for example, Roskosi
(1983) Methods Enzymol-i, 99:3-6.
[0114] In addition to the prenylation and other enzymatic
reaction-based assays, it is contemplated that any of the novel
protein-protein interactions described herein could be directly be
the target of a drug screening assay. For example, in one
embodiment, the interaction between a GTPase and a catalytic
subunit of a fungal glucan synthase, such as Gsc1/Fsk1 homologs,
can be detected in the presence and the absence of a test compound.
In another embodiment, the ability of a compound to inhibit the
binding of a GTPase protein with a fungal PKC-like protein, such as
PKC1, can be assessed in the subject assay. A variety of assay
formats for detecting non-enzymatic protein interactions will
suffice and, in light of the present invention, will be
comprehended by a skilled artisan.
[0115] Complex formation between the GTPase polypeptide and a
"target polypeptide" (e.g., a PKC polypeptide, a GS subunit, or a
GGPTase) may be detected by a variety of techniques. Modulation of
the formation of complexes can be quantitated using, for example,
detectably labeled proteins such as radiolabeled, fluorescently
labeled, or enzymatically labeled GTPase polypeptides, by
immunoassay, by chromatographic detection, or by detecting the
intrinsic activity of either the GTPase or target polypeptide.
[0116] Typically, it will be desirable to immobilize either the
GTPase or the target polypeptide to facilitate separation of
complexes from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay. Binding of a GTPase
polypeptide to the target polypeptide, in the presence and absence
of a candidate agent, can be accomplished in any vessel suitable
for containing the reactants. Examples include microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows the protein
to be bound to a matrix. For example,
glutathione-S-transferase/GTPase (GST/GTPase) fusion proteins can
be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, Mo.) or glutathione derivatized microtitre plates, which are
then combined with a preparation of a target polypeptide, e.g. a
labeled target polypeptide, along with the test compound, and the
mixture incubated under conditions conducive to complex formation,
e.g. at physiological conditions for salt and pH, though slightly
more stringent conditions may be desired. Following incubation, the
beads are washed to remove any unbound label, and the matrix
immobilized and labeled target polypeptide retained on the matrix
determined directly, or in the supernatant after the complexes are
subsequently dissociated. Alternatively, the complexes can be
dissociated from the matrix, separated by SDS-PAGE, and the level
of target polypeptide found in the bead fraction quantitated from
the gel using standard electrophoretic techniques.
[0117] Other techniques for immobilizing proteins on matrices are
also available for use in the subject assay. For instance, either
the GTPase or target polypeptide can be immobilized utilizing
conjugation of biotin and streptavidin. For instance, biotinylated
GTPase molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with GTPase,
but which do not interfere with the interaction between the GTPase
and target polypeptide, can be derivatized to the wells of the
plate, and GTPase trapped in the wells by antibody conjugation. As
above, preparations of a target polypeptide and a test compound are
incubated in the GTPase-presenting wells of the plate, and the
amount of complex trapped in the well can be quantitated. Other
exemplary methods for detecting such complexes, in addition to
those described above, include detection of a radiolabel or
fluorescent label; immunodetection of complexes using antibodies
reactive with the target polypeptide, or which are reactive with
GTPase protein and compete with the target polypeptide; as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the target polypeptide, e.g., either intrinsic or
extrinsic activity. In the instance of the latter, the enzyme can
be chemically conjugated or provided as a fusion protein with the
target polypeptide. To illustrate, the target polypeptide can be
chemically cross-linked or genetically fused with horseradish
peroxidase, and the amount of polypeptide trapped in the complex
can be assessed with a chromogenic substrate of the enzyme, e.g.
3,3'-diamino-benzadine terahydrochloride or 4-chloro-1-napthol.
Likewise, a fusion protein comprising the target polypeptide and
glutathione-S-transferase can be provided, and complex formation
quantitated by detecting the GST activity using
1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem
249:7130). Alternatively, using such substrates as described above,
an intrinsic activity of the target polypeptide can be used to
facilitate detection.
[0118] For processes which rely on immunodetection for quantitating
one of the proteins trapped in the complex, antibodies against the
target protein or GTPase protein, can be used. Alternatively, the
protein to be detected in the complex can be "epitope tagged" in
the form of a fusion protein which includes a second polypeptide
for which antibodies are readily available (e.g. from commercial
sources). For instance, the GST fusion proteins described above can
also be used for quantification of binding using antibodies against
the GST moiety. Other useful epitope tags include myc-epitopes
(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) which
includes a 10-residue sequence from c-myc, as well as the pFLAG
system (International Biotechnologies, Inc.) or the pEZZ-protein A
system (Pharamacia, N.J.).
[0119] Cell-based Assay Formats
[0120] In yet further embodiments, the drug screening assay is
derived to include a whole cell expressing a fungal GTPase protein,
along with one or more of a GGPTase, a PKC or a glucan synthase
catalytic subunit. In preferred embodiments, the reagent cell is a
non-pathogenic cell which has been engineered to express one or
more of these proteins from recombinant genes cloned from a
pathogenic fungus. For example, non-pathogenic fungal cells, such
as S. cerevisae, can be derived to express a Rho-like GTPase from a
fungal pathogen such as Candida albicans. Furthermore, the reagent
cell can be manipulated, particularly if it is a yeast cell, such
that the recombinant gene(s) complement a loss-of-function mutation
to the homologous gene in the reagent cell. In an exemplary
embodiment, a non-pathogenic yeast cell is engineered to express a
Rho-like GTPase, e.g. Rho1, and at least one of the subunits of a
GGPTase, e.g. RAM2 and/or Cdc43, derived from a fungal protein. One
salient feature to such reagent cells is the ability of the
practitioner to work with a non-pathogenic strain rather than the
pathogen itself. Another advantage derives from the level of
knowledge, and available strains, when working with such reagent
cells as S. cerevisae.
[0121] The ability of a test agent to alter the activity of the
GTPase protein can be detected by analysis of the cell or products
produced by the cell. For example, agonists and antagonists of the
GTPase biological activity can be detected by scoring for
alterations in growth or viability of the cell. Other embodiments
will permit inference of the level of GTPase activity based on, for
example, detecting expression of a reporter, the induction of which
is directly or indirectly dependent on the activity of a Rho-like
GTPase. General techniques for detecting each are well known, and
will vary with respect to the source of the particular reagent cell
utilized in any given assay.
[0122] For example, quantification of proliferation of cells in the
presence and absence of a candidate agent can be measured with a
number of techniques well known in the art, including simple
measurement of population growth curves. For instance, where the
assay involves proliferation in a liquid medium, turbidimetric
techniques (i.e. absorbence/transmittance of light of a given
wavelength through the sample) can be utilized. For example, in the
instance where the reagent cell is a yeast cell, measurement of
absorbence of light at a wavelength between 540 and 600 nm can
provide a conveniently fast measure of cell growth. Likewise,
ability to form colonies in solid medium (e.g. agar) can be used to
readily score for proliferation. In other embodiments, a GTPase
substrate protein, such as a histone, can be provided as a fusion
protein which permits the substrate to be isolated from cell
lysates and the degree of acetylation detected. Each of these
techniques are suitable for high through-put analysis necessary for
rapid screening of large numbers of candidate agents.
[0123] Additionally, visual inspection of the morphology of the
reagent cell can be used to determine whether the biological
activity of the targeted GTPase protein has been affected by the
added agent. To illustrate, the ability of an agent to create a
lytic phenotype which is mediated in some way by a recombinant
GTPase protein can be assessed by visual microscopy.
[0124] The nature of the effect of test agent on reagent cell can
be assessed by measuring levels of expression of specific genes,
e.g., by reverse transcription-PCR. Another method of scoring for
effect on GTPase activity is by detecting cell-type specific marker
expression through immunofluorescent staining. Many such markers
are known in the art, and antibodies are readily available.
[0125] In yet another embodiment, in order to enhance detection of
cell lysis, the target cell can be provided with a cytoplasmic
reporter which is readily detectable, either because it has
"leaked" outside the cell, or substrate has "leaked" into the cell,
by perturbations in the cell wall. Preferred reporters are proteins
which can be recombinantly expressed by the target cell, do not
interfere with cell wall integrity, and which have an enzymatic
activity for which chromogenic or fluorogenic substrates are
available. In one example, a fungal cell can be constructed to
recombinantly express the .beta.-galactosidase gene from a
construct (optionally) including an inducible promoter. At some
time prior to contacting the cell with a test agent, expression of
the reporter protein is induced. Agents which inhibit prenylation
of a Rho-like GTPase in the cell, or the subsequent involvement of
a Rho-like GTPase in cell wall integrity, can be detected by an
increase in the reporter protein activity in the culture
supernatant or from permeation of a substrate in the cell. This,
for example, .beta.-galactosidase activity can be scored using such
calorimetric substrates as
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside or
fluorescent substrates such as
methylumbelliferyl-.beta.-D-galactopyranoside. Permeation of the
substrate into the cell, or leakage of the reporter into the
culture media, is thus readily detectable.
[0126] In yet another embodiment, the alteration of expression of a
reporter gene construct provided in the reagent cell provides a
means of detecting the effect on GTPase activity. For example,
reporter gene constructs derived using the transcriptional
regulatory sequences, e.g. the promoters, for genes regulated by
signal transduction processes downstream of the target Rho-like
GTPase can be used to drive the expression of a detectable marker,
such as a luciferase gene or the like. In an illustrative
embodiment, the construct is derived using the promoter sequence
from a gene expressed in PCK1-dependent heat shock response.
[0127] In still another embodiment, the membrane localization
resulting from prenylation of the fungal GTPase can be exploited to
generate the cell-based assay. For instance, the subject assay can
be derived with a reagent cell having: (i) a reporter gene
construct including a transcriptional regulatory element which can
induce expression of the reporter upon interaction of the
transcriptional regulatory protein portion of the above fusion
protein. For example, a gal4 protein can be fused with a Rho1
polypeptide sequence which includes the CAAX prenylation target.
Absent inhibitors of GGPTase activity in the reagent cell,
prenylation of the fusion protein will result in partitioning of
the fusion protein at the cell surface membrane. This provides a
basal level of expression of the reporter gene construct. When
contacted with an agent that inhibits prenylation of the fusion
protein, partitioning is lost and, with the concomitant increase in
nuclear concentration of the protein, expression from the reporter
construct is increased.
[0128] In a preferred embodiment, the cell is engineered such that
inhibition of the GGPTase activity does not result in cell lysis.
For example, as described in Ohya et al. (1993) Mol Cell Biol
4:1017-1025, mutation of the C-terminus of Rho1 and cdc42 can
provide proteins which are targets of farsenyl transferase rather
than geranylgeranyl transferase. As Ohya et al. describe, such
mutants can be used to render the GGPTase I activity dispensable.
Accordingly, providing a reporter gene construct and an expression
vector for the GGPTase substrate/transcription factor fusion
protein in such cells as YOT35953 cells (Ohya et al., supra)
generates a cell whose viability vis--vis the GGPTase activity is
determined by the reporter construct, if at all, rather than by
prenylation of an endogenous Rho-like GTPase by the GGPTase. Of
course, the reporter gene product can be derived to have no effect
on cell viability, providing for example another type of detectable
marker (described, infra). Such cells can be engineered to express
an exogenous GGPTase activity in place of an endogenous activity,
or can rely on the endogenous activity. To further illustrate, the
Call mutant YOT35953 cell can be further manipulated to express a
Call homolog from, e.g., a fungal pathogen or a mammalian cell.
[0129] Alternatively, where inhibition of a GGPTase activity causes
cell lysis and reporter gene expression, the leakage assay provided
above can be utilized to detect expression of the reporter protein.
For instance, the reporter gene can encode .beta.-galactosidase,
and inhibition of the GGPTases activity scored for by the presence
of cells which take up substrate due to loss of cell wall
integrity, and convert substrate due to the expression of the
reporter gene.
[0130] In preferred embodiments, the reporter gene is a gene whose
expression causes a phenotypic change which is screenable or
selectable. If the change is selectable, the phenotypic change
creates a difference in the growth or survival rate between cells
which express the reporter gene and those which do not. If the
change is screenable, the phenotype change creates a difference in
some detectable characteristic of the cells, by which the cells
which express the marker may be distinguished from those which do
not.
[0131] The marker gene is coupled to GTPase-dependent activity, be
it membrane association, or a downstream signaling pathway induced
by a GTPase complex, so that expression of the marker gene is
dependent on the activity of the GTPase. This coupling may be
achieved by operably linking the marker gene to a promoter
responsive to the therapeutically targeted event. The term
"GTPase-responsive promoter" indicates a promoter which is
regulated by some product or activity of the fungal GTPase. By this
manner, the activity of a GGPTase can be detected by its effects on
prenylation of GTPase and, accordingly, the downstream targets of
the prenylated protein. Thus, transcriptional regulatory sequences
responsive to signals generated by PKC/GTPase, GS/GTPase and/or
other GTPase complexes, or to signals by other proteins in such
complexes which are interupted by GTPase binding, can be used to
detect function of Rho-like GTPases such as Rho1 and cdc42.
[0132] In the case of yeast, suitable positively selectable
(beneficial) genes include the following: URA3, LYS2, HIS3, LEU2,
TRP1; ADE1,2,3,4,5,7,8; ARG1, 3, 4, 5, 6, 8; HIS1, 4, 5; ILV1, 2,
5; THR1, 4; TRP2, 3, 4, 5; LEU1, 4; MET2,3,4,8,9,14,16,19;
URA1,2,4,5,10; HOM3,6; ASP3; CHO1; ARO 2,7; CYS3; OLE1; IN012,4;
PR013. Countless other genes are potential selective markers. The
above are involved in well-characterized biosynthetic pathways. The
imidazoleglycerol phosphate dehydratase (IGP dehydratase) gene
(HIS3) is preferred because it is both quite sensitive and can be
selected over a broad range of expression levels. In the simplest
case, the cell is auxotrophic for histidine (requires histidine for
growth) in the absence of activation. Activation of the gene leads
to synthesis of the enzyme and the cell becomes prototrophic for
histidine (does not require histidine). Thus the selection is for
growth in the absence of histidine. Since only a few molecules per
cell of IGP dehydratase are required for histidine prototrophy, the
assay is very sensitive.
[0133] The marker gene may also be a screenable gene. The screened
characteristic may be a change in cell morphology, metabolism or
other screenable features. Suitable markers include
beta-galactosidase (Xgal, C.sub.12FDG, Salmon-gal, Magenta-Gal
(latter two from Biosynth Ag)), alkaline phosphatase, horseradish
peroxidase, exo-glucanase (product of yeast exbl gene;
nonessential, secreted); luciferase; bacterial green fluorescent
protein; (human placental) secreted alkaline phosphatase (SEAP);
and chloramphenicol transferase (CAT). Some of the above can be
engineered so that they are secreted (although not
.beta.-galactosidase). A preferred screenable marker gene is
beta-galactosidase; yeast cells expressing the enzyme convert the
colorless substrate Xgal into a blue pigment.
[0134] In another embodiment, the present invention provides a
cell-based assay which is based on our finding that the Cal1-1
mutant (see Example 3), e.g., a mutant of the GGPTase subunit
cdc43, results in supersensitivity to echinocandin. This
observation suggests to us that GGPTase I inhibitors can enhance
sensitivity to GS inhibitors, a phenotype which can be easily
detected. In an exemplary embodiment, a fungal cell can be
contacted with a test agent, and a GS inhibitor such as
echinocandin B (other congeners of the echinocandin class of
agents, such as cilofungin, certain pneumocandins, and WF11899A, B
and C). The amount of cell lysis is determined and compared to the
amount of cell lysis is the absence of the GS inhibitor. Synergism,
e.g., a statistically significant increase in lysis of the GS
inhibitor treated cell relative to the cell contacted only with the
test agent, suggests that the test agent is likely to be a
cytotoxic agent which targets prenylation of Rho-like GTPases, or
the association of prenylated Rho-like GTPases with proteins
critical to cell wall integrity. The fungal cell can be a wild-type
or recombinant cell, e.g., such as an S. cerevisiae cell engineered
to express Candida proteins.
[0135] It has also been observed in the art that mutations to Gsc1
(Fks1) confer hypersensitivity to the immunosuppressants FK506 and
cyclosporin A (Douglas et al. (1994) PNAS 91:12907). The mechanism
of action of such agents is understood to involve inhibition of
expression of the Fks2 gene (Mazur et al. (1995) Mol Cell Biol
15:5671). Similar to the echinocandin-sensitivity assay embodiments
provided above, another assay format provides a cell in which Fks2
activity is compromised. Synergism of the Fks2 impairment with a
test compound can be used to identify inhibitors of, for example,
the glucan synthase subunit Gsc1. For instance, FK506 or
cyclosporin A can be used to impair Fks2 activity, as can mutations
to calcineurin or to the Fks2 gene.
[0136] These observations also suggest that Cal1-1 cells or the
like, e.g., impaired for certain GGPTase activities, are suitable
for use in assay to detect GS inhibitors, as such cells are more
sensitive to the effects of GS inhibitors. The benefits to enhanced
sensitivity include speedier development of assay readouts, and the
further prejudicing of the assay towards GS inhibitors rather than
other targets which may not provide cytotoxicity. The latter can
provide the ability to identify potential hits which may not
themselves be potent GS inhibitors, but which can be manipulated,
e.g., by combinatorial chemistry approaches, to provide potent and
specific GS inhibitors.
[0137] In yet another embodiment, fungal proteins involved in the
various interactions set out as targets above can be used to
generate an interaction trap assay (see also, U.S. Pat. No.
5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.
(1993) J Biol Chem 268:12046-12054; Bartel et al. (1993)
Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene
8:1693-1696), for subsequently detecting agents which disrupt
binding of the proteins to one and other.
[0138] In particular, the method makes use of chimeric genes which
express hybrid proteins. To illustrate, a first hybrid gene
comprises the coding sequence for a DNA-binding domain of a
transcriptional activator fused in frame to the coding sequence for
a "bait" protein, e.g., a fungal Rho1. The second hybrid protein
encodes a transcriptional activation domain fused in frame to a
gene encoding a "fish" protein which interacts with the Rho1
protein, e.g. a Gsc1 protein. If the bait and fish proteins are
able to interact, e.g., form a Rho1/Gsc1 complex, they bring into
close proximity the two domains of the transcriptional activator.
This proximity is sufficient to cause transcription of a reporter
gene which is operably linked to a transcriptional regulatory site
responsive to the transcriptional activator, and expression of the
reporter gene can be detected and used to score for the interaction
of the bait and fish proteins.
[0139] In accordance with the present invention, the method
includes providing a host cell, preferably a yeast cell, most
preferably Saccharomyces cerevisiae or Schizosaccharomyces pombe.
The host cell contains a reporter gene having a binding site for
the DNA-binding domain of a transcriptional activator, such that
the reporter gene expresses a detectable gene product when the gene
is transcriptionally activated. Such activation occurs when the
activation domain of the transcriptional activator is brought into
sufficient proximity to the DNA-binding domain of a transcriptional
activator bound to the regulatory element of the reporter gene. The
first chimeric gene may be present in a chromosome of the host
cell, or as part of an expression vector.
[0140] A first chimeric gene is provided which is capable of being
expressed in the host cell. The gene encodes a chimeric protein
which comprises (i) a DNA-binding domain that recognizes the
responsive element on the reporter gene in the host cell, and (ii)
bait protein, such as Rho1.
[0141] A second chimeric gene is provided which is capable of being
expressed in the host cell. In one embodiment, both the first and
the second chimeric genes are introduced into the host cell in the
form of plasmids. Preferably, however, the first chimeric gene is
present in a chromosome of the host cell and the second chimeric
gene is introduced into the host cell as part of a plasmid. The
second chimeric gene includes a DNA sequence that encodes a second
hybrid protein comprising a transcriptional activation domain fused
to a fish protein, or a fragment thereof, which is to be tested for
interaction with the bait protein. The fish protein can be a
subunit of a GGPTase which interacts with Rho1, or a subunit of a
glucan synthase which interacts with Rho1, or Pkc1.
[0142] Preferably, the DNA-binding domain of the first hybrid
protein and the transcriptional activation domain of the second
hybrid protein are derived from transcriptional activators having
separable DNA-binding and transcriptional activation domains. For
instance, these separate DNA-binding and transcriptional activation
domains are known to be found in the yeast GAL4 protein, and are
known to be found in the yeast GCN4 and ADR1 proteins. Many other
proteins involved in transcription also have separable binding and
transcriptional activation domains which make them useful for the
present invention, and include, for example, the LexA and VP16
proteins. It will be understood that other (substantially)
transcriptionally -inert DNA-binding domains may be used in the
subject constructs; such as domains of ACE1, .lambda.cI, lac
repressor, jun or fos. In another embodiment, the DNA-binding
domain and the transcriptional activation domain may be from
different proteins. The use of a LexA DNA binding domain provides
certain advantages. For example, in yeast, the LexA moiety contains
no activation function and has no known effect on transcription of
yeast genes. In addition, use of LexA allows control over the
sensitivity of the assay to the level of interaction (see, for
example, the Brent et al. PCT publication WO94/10300).
[0143] In preferred embodiments, any enzymatic activity associated
with the bait or fish proteins is inactivated, e.g., dominant
negative mutants of Rho1 and the like can be used. Where the
interacting proteins are of the enzyme-substrate relationship,
mutation of one or more catalytic residues of the enzyme can
provide a mutant protein which retains the ability to bind the
substrate but not catalytically convert it to product.
[0144] Continuing with the illustrated example, the
Rho1/Gsc1-mediated interaction, if any, between the first second
fusion proteins in the host cell, therefore, causes the activation
domain to activate transcription of the reporter gene. The method
is carried out by introducing the first chimeric gene and the
second chimeric gene into the host cell, and subjecting that cell
to conditions under which the first hybrid protein and the second
hybrid protein are expressed in sufficient quantity for the
reporter gene to be activated. The formation of a Rho1/Gsc1 complex
results in a detectable signal produced by the expression of the
reporter gene. Accordingly, the formation of a complex in the
presence of a test compound to the level of Rho1/GSC1 complex in
the absence of the test compound can be evaluated by detecting the
level of expression of the reporter gene in each case.
[0145] In an illustrative embodiment, Saccharomyces cerevisiae YPB2
cells are transformed simultaneously with a plasmid encoding a
GAL4db-Rho1 fusion and with a plasmid encoding the GAL4ad domain
fused to a fungal Gsc1 gene. Moreover, the strain is transformed
such that the GAL4-responsive promoter drives expression of a
phenotypic marker. For example, the ability to grow in the absence
of histidine can depend on the expression of the LacZ gene. When
the LacZ gene is placed under the control of a GAL4-responsive
promoter, the yeast cell will turn blue in the presence of
.beta.-gal if a functional GAL4 activator has been reconstituted
through the interaction of Rho1 and Gsc1. Thus, a convenient
readout method is provided. Other reporter constructs will be
apparent, and include, for example, reporter genes which produce
such detectable signals as selected from the group consisting of an
enzymatic signal, a fluorescent signal, a phosphorescent signal and
drug resistance.
[0146] A similar method modifies the interaction trap system by
providing a "relay gene" which is regulated by the transcriptional
complex formed by the interacting bait and fish proteins. The gene
product of the relay gene, in turn, regulates expression of a
reporter gene, the expression of the latter being what is scored in
the modified ITS assay. Fundamentally, the relay gene can be seen
as a signal inverter.
[0147] As set out above, in the standard ITS, interaction of the
fish and bait fusion proteins results in expression of a reporter
gene. However, where inhibitors of the interaction are sought, a
positive readout from the reporter gene nevertheless requires
detecting inhibition (or lack of expression) of the reporter
gene.
[0148] In the inverted ITS system, the fish and bait proteins
positively regulate expression of the relay gene. The relay gene
product is in turn a repressor of expression of the reporter gene.
Inhibition of expression of the relay gene product by inhibiting
the interaction of the fish and bait proteins results in
concomitant relief of the inhibition of the reporter gene, e.g.,
the reporter gene is expressed. For example, the relay gene can be
the repressor gene under control of a promoter sensitive to the
Rho1/Gsc1 complex described above. The reporter gene can
accordingly be a positive signal, such as providing for growth
(e.g., drug selection or auxotrophic relief), and is under the
control of a promoter which is constitutively active, but can be
suppressed by the repressor protein. In the absence of an agent
which inhibits the interaction of the fish and bait protein, the
repressor protein is expressed. In turn, that protein represses
expression of the reporter gene. However, an agent which disrupts
binding of the Rho1 and Gsc1 proteins results in a decrease in
repressor expression, and consequently an increase in expression of
the reporter gene as repression is relieved. Hence, the signal is
inverted.
[0149] Returning to the teachings of Ohya et al. (1993) Mol Cell
Biol 4:1017-1025, it is noted that there are only two essential
targets of GGPTase in S. cerevisae, the Rho-like GTPases Rho1 and
cdc42. With such observations in mind, yet another embodiment of
the subject assay utilizes a side-by-side comparison of the effect
of a test agent on (i) a cell which prenylates a Rho-like GTPase by
adding geranylgeranyl moieties, and (ii) a cell which prenylates an
equivalent Rho-like GTPase by adding farnesyl moieties. In
particular, the assay makes use of the ability to suppress GGPTase
I defects in yeast by altering the C-terminal tail of Rho1 and
cdc42 to become substrate targets of farnesyl transferase (see Ohya
et al., supra). According to the present embodiment, the assay is
arranged by providing a yeast cell in which the target Rho-like
GTPases is prenylated by a GGPTase activity of the cell. Both the
GGPTase and GTPase can be endogenous to the "test" cell, or one or
both can be recombinantly expressed in the cell. The level of
prenylation of the GTPase is detected, e.g., cell lysis or other
means described above. The ability of the test compound to inhibit
the addition of geranylgeranyl groups to the GTPase in the first
cell is compared against the ability of test compound to inhibit
the farnesylation of the GTPase in a control cell. The "control"
cell is preferably identical to the test cell, with the exception
that the targeted GTPase(s) are mutated at their CAAX sequence to
become substrates for FPTases rather than GGPTases. Agents which
inhibit prenylation in the test cell but not the control cell are
selected as potential antifungal agents. Such differential screens
can be exquisitely sensitive to inhibitors of GGPTase I prenylation
of Rho-like GTPases. In a preferred embodiment, the test cell is
derived from the S. cerivisae cell YOT35953 (Ohya et al., supra) or
the like which is defective in GGPTase subunit cdc43. The cell is
then engineered with a cdc43 subunit from a fungal pathogen such as
Candida albicans to generate the test cell, and additionally with
the mutated Rho-like GTPases to generate the control cell.
[0150] Differential Screening Formats
[0151] In a preferred embodiment, assays can be used to identify
compounds that have therapeutic indexes more favorable than such
antifungal as, for example, papulacandins or echinocandins or the
like. For instance, antifungal agents can be identified by the
present assays which inhibit proliferation of yeast cells or other
lower eukaryotes, but which have a substantially reduced effect on
mammalian cells, thereby improving therapeutic index of the drug as
an anti-mycotic agent.
[0152] In one embodiment, the identification of such compounds is
made possible by the use of differential screening assays which
detect and compare the ability of the test compound to inhibit an
activity associated with a fungal GTPase, relative to its ability
to inhibit an analogous activity of a human GTPase. To illustrate,
the assay can be designed for side-by-side comparison of the effect
of a test compound on the prenylation activity or protein
interactions of fungal and human GGPTase and GTPase proteins. Given
the apparent diversity of GGPTase proteins, it is probable that the
fungal and human GGPTases differ both in substrate specificity and
mechanistic action which can be exploited in the subject assay.
Running the fungal and human prenylation systems side-by-side
permits the detection of agents which have a greater inhibitory
effect (e.g. statistically significant) on the prenylation reaction
mediated by the fungal GGPTase than the human enzyme.
[0153] Accordingly, differential screening assays can be used to
exploit the difference in protein interactions and/or catalytic
mechanism of mammalian and fungal GGPTases in order to identify
agents which display a statistically significant increase in
specificity for inhibiting the fungal prenylation reaction relative
to the mammalian prenylation reaction. Thus, lead compounds which
act specifically on the prenylation reaction in pathogens, such as
fungus involved in mycotic infections, can be developed. By way of
illustration, the present assays can be used to screen for agents
which may ultimately be useful for inhibiting the growth of at
least one fungus implicated in such mycosis as candidiasis,
aspergillosis, mucormycosis, blastomycosis, geotrichosis,
cryptococcosis, chromoblastomycosis, coccidioidomycosis,
conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis,
nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, or
sporotrichosis. For example, if the mycotic infection to which
treatment is desired is candidiasis, the present assay can comprise
comparing the relative effectiveness of a test compound on
inhibiting the prenylation of a mammalian GTPase protein with its
effectiveness towards inhibiting the prenylation of a GTPase from a
yeast selected from the group consisting of Candida albicans,
Candida stellatoidea, Candida tropicalis, Candida parapsilosis,
Candida krusei, Candida pseudotropicalis, Candida quillermondii, or
Candida rugosa. Likewise, the present assay can be used to identify
anti-fungal agents which may have therapeutic value in the
treatment of aspergillosis by selectively targeting, relative to
human cells, GTPase homologs from yeast such as Aspergillus
fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus
nidulans, or Aspergillus terreus. Where the mycotic infection is
mucormycosis, the GTPase system to be screened can be derived from
yeast such as Rhizopus arrhizus, Rhizopus oryzae, Absidia
corymbifera, Absidia ramosa, or Mucor pusillus. Sources of other
assay reagents for includes the pathogen Pneumocystis carinii.
[0154] Thus, it is also deemed to be within the scope of this
invention that the recombinant GTPase cells of the present assay
can be generated so as to comprise heterologous GTPase proteins
from metazoan sources such as humans (i.e. cross-species
expression). For example, GTPase proteins from humans can be
expressed in the reagent cells under conditions wherein the
heterologous protein is able to rescue loss-of-function mutations
in the host cell. For example, the reagent cell can be a yeast cell
in which a human GTPase protein (e.g. exogenously expressed) is to
be a counter-screen for identifying agents which selectively
inhibit yeast GTPase activities. To illustrate, the YOC706 strain,
described by Qadota et al. (1994) Genetics 91:9317-9321, lacks a
functional endogenous Rho1 gene, and can be transfected with an
expression plasmid including a human GTPase gene such as RHoA in
order to complement the Rho1 loss-of-function. For example, the
coding sequence for RHoA can be cloned into a pRS integrative
plasmid containing a selectable marker (Sikorski et al. (1989)
Genetics 122:19-27), and resulting construct used to transform the
YOC706 strain. The resulting cells should produce a human RHoA
protein which is capable of performing at least some of the
functions of the yeast Rho1 protein. The GTPase transformed yeast
cells can be easier to manipulate than mammalian cells, and can
also provide access to certain assay formats, such as turbidity
detection, which may not be obtainable with mammalian cells.
[0155] Reagents
[0156] If yeast cells are used, the yeast may be of any species
which are cultivable and, preferably, in which an exogenous
Rho1-like protein can be made to engage the appropriate prenylation
enzyme and/or participate in protein complexes such as with glucan
synthesase subunits or PKC homologs of the host cell. Suitable
species include Kluyverei lactis, Schizosaccharomyces pombe, and
Ustilaqo maydis; Saccharomyces cerevisiae is preferred. Other yeast
which can be used in practicing the present invention. The term
"yeast", as used herein, includes not only yeast in a strictly
taxonomic sense, i.e., unicellular organisms, but also yeast-like
multicellular fungi or filamentous fungi.
[0157] The choice of appropriate host cell can be influenced by the
choice of detection signal. For instance, reporter constructs, as
described below, can provide a selectable or screenable trait upon
transcriptional activation (or inactivation) in response to a
signal provided by the GTPase target. Suitable genes and promoters
can be dependent on the reagent cell. Likewise, ease of
complementation, genetic manipulation, etc., may also affect the
choice of reagent cell.
[0158] With respect to sources for constituting recombinant
proteins of the subject assays, various GGPTases, GTPases, glucan
synthase subunits, and PKC homologs have been identified from a
variety of fungal species, and in a significant number of
instances, have been cloned so that recombinant sources exist.
[0159] For example, identification of enzymes involved in the
prenylation pathway from different sources have facilitated the
cloning of corresponding genes. For instance, genes GGPTase
enzymes, PKC homologs and GTPase homologs have been cloned from
various fungal organisms, and are generally described in the
literature and available on GenBank or other such databases.
Complementation of defects in yeast cells such as S. cereviae also
constitute a standard protocol for isolating genes encoding fungal
and mammalian homologs (as appropriate) of such target proteins as
GGPTase subunits, Rho-like GTPases, PKC homologs and glucan
synthase subunits.
[0160] The proteins provided in the subject assay can be derived by
purification from a cell in which it is endogenously expressed, or
from a recombinant source of the protein. In each instance where a
recombinant source of a protein is used in the subject assay, the
manipulation of the gene encoding the protein and the subsequent
expression of the protein can be carried out by standard molecular
biological techniques. Ligating the polynucleotide sequence
encoding the recombinant protein into a gene construct, such as an
expression vector, and transforming or transfecting into host
cells, either eukaryotic (yeast, avian, insect or mammalian) or
prokaryotic (bacterial cells), are standard procedures used in
producing other well-known proteins, including the S. cerevisae
proteins PCK1, GGPTase, Rho1 and the like. Similar procedures, or
obvious modifications thereof, can be employed to prepare and
purify recombinant proteins of the prenylation system from other
sources.
[0161] The recombinant protein can be produced by ligating the
cloned gene, or a portion thereof, into a vector suitable for
expression in either prokaryotic cells, eukaryotic cells, or both.
Expression vehicles for production of recombinant proteins include
plasmids and other vectors. For instance, suitable vectors for the
expression of these proteins include plasmids of the types:
pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived
plasmids, pBTac-derived plasmids and pUC-derived plasmids for
expression in prokaryotic cells, such as E. coli.
[0162] In general, it will be desirable that the gene construct be
capable of replication in the host cell. It may be a DNA which is
integrated into the host genome, and thereafter is replicated as a
part of the chromosomal DNA, or it may be DNA which replicates
autonomously, as in the case of a plasmid. In the latter case, the
vector will include an origin of replication which is functional in
the host. In the case of an integrating vector, the vector may
include sequences which facilitate integration, e.g., sequences
homologous to host sequences, or encoding integrases.
[0163] Appropriate cloning and expression vectors for use with
bacterial, fungal, yeast, and mammalian cellular hosts are known in
the art, and are described in, for example, Powels et al. (Cloning
Vectors: A Laboratory Manual, Elsevier, N.Y., 1985). Mammalian
expression vectors may comprise non-transcribed elements such as an
origin of replication, a suitable promoter and enhancer linked to
the gene to be expressed, and other 5' or 3' flanking
nontranscribed sequences, and 5' or 3' nontranslated sequences,
such as necessary ribosome binding sites, a poly-adenylation site,
splice donor and acceptor sites, and transcriptional termination
sequences.
[0164] The preferred mammalian expression vectors contain both
prokaryotic sequences, to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine
papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived
and p205) can be used for transient expression of proteins in
eukaryotic cells. The various methods employed in the preparation
of the plasmids and transformation of host organisms are well known
in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells, as well as general recombinant
procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989) Chapters 16 and 17.
[0165] A number of vectors exist for the expression of recombinant
proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2,
and YRP17, as well as the pRS vectors, e.g., pRS303, pRS304,
pRS305, pRS306, etc., are cloning and expression vehicles useful in
the introduction of genetic constructs into S. cerevisiae (see, for
example, Broach et al. (1983) in Experimental Manipulation of Gene
Expression, ed. M. Inouye Academic Press, p. 83, incorporated by
reference herein). These vectors can replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the
replication determinant of the yeast 2 micron plasmid. In addition,
drug resistance markers such as ampicillin can be used.
[0166] Moreover, when yeast are used as the reagent cell, it will
be understood that the expression of a gene in a yeast cell
requires a promoter which is functional in yeast. Suitable
promoters include the promoters for gall, metallothionein,
3-phosphoglycerate kinase (Hitzeman et al, J. Biol. Chem. 255, 2073
(1980) or other glycolytic enzymes (Hess et al, J. Adv. Enzyme Req.
7, 149 (1968); and Holland et al. Biochemistry 17, 4900 (1978)),
such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phospho-fructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phospho-glucose isomerase, and
glucokinase. Suitable vectors and promoters for use in yeast
expression are further described in R. Hitzeman et al, EPO Publn.
No. 73,657. Other promoters, which have the additional advantage of
transcription controlled by growth conditions, are the promoter
regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned metallothionein and
glyceraldehyde -3-phosphate dehydrogenase, as well as enzymes
responsible for maltose and galactose utilization. Finally,
promoters that are active in only one of the two haploid mating
types may be appropriate in certain circumstances. Among these
haploid-specific promoters, the pheromone promoters MFa1 and
MF.alpha.1 are of particular interest.
[0167] In some instances, it may be desirable to derive the host
cell using insect cells. In such embodiments, recombinant
polypeptides can be expressed by the use of a baculovirus
expression system. Examples of such baculovirus expression systems
include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),
pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived
vectors (such as the .beta.-gal containing pBlueBac III).
[0168] Furthermore, the recombinant protein can be encoded by a
fusion gene created to have additional sequences coding for a
polypeptide portion of a fusion protein which would facilitate its
purification. For instance, a fusion gene coding for a purification
leader sequence comprising a poly-(His)/enterokinase cleavage site
sequence can be engineered at the a terminus of the protein,
thereby enabling purification of the expressed fusion protein by
affinity chromatography using a Ni.sup.2+ metal resin. The
purification leader sequence can then be subsequently removed by
treatment with enterokinase (e.g., see Hochuli et al. 1987 J.
Chromatography 411:177; and Janknecht et al. PNAS 88:8972).
[0169] Exemplary Construction of the Expression Plasmid For
Recombinant GGPTase-I.
[0170] Polymerase chain reaction (PCR) can be carried out to
isolate the CDC43 coding sequence from S cerevisiae. Using a sense
strand primer (5'-CCATCGATCATATGTGTCAAGCTAGGAAT-3') can introduce a
unique ClaI restriction site upstream of the CDC43 start codon and
an NdeI site that overlaps the ATG initiation codon. An antisense
strand PCR primer (5'-GCGGGTACCCTGCAGTCAAAAACAGCACCTTTT-3')
introduces unique PstI and KpnI restriction sites downstream of the
CDC43 stop codon. The PCR product is ligated into a convenient
vector, such as bluescript II SK-(+) using ClaI and KpnI. An
XbaI-ClaI fragment containing RAM2 (Mayer et al., (1993) Gene
132:41-47) can be cloned into the CDC43 containing vector, upstream
of the CDC43 sequence, to produce a bicistronic construct. The RAM2
and CDC43 orfs are then coupled by deletion mutagenesis with the
antisense strand primer
(5'-GGTAGCTTGAVACATCAAAACTCCTCCTGCAGATTTATTTTG-3'), which overlaps
the RAM2 translation termination codon with the CDC43 initiation
codon. The RAM2-CDC43 cassette can then be cloned into an
appropriate expression vector and used to transform E coli.
[0171] Recombinant GGPTase-I can be purified from the resulting
cultures as described for recombinant yeast FPTase (Mayer et al.,
supra), with minor modifications (Stirtan et al. (1995) Arch
Biochem Biophys 321:182-190). Wet cell paste is resuspended in 16
ml of lysis buffer (50 mM Tris-HCl, pH 7.0, 10 mM BME, 10 mM
MgCl.sub.2, 50 .mu.M ZnCl.sub.2, 1 mM PMSF) and disrupted by
sonication. The cell-free homogenate is clarified by centrifugation
and chromatographed on DE52 ion-exchange resin (1.5.times.14 cm) at
4.degree. C., preequilibrated with low-salt buffer (50 mM Tris-Hcl,
pH 7.0, 10 mM MgCl.sub.2, 50 .mu.M ZnCl.sub.2, 10 mM BME). Protein
is eluted with a stepwise gradient of 0 to 800 mM NaCl in low-salt
buffer. Recombinant PGGPTase-I is expected to elute at 200 mM NaCl.
The DE52-purified material is dialyzed at 4.degree. C. against
low-salt buffer, diluted to .about.1 mg/ml with the same buffer,
and loaded onto an anti-.alpha.-tubulin immunoaffinity column
(Mayer et al., supra) preequilibrated with binding buffer (20 mM
Tris-HCl, pH 7.5, 1 mM MgCl.sub.2, 10 .mu.M ZnCl.sub.2, 5 mM BME,
50 mM NaCl). The column is washed with binding buffer (.about.25
ml) and then eluted with binding buffer containing 5 mM Asp-Phe.
Fractions containing GGPTase-I activity are combined. Recombinant
GGPTase-I has been demonstrated to be stable for several months at
-80.degree. C. and for several days at 0.degree. C.
[0172] Preparation of Dansyl-Gly-Cys-Ile-Ile-Leu.
[0173] Dansyl-Gly-Cys-Ile-Ile-Leu is prepared essentially as
described previously for dansyl-Gly-Cys-Val-Ile-Ala (Cassidy et
al., (1985) Methods Enzymol. 250: 30-43), the farnesylated
substrate corresponding to Cys-Val-Ile-Ala.
Dansyl-Gly-Cys-Ile-Ile-Leu can be purified by preparative HPLC on a
Vydac protein and peptide C18 reversed-phase column (22 mm.times.25
cm) by elution with a gradient of 85-92% CH.sub.3CN/0.1% TFA in
H.sub.2O/0.1% TFA over 20 min, followed by a gradient of 92-100%
CH.sub.3CN/0.1% TFA over 5 min, and finally with 100%
CH.sub.3CN/0.1% TFA for 10 min. Organic materials are removed by
rotary evaporation, and the resulting aqueous suspension is
lyophilized to afford dansyl-Gly-Cys-Ile-Ile-Leu.
[0174] Pharmaceutical Preparations of Identified Agents
[0175] After identifying certain test compounds as potential
antifungal agents, the practioner of the subject assay will
continue to test the efficacy and specificity of the selected
compounds both in vitro and in vivo. Whether for subsequent in vivo
testing, or for administration to an animal as an approved drug,
agents identified in the subject assay can be formulated in
pharmaceutical preparations for in vivo administration to an
animal, preferably a human.
[0176] The subject compounds selected in the subject, or a
pharmaceutically acceptable salt thereof, may accordingly be
formulated for administration with a biologically acceptable
medium, such as water, buffered saline, polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycol and the
like) or suitable mixtures thereof. The optimum concentration of
the active ingredient(s) in the chosen medium can be determined
empirically, according to procedures well known to medicinal
chemists. As used herein, "biologically acceptable medium" includes
any and all solvents, dispersion media, and the like which may be
appropriate for the desired route of administration of the
pharmaceutical preparation. The use of such media for
pharmaceutically active substances is known in the art. Except
insofar as any conventional media or agent is incompatible with the
activity of the compound, its use in the pharmaceutical preparation
of the invention is contemplated. Suitable vehicles and their
formulation inclusive of other proteins are described, for example,
in the book Remington's Pharmaceutical Sciences (Remington's
Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA
1985). These vehicles include injectable "deposit formulations".
Based on the above, such pharmaceutical formulations include,
although not exclusively, solutions or freeze-dried powders of the
compound in association with one or more pharmaceutically
acceptable vehicles or diluents, and contained in buffered media at
a suitable pH and isosmotic with physiological fluids. In preferred
embodiment, the compound can be disposed in a sterile preparation
for topical and/or systemic administration. In the case of
freeze-dried preparations, supporting excipients such as, but not
exclusively, mannitol or glycine may be used and appropriate
buffered solutions of the desired volume will be provided so as to
obtain adequate isotonic buffered solutions of the desired pH.
Similar solutions may also be used for the pharmaceutical
compositions of compounds in isotonic solutions of the desired
volume and include, but not exclusively, the use of buffered saline
solutions with phosphate or citrate at suitable concentrations so
as to obtain at all times isotonic pharmaceutical preparations of
the desired pH, (for example, neutral pH).
Exemplification
[0177] The invention, now being generally described, will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention and are not intended to
limit the invention.
EXAMPLE 1
Activation of Yeast Protein Kinase C by Rho1 GTPase
[0178] The abbreviations used in Example 1 are: PKC, protein kinase
C; MAPK, mitogen-activated protein kinase; MEK, MAPK-activating
kinase; MEKK, MEK-activating kinase; DAG, diacylglycerol; SRF,
serum response factor; JNK, Jun NH.sub.2-terminal kinase (also
known as SAPK, stress-activated protein kinase); PCR, polymerase
chain reaction; HA, influenza hemagglutinin; PAGE, polyacrylamide
gel electrophoresis; GST, glutathione-S-transferase; PS,
phosphatidylserine; PMA, phorbol myristate acetate; GS,
1,3-.beta.-glucan synthase; MBP, myelin basic protein.
[0179] A. Overview
[0180] We have investigated the role of the essential Rho1 GTPase
in cell integrity signaling in budding yeast. Conditional rho1
mutants display a cell lysis defect that is similar to that of
mutants in the cell integrity signaling pathway mediated by protein
kinase C (PKC1), which is suppressed by overexpression of PKC1.
rho1 mutants are also impaired in pathway activation in response to
growth at elevated temperature. PKC1 co-immuneprecipitates with
Rho1 in yeast extracts, and recombinant Rho1 associates with PKC1
in vitro in a GTP-dependent manner. Recombinant Rho1 confers upon
PKC1 the ability to be stimulated by phosphatidylserine (PS),
indicating that Rho1 controls signal transmission through PCK1.
[0181] The PKC1 gene of the budding yeast Saccharomyces cerevisiae
encodes a homolog of mammalian protein kinase C (PKC) (ref. 1) that
regulates a MAP kinase (MAPK)-activation cascade comprised of a
MEKK (Bck1), a redundant pair of MEKs (Mkk1/2), and a MAPK (Mpk1)
(2, 3). Mutants in this signaling cascade, called the cell
integrity pathway, undergo cell lysis resulting from a deficiency
in cell wall construction that is exacerbated by growth at elevated
temperatures. We have reported that thermal stress activates the
cell integrity pathway, and proposed that weakness in the cell wall
that develops during growth at high temperature induces the signal
for pathway activation (4).
[0182] PCK1 most closely resembles the conventional isoforms of
mammalian PKC, which require phospholipids, Ca.sup.2+, and
diacylglycerol (DAG) as cofactors to stimulate their catalytic
activity (1). However, in vitro studies of this yeast protein
kinase have failed to demonstrate stimulation by cofactors, despite
the finding that mutations in PKC1 predicted to relieve cofactor
dependence have an activating effect on the enzyme (5, 6). This
suggested that one or more components required for
cofactor-dependent stimulation of PCK1 was missing from in vitro
reconstitution experiments.
[0183] Members of the Rho family of small GTPases (RhoA, Cdc42, and
Rac) regulate various aspects of actin cytoskeleton organization
and activation of the SRF transcription factor in mammalian cells
(7-10). Cdc42 and Rac, but not RhoA, stimulate the signaling
pathway that contains the JNK/SAPK (Jun NH.sub.2-terminal kinase or
stress-activated protein kinase) MAPK homolog in mammalian cells
(11-13). Downstream effectors of RhoA have not been identified (14,
15). The yeast RHO1 gene encodes a homolog of mammalian RhoA that
resides at sites of cell growth (16) and whose function is
essential for viability (17). A rho1.DELTA. mutant is partially
suppressed by expression of human RhoA, but a residual cell lysis
defect is apparent at high temperature (18), suggesting that RHO1
may function within the cell integrity pathway. Additionally, an
activated allele of PKC1 was isolated recently as a dominant
mutational suppressor of this defect (19), further supporting the
notion that these signaling molecules act through a common pathway.
In this communication, we demonstrate that Rho1 associates with
PKC1 in a GTP-dependent manner, and confers upon this protein
kinase the ability to respond to phosphatidylserine as an
activating cofactor.
[0184] B. Experimental Procedures
[0185] Yeast strains and mutant construction--All strains used in
this study were derived from YPH500 (See reference of Example 3).
Error-prone PCR (21) was used to introduce random mutations into
the RHO1 sequence. The PCR-amplified RHO1 fragment was inserted
into the EcoRI/BglII gap of pYO701, and introduced into yeast
strain YOC706, which harbors a rho1.DELTA. and a plasmid expressing
RHO1 under the control of the GAL1 promoter (18). We examined 4000
transformants for growth on YPD (yeast extract/peptone/dextrose)
plates at 23.degree. C. and 37.degree. C., and identified 41
rho1.sup.ts mutations. Among these, 11 rho1 alleles (designated
rho1-1-rho1-11) contained single or double base changes. All of
these alleles were reconstructed by site-directed mutagenesis, and
integrated at the ADE3 locus (See reference of Example 3) of
diploid strain YOC701 (RHO1/rho1.DELTA.::HIS3). Haploid strains
used in this study (YOC764 [RHO1], YOC729 [rho1-3], and YOC755
[rho1-5]) were derived from YOC701 integrants by standard genetic
techniques. A single copy plasmid (pYO904) that carries HA-tagged
RHO1 was constructed in vector pRS314, as described previously
(16), and introduced into yeast strain YOC701. A segregant bearing
rho1.DELTA.::HIS3 and pYO904, and a wild-type (RHO1) segregant
lacking the plasmid were used for coimmunoprecipitation
experiments.
[0186] Antibodies, extracts, immunoprecipitation, protein kinase
assays and immunodetection--Anti-HA antibodies (12CA5; BAbCo, Inc.)
were used for immunoprecipitation and immunodetection of
.sup.HARho1, Mpk1.sup.HA, and PKC1.sup.HA. Polyvalent PKC1
antibodies (used for immunodetection of PKC1) were raised by
Cocalico Biologicals (Reamstown, Pa.) in New Zealand white rabbits
against a TrpE::PKC1 fusion protein that contains amino acids
470-664 of PKC1. This antiserum was used (at 1:3000 dilution) for
immunodetection of PKC1. Secondary antibodies used were horseradish
peroxidase-conjugated donkey anti-rabbit (Amersham; at 1:10,000
dilution).
[0187] Yeast extract preparation, immunoprecipitation,
immunodetection and protein kinase assays of Mpk1.sup.HA were
conducted as described previously (4). Preparation of cell extracts
and immunoprecipitations for experiments with .sup.HARho1 were
carried out as in (4) with some modifications. Lysis buffer without
p-nitrophenyl phosphate and with 1% NP-40 was used. The extract
(700 .mu.g protein) was precleared by incubation with 20 .mu.l of a
50% suspension of protein A-sepharose for 1 h prior to
immunoprecipitation to eliminate non-specific binding of proteins
to immunecomplexes. Beads were boiled in SDS-PAGE sample buffer,
and samples were applied to 7.5% (for PKC1 blots) or 15% (for
.sup.HARho1 blots) SDS-PAGE gels. For PKC1 kinase assays, all as
described previously (5), except for the addition of recombinant
GTPases (see below). A synthetic peptide corresponding to the
sequence surrounding Ser939 of Bck1, a phosphorylation site for PKC
1, was used as substrate in PKC1 kinase assays (5).
[0188] Recombinant Rho1 and Cdc42. Recombinant GST-Rho1 and
GST-Cdc42 were expressed and purified from baculovirus-infected
insect (Sf9) cells, as described (23). For in vitro association
with PKC1.sup.HA, GST-Rho1 was not eluted from the glutathione
agarose beads used for purification. GST-Rho1-bound beads were
incubated with cell extract in immunoprecipitation buffer (4) for 5
h at 4.degree. C., followed by 3 washes with this buffer. For use
in PKC1.sup.HA protein kinase assays, GST-Rho1 and GST-Cdc42 were
eluted from the beads with reduced glutathione. Purified GST-Rho1
displayed no protein kinase activity against the Bck1 peptide in
the absence of PKC1 (not shown).
[0189] C. Results and Discussion
[0190] To examine the role of RHO1 in the cell integrity signaling
pathway, we isolated a set of 11 temperature-sensitive rho1 alleles
by in vitro random mutagenesis. Some of these mutants displayed
cell lysis defects at the restrictive temperature (eg. rho1-5), but
others did not (eg. rho1-3; FIG. 1A). Additionally, overexpression
of PKC1 suppressed exclusively rho1-5 (FIG. 1B). Because of this
allele-specific behavior, we chose rho1-3 and rho1-5 for further
study.
[0191] The Mpk1 MAPK is activated via PKC1 in response to brief
heat shock treatment (4). To determine if RHO1 is required for cell
integrity pathway signaling, we tested the ability of rho1.sup.ts
mutants to activate Mpk1 upon heat shock. Mpk1, tagged at its
COOH-terminus with the influenza hemagglutinin (HA) epitope (Mpk1
HA), was immunoprecipitated from extracts of heat shock-treated
cells, and assayed for protein kinase activity in vitro using
myelin basic protein (MBP) as substrate. Heat shock-induced
activation of Mpk1 was completely blocked in the rho1-3 mutant
(FIG. 2), indicating that RHO1 function is essential for Mpk1
activation. The rho1-5 mutant allowed some Mpk1 activation,
suggesting that this allele retains some function at restrictive
temperature. Residual function of the rho1-5 allele at high
temperature might also explain the allele-specific suppression of
this mutant by PKC1 overexpression if Rho1 function is required for
PCK1 activation.
[0192] The yeast Cdc42 GTPase interacts with and stimulates the
Ste20 protein kinase, which regulates the MAPK-activation cascade
of the yeast pheromone response pathway (24, 25). Additionally,
both recombinant human Cdc42 and Rac stimulate a mammalian protein
kinase that is closely related to Ste20 (PAK65) (26, 27). Because
Ste20 and PKC1 function at analogous positions in their respective
MAPK signaling pathways (2, 3), we examined the possibility that
Rho1 interacts directly with PKC1 in vivo. Rho1, tagged at its
NH.sub.2-terminus with the HA epitope (.sup.HARho1), was
immunoprecipitated from yeast extracts, and the resultant
immunoprecipitates were analyzed by SDS-PAGE and immunoblotting
with anti-PKC1 antibody. PKC1 was co-immunoprecipitated with
.sup.HARho1 (FIG. 3A, lanes 4 and 6), suggesting that PKC1
associates with Rho1 in vivo. This interaction was observed both in
cells growing at 23.degree. C. and after heat shock.
[0193] To determine if the association between Rho1 and PKC1
depends on the activation state of Rho1, we examined the effect of
different guanine nucleotides on this interaction in vitro.
Recombinant glutathione-S-transferase-(GST)-Rho1, immobilized on
glutathione agarose beads, was preloaded with either GTP.gamma.S or
GDP prior to incubation with a yeast extract containing soluble
PKC1 tagged at its COOH-terminus with the HA epitope (PKC1.sup.HA).
After washing the beads, bound PKC1.sup.HA was detected by SDS-PAGE
and immunoblotting with anti-HA antibody. FIG. 3B shows that
GTP.gamma.S-bound GST-Rho1 associated with PCK1 (lane 5), but
GDP-bound protein did not (lane 3).
[0194] We also tested the possibility that PKC1 activity is
stimulated by Rho1. PKC1.sup.HA was immunoprecipitated from yeast
extracts, and its protein kinase activity was measured in the
presence or absence of GST-Rho1 using a synthetic Bck1 peptide as
substrate. FIG. 4A shows that GST-Rho1 did not stimulate PKC1
activity alone but, when bound to GTP.gamma.S, conferred upon the
protein kinase the ability to respond to activating cofactors (PS,
DAG, and Ca.sup.2+). This stimulatory effect is specific to Rho1,
because GST-Cdc42 did not confer cofactor-dependent stimulation on
PCK1. In the presence of GTP-bound GST-Rho1, PCK1 was strongly
activated by phosphatidylserine (PS) as a lone cofactor (FIG. 4B).
The conventional isoforms of mammalian PKC are not stimulated by PS
alone (28, 29). In contrast, this behavior is characteristic of the
atypical .zeta. isoform of PKC (28, 30). No additional stimulation
was observed by addition of Ca.sup.2+, DAG, or phorbol ester (PMA)
as a DAG substitute. This behavior is also exclusively
characteristic of PKC.zeta. (28, 30). Interestingly, the cys-rich
region of PKC1, which is predicted to be a DAG-binding domain, has
been reported to interact with Rho1 in two-hybrid experiments (19).
Therefore, Rho1 may replace DAG in the activation of PKC1.
[0195] This study provides the first example of a PKC isoform whose
stimulation by cofactors is dependent on a GTPase. We have
identified recently a second role for Rho1 in the maintenance of
cell integrity. Specifically, Rho1 is an essential component of the
1,3-.beta.-glucan synthase (GS) complex (see Example 2, infra), the
enzyme responsible for constructing polymers of 1,3-.beta.-glucan
in the cell wall. We have found that thermal induction of the FKS2
gene, which encodes another component of the GS (32, 33), is under
the control of PKC1 and MPK1. Based on these findings, we propose
the following model. A signal induced by weakness created in the
cell wall during growth (and exacerbated at high temperature)
stimulates guanine nucleotide exchange of Rho1 at the growth site.
The GTP-bound Rho1 stimulates cell wall construction directly by
activating GS and indirectly by stimulating PCK1-dependent gene
expression in support of this process (FIG. 5).
[0196] D. References For Example 1
[0197] 1. D. E. Levin et al., (1990) Cell 62: 213-224
[0198] 2. I. Herskowitz (1995) Cell 80: 187-197
[0199] 3. D. E. Levin and B. Errede (1995) Curr. Opin. Cell. Biol.
7: 197-202
[0200] 4. Y. Kamada et al., (1995) Genes Dev. 9: 1559-1571
[0201] 5. M. Watanabe et al., (1994) J. Biol. Chem. 269:
16829-16836
[0202] 6. B. Antonsson et al., (1994) J. Biol. Chem. 269:
16821-16828
[0203] 7. A. J. Ridley and A. Hall (1992) Cell 70: 389-399
[0204] 8. A. J. Ridley et al., (1992) Cell 70: 401-410
[0205] 9. C. D. Nobes and A. Hall (1995) Cell 81: 53-62
[0206] 10. C. S. Hill et al., (1995) Cell 81: 1159-1170
[0207] 11. M. F. Olson et al., (1995) Science 269: 1270-1272
[0208] 12. A. Minden et al., (1995) Cell 81: 1147-1157
[0209] 13. O. A. Coso et al., (1995) Cell 81: 1137-1146
[0210] 14. A. B. Vojtek and J. A. Cooper (1995) Cell 82:
527-529
[0211] 15. R. Treisman (1995) EMBO J. 14: 4905-4913
[0212] 16. W. Yamochi et al., (1994) J. Cell Biol. 125:
1077-1093
[0213] 17. P. Madaule et al., (1987) PNAS USA 84: 779-783
[0214] 18. H. Qadota et al., (1994)PNAS USA 91: 9317-9321
[0215] 19. H. Nonaka et al., (1995) EMBO J. 14: 5931-5938
[0216] 20. R. S. Sikorski and P. Hieter (1989) Genetics 122:
19-27
[0217] 21. R. C. Cadwell and G. F. Joyce (1992) PCR Meth. Appl. 2:
28-32
[0218] 22. Y. Ohya and D. Botstein (1994) Genetics 138:
1041-1054
[0219] 23. Y. Zheng et al., (1994) J. Biol. Chem. 269:
2369-2372
[0220] 24. M-N. Simon et al., (1995) Nature 376: 702-705
[0221] 25. Z-S. Zhao et al., (1995) Mol. Cell. Biol. 15:
5246-5257
[0222] 26. E. Manser et al., (1994) Nature, 367: 40-46
[0223] 27. U. G. Knaus et al., (1995) Science, 269: 221-223
[0224] 28. Y. Ono et al., (1989) PNAS USA 86: 3099-3103
[0225] 29. D. J. Burns et al., (1990) J. Biol. Chem. 265:
12044-12051
[0226] 30. A. Toker et al., (1994) J. Biol. Chem. 269: 32358-32367
et al.
[0227] 32. P. Mazur et al., (1995) Mol. Cell. Biol. 15:
5671-5681
[0228] 33. S. B. Inoue et al., (1995) Eur. J. Biochem. 231:
845-854
EXAMPLE 2
Identification of Yeast Rho1 GTPase as a Regulatory Subunit of
1,3-.beta.-glucan Synthase
[0229] A. Overview
[0230] 1,3-.beta.-glucan synthase is a multi-enzyme complex that
catalyzes the synthesis of 1,3-.beta.-linked glucan, a major
structural component of the yeast cell wall. Temperature-sensitive
mutants in the essential Rho-type GTPase, Rho1, displayed
thermolabile glucan synthase activity, which was restored by the
addition of recombinant Rho1. Glucan synthase from mutants
expressing constitutively active Rho1 did not require exogenous GTP
for activity. Rho1 copurified with 1,3-.beta.-glucan synthase and
was found to associate with the Gsc1/Fks1 subunit of this complex
in vivo. Both proteins were found to reside predominantly at sites
of cell wall remodeling. Therefore, it appears that Rho1 is a
regulatory subunit of 1,3-.beta.-glucan synthase.
[0231] The cell wall of the budding yeast Saccharomyces cerevisiae
is required to maintain cell shape and integrity (1). Vegetative
proliferation requires that the cell remodel its wall to accomodate
growth, which during bud formation, is polarized to the bud tip.
The main structural component responsible for the rigidity of the
yeast cell wall is 1,3-.beta.-linked glucan polymers with some
branches through 1,6-.beta.-linkages. The biochemistry of the yeast
enzyme catalyzing the synthesis of 1,3-.beta.-glucan chains has
been studied extensively (2,3), but little is known at the
molecular level about the genes encoding subunits of this enzyme.
Only a pair of closely related proteins (Gsc1/Fks1 and Gsc2/Fks2)
are known to be subunits of the 1,3-.beta.-glucan synthase (GS)
(3-5). GS activity in many fungal species, including S. cerevisiae,
requires GTP or a non-hydrolyzable analog (eg. GTP.gamma.S) as a
cofactor, suggesting that a GTP-binding protein stimulates this
enzyme (2,6). In this report, we demonstrate that the Rho1 GTPase
is an essential regulatory component of the GS complex.
[0232] The Saccharomyces RHO1 (Ras homologous) gene encodes a small
GTPase that resides at sites of growth (7), and whose function is
essential for viability (M. S. Boguski et al. (1992) New Biol.
4:408). Based on phenotypic analyses of conditional rho1 mutants,
we and others have suggested that the normal function of Rho1 is to
maintain cell integrity (7,9). Conditional rho1 mutants are
hypersensitive to Calcofluor white and echinocandin B, drugs that
interfere with cell wall assembly, suggesting that this gene is
involved in wall construction (10). To determine if Rho1 is
required for glucan synthesis, we measured GS activity in extracts
of temperature-sensitive rho1 mutants grown at permissive
temperature. GS activity from wild-type cells increased as a
function of assay temperature from 23.degree. C. to 30.degree. C.
to 37.degree. C. (FIG. 6A). All of the rho1 mutants tested
displayed reduced levels of activity at each temperature relative
to wild-type. Moreover, the enzyme from all but one mutant (rho1-5)
exhibited some level of thermolability, suggesting that RHO1
function is required for GS activity. Therefore, we tested the
ability of purified, recombinant glutathione-S-transferase
(GST)-Rho1 to restore GS activity to membrane fractions from the
most impaired rho1 mutant (rho1-3). Membranes from this mutant were
virtually devoid of activity at 37.degree. C. FIG. 6B shows that GS
activity was restored fully by the addition of GTP.gamma.S-bound
GST-Rho1, but not GST-Cdc42, another member of the Rho-family of
GTPases. GTP.gamma.S could be replaced with GTP, but not GDP (FIG.
6C). These results indicate that the GS-deficient mutant membranes
lack only Rho1 function.
[0233] We also examined GS activity from yeast cells expressing an
constitutively active RHO1 allele (RHO1-Q68H). The analogous
mutation in Ras results in a protein that is impaired for the
ability to hydrolyze GTP and has transforming potential in
mammalian cells (11). The GTP requirement of GS activity was
examined in membranes obtained from rho1-3 cells overexpressing
RHO1 or RHO1-Q68H under the inducible control of the GAL1 promoter
(FIG. 7). Under inducing conditions (galactose), expression of
RHO1-Q68H resulted in GS activity that was independent of exogenous
GTP. By contrast, GS activity in membranes from cells
overexpressing RHO1 was largely dependent on GTP. Similar results
were obtained with another activated allele (RHO1-G19V; 12). Taken
together, these results indicate that GS activity requires
functional Rho1 in the GTP-bound state.
[0234] To determine if Rho1 is a component of the GS complex, we
monitored the levels of Rho1 during purification of GS activity.
The enzyme was purified by successive product entrapments following
extraction from membranes (3). FIG. 8 shows that both Rho1 and
Gsc1/Fks1 were enriched in the partially purified GS. The specific
activity of GS was increased approximately 700-fold through
purification, whereas Rho1 was enriched approximately 400-fold. GS
purified from the rho1-5 mutant was deficient in GS activity
despite normal levels of Rho1 and Gsc1/Fks1 proteins (data not
shown). To determine if Rho1 copurifies with GS because it
physically associates with the GS complex, the partially purified
enzyme was immunoprecipitated with either of two monoclonal
antibodies against Gsc1/Fks1. The resultant immunoprecipitates were
analyzed by SDS-PAGE and immunoblotting with anti-Rho1 antibody.
FIG. 9A shows that Rho1 coimmunoprecipitates with Gsc1/Fks1.
[0235] Finally, we examined the localization of Rho1, tagged at its
NH.sub.2-terminus with the influenza hemagglutinin (HA) epitope
(.sup.HARho1), and Gsc1/Fks1 in growing yeast cells. Rho1 is known
to be located at the bud tip (the site of polarized growth) during
bud formation, and at the mother/bud neck (the site of septum
formation) during cytokinesis (7). Indirect immunofluorescence of
cells double labeled with anti-HA and anti-Gsc1/Fks1 antibodies
revealed that Gsc1/Fks1 colocalizes with .sup.HARho1 (FIG. 9B).
These results strongly suggest that Rho1, like Gsc1/Fks1, is a
component of the GS complex. This complex is redistributed through
the cell cycle so as to reside at sites of cell wall
remodeling.
[0236] We have shown recently that Rho1 interacts with and
activates the PKC1 protein kinase (see Example 1, supra). Like rho1
mutants, pkc1 mutants display cell integrity defects that result
from a deficiency in cell wall construction. However, several
observations indicate that PKC1 is not involved in the activation
of GS. First, mutants in PKC1 display no defect in GS activity
(14). Second, overexpression of PKC1 did not restore GS activity to
rho1 mutants (15). Third, PKC1 was not detected in the purified GS
complex (16). Therefore, we propose that Rho1 plays at least two
distinct regulatory roles in the maintenance of cell integrity. One
is the activation of GS and the other is the stimulation of PKC1
for signal transduction. Rho1 may serve to coordinate, both
spacially and temporally, several events required for effective
cell wall remodeling. Both the GTP requirement for GS activity, and
the structure of fungal PKCs are evolutionarily conserved (6,17),
suggesting that the dual function of Rho1 may be conserved as
well.
[0237] C. References and Notes For Example 2
[0238] 1. V. J. Cid et al., (1995) Microbiol. Rev. 59:345; F. M.
Klis, (1994) Yeast 10:851
[0239] 2. P. C. Mol et al., (1994) J. Biol. Chem. 269:31267
[0240] 3. S. B. Inoue et al., (1995) Eur. J. Biochem. 231:845
[0241] 4. C. M. Douglas et al., (1994) PNAS USA 91:12907; A. F. J.
Ram et al.,(1995) FEBS Lett. 358:165; P. Garett-Engele et al.,
(1995) Mol. Cell. Biol. 15:4103
[0242] 5. P. Mazur et al., ibid, p. 5671.
[0243] 6. P. J. Szaniszlo et al., (1985) J. Bacteriol. 161:1188
[0244] 7. W. Yamochi et al., (1994) J. Cell. Biol. 125:1077
[0245] 8. P. Madaule et al., (1987) PNAS USA 84:779
[0246] 9. H. Qadota et al., (1994) PNAS USA 91:9317
[0247] 10. Yeast strains YOC752 (rho1-2), YOC729 (rho1-3), YOC754
(rho1-4), YOC755 (rho1-5) and YOC764 (wild-type) were used in this
study. YOC752, YOC729, and YOC755 displayed hypersensitivity to
Calcofluor white and echinocandin B at 23.degree. C.
[0248] 11. C. J. Der et al., (1986) Cell 44:167
[0249] 12. YPH499 cells carrying plasmids with wild-type RHO1
(pYO762), RHO1-G19V (pYO906) under the control of the GAL1
promoter, or vector alone (pYO761) were used. Cells were incubated
in galactose medium for 10 h, and GS activity associated with the
membrane fraction was measured (3). Most of the GS activity from
cells with pYO906 was GTP.gamma.S-independent, whereas only 15-20%
of the activity was GTP.gamma.S-independent in the control
strains.
[0250] 14. A temperature-sensitive pkc1 strain (SYT11-12A) and its
isogenic wild-type strain (YS3-6D) [S. Yoshida et al., (1992) Mol.
Gen. Genet. 231:337] were grown in YPD (yeast
extract/peptone/dextrose) at 23.degree. C. A pkc1.DELTA. strain
(DL376) and its isogenic wild-type (DL100) [D. E. Levin and E.
Bartlett-Heubusch, (1992) J. Cell Biol. 116:1221] were grown at
23.degree. C. in YPD containing 10% sorbitol. GS activities were
assayed at 23.degree. C. and at 37.degree. C.
[0251] 15. Mutants used were rho1-3 and rho1-5 carrying PKC1 on a
multicopy plasmid (pYO910), or vector alone (pYO324).
[0252] 16. Partially purified enzyme fraction (second product
entrapment) was analyzed by immunoblotting with anti-PCK1 antibody
(S. Yoshida, unpublished).
[0253] 17. T. Toda, et al., (1993) EMBO J. 12: 1987; G. Paravicini
et al., Yeast, in press.
[0254] 18. Crude yeast extracts were prepared as described [Y.
Kamada et al., (1995) Genes Dev. 9:1559], and stored at -80.degree.
C. in lysis buffer supplemented with 33% glycerol. Membrane
fractions, where indicated, were obtained from crude extracts and
1,3-.beta.-glucan synthase (GS) activity was measured as described
in (2) with the following modifications: UDP-[.sup.3H]glucose was
used as the substrate and .alpha.-amylase (1U/40 .mu.l) was added
to reaction mixtures to eliminate the contribution of
[.sup.3H]glucose incorporation into glycogen. For all GS assays,
the mean and standard error for four experiments is shown.
[0255] 19. Recombinant GST-Rho1 and GST-Cdc42 were expressed in Sf9
insect cells, and purified as described previously [Y. Zheng et
al., (1994) J. Biol. Chem. 269:2369].
[0256] 20. A series of protein sample dilutions was analyzed by
immunoblotting with guinea pig anti-Rho1 antiserum or mouse
anti-Gsc1/Fks1 monoclonal antibodies (T2B8; 3). The amount of
antigens was estimated by densitometry.
[0257] 21. Goat anti-mouse IgG-agarose (20 .mu.l; Sigma) was
incubated with 500 .mu.l media from monoclonal antibody cultures
for 5 h at 37.degree. C. The agarose beads were washed 5 times with
phosphate-buffered saline and twice with Buffer A (0.4 CHAPS, 0.08%
cholesteryl hemisuccinate, 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 8
.mu.M GTP.gamma.S and 33% glycerol). Partially purified GS (1.8
.mu.g) was added and the reaction mixtures were further incubated
for 2 h at 37.degree. C. After washing the beads four times with
Buffer A, the bound complexes were analyzed by immunoblotting with
anti-Rho1 antiserum or anti-Gsc1/Fks1 monoclonal antibodies
(T2B8).
[0258] 22. Cells of haploid strain YOC785, which bears a
rho1.DELTA. and the HA-tagged RHO1 gene (13) on a centromere
plasmid (pYO904) were double stained with mouse monoclonal antibody
against Gsc1/Fks1 (T2B8) and rabbit anti HA-antibody (Boehringer),
as described previously [J. R. Pringle et al., (1989) Methods Cell
Biol., 31:357]. Secondary antibodies were FITC-conjugated
anti-mouse IgG (Cappel) and TRITC-conjugated anti-rabbit IgG
(Cappel). Control strains (YPH499 for .sup.HARho1 and gsc1.DELTA.
for Gsc1/Fks1) produced no signals in single staining experiments.
The secondary antibodies did not cross-react with the heterologous
primary antibodies. Some internal punctate staining of Gsc1/Fks1
that did not colocalize with .sup.HARho1 may represent secretory
intermediates.
EXAMPLE 3
Yeast Geranylgeranyl Protein Transferase I is Essential for
Membrane Localization of Rho1 GTPase and 1,3-.beta.-glucan Synthase
Activity
[0259] The abbreviations used in Example 3 are: GGPTase I,
geranylgeranyl protein transferase I; GST,
glutathone-S-transferase; HA, influenza hemagglutinin; ORF open
reading frame; GS, 1,3-.beta.-glucan synthase.
[0260] A. Overview
[0261] Protein prenylation, farnesylation and geranylgeranylation,
is a posttranslational reaction which requires the covalent
attachment of a hydrophobic tail, isoprenoid (C15 or C20), to the
C-terminal cysteine residue of the substrate proteins (1).
Prenylation is necessary for many proteins to interact with
membranes and to locate at proper intracellular places. Many lines
of evidence have been accumulated to show that small GTPases
require prenylation to gain full functionality (1, 2).
[0262] Genes encoding subunits of each prenyltransferase have been
cloned in the yeast Saccharomyces cerevisiae. The genes CAL1 (3)
(also known as CDC43 (4)) and DPR1 (5) (also known as RAM1) encode
.beta. subunits of the yeast GGPTase I and FTase, respectively, and
RAM2 encodes the common .alpha. subunit (6). The .alpha. subunit,
.beta. subunit and component A of the yeast GGPTase II are encoded
by BET4, BET2 and MSI4, respectively (7). An alignment of the
homologous regions of the three .beta. subunit sequences (positions
159-350 of the Cal1/Cdc43 sequence) reveals 32-40% identity each
other (3). This region contains novel repeat motifs .(M. S. Boguski
et al. (1992) New Biol. 4:408). The repeats have a length of 44-45
residues and there are three repeats in the Cal1p/Cdc43p sequence.
The repeats are conserved in the central Gly-Gly-Phe-Gly-Gly
sequence region. The .alpha. subunit of isoprenyl transferases also
possesses distinct internal repetitive sequence containing
tryptophan. Hydrophobic bonds between the side chains of the
conserved tryptophan and phenylalanine may be important for forming
heterodimer.(M. S. Boguski et al. (1992) New Biol. 4:408).
[0263] Among prenyltransferase mutants, a mutation in the GGPTase I
.beta. subunit gene was the first to be isolated and characterized.
cal1-1 was identified originally as a mutation resulting in a
Ca.sup.2+ -dependent phenotype.(9). The cal1-1 mutant
simultaneously exhibits a homogeneous terminal phenotype with a
G2/M nucleus and a small bud at 37.degree. C. (9). Independent
screening of yeast cell cycle mutants which accumulated enlarged
unbudded cells identified six other alleles, cdc43-2.about.cdc43-7
(10). Yeast GGPTase I is essential for yeast cell growth, since
deletions of the CAL1 gene result in a lethal phenotype (3).
However, GGPTase I is no longer essential, when the dosage of the
two GTPases, Rho1p (11, 12) and Cdc42p (13), are artificially
elevated (14). Since the yeast GGPTase I prenylates these two
GTPases, Cdc42p and Rho1p are implicated genetically as the only
two essential substrates of GGPTase I (14). CAL1/CDC43 is necessary
not only for the function of the small GTPases but also for
membrane localization of the small GTPases. An increase in soluble
Cdc42p is observed in the cdc43-2 strain grown at the restrictive
temperature (15).
[0264] This study was undertaken to understand the molecular
lesions caused by loss of the GGPTase I function, using the seven
temperature-sensitive mutations in the CAL1/CDC43 gene. All of the
mutation sites were determined at the nucleotide level. An increase
in soluble Rho1p was observed in the cal1-1 strain grown at the
restrictive temperature. Futhermore, GS activity was dramatically
reduced in the cal1-1 mutant strains. Several phenotypic
differences were observed among the cal1/cdc43 mutations, possibly
due to the alteration of substrate specificity caused by the
mutations.
[0265] B. Experimental Procedures
[0266] Materials.--YPD medium contained 1% Bacto-yeast extract
(Difco Laboratories, Detroit, Mich.), 2% polypeptone (Nihon
Chemicals, Osaka), and 2% glucose (Wako Chemicals, Tokyo). YPD
supplemented with 100 mM or 300 mM CaCl.sub.2 was used as
Ca.sup.2+-rich medium. Other standard media are described elsewhere
(16).
[0267] DNA manipulation--DNA fragments containing the cdc43
mutations were cloned by gap repair (17). The pCAL-F9 plasmid
containing the 2.8 kb SphI-PstI fragment of the CAL1/CDC43 gene was
digested with Nsp(7524)V and EcoT22I and introduced into the cdc43
strains (cdc43-2.about.cdc43-7)- . Transformation of the plasmid
containing the Nsp(7524)V-EcoT22I gap resulted in repair of the gap
to yield plasmids in which the gap was repaired by gene conversion
with the chromosomal sequences. The gap-repaired plasmids were
recovered from yeast, and its Nsp(7524)V-EcoT22I fragment was
subcloned into the Nsp(7524)V-EcoT22I gap of pCAL-F9. Then, the
resulting plasmids YCpT-cdc43-2.about.YCpT-cdc43-7 were introduced
into the cdc43 strains. Because the transformants showed a
temperature-sensitive phenotype, we concluded that all of the cdc43
mutations resided within the region between the Nsp(7524)V and
EcoT22I. Nucleotide sequencing of the 1.0-kb Nsp(7524)V-EcoT22I
fragment from the YCpT-cdc43-2.about.YCpT-cdc43-7 revealed that
each of the cdc43 mutants possessed a single base pair change
within the ORF.
[0268] Production of the anti-Rho1p antibody--The purified
GST-Rho1p (64-209) which is a fusion protein of GST with Rho1p from
amino acid positions 64 to 209 was minced and emulsified with R-700
(RIBI ImmunoChem Research, Hamilton, Mont.) and the resulting
emulsion was used to immunize four guinea pigs. After boost was
repeated five times with three-weeks intervals, blood was collected
from the animals and one of the immune serum was used in this
study. The anti-Rho1p antibody specifically recognized Rho1p.
Western blotting analysis showed that there was no other protein
band detected in the lysates of cells expressing human rhoA in
place of RHO1.
[0269] Cell Fractionation Experiments. Cell fractionation
experiments were performed using techniques described by Ziman et
al. (15). Briefly, cells were grown at 23.degree. C. to mid log
phase, and approximately 5.times.10.sup.8 cells were collected,
washed with water, resuspended in 0.1 ml of lysis buffer (0.8 M
sorbitol, 1 mM EDTA, 10 mM
N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid pH 7.0) with
0.5 mM PMSF, and lysed on ice by vortexing with 400-500 mm
acid-washed glass beads (Sigma). Greater than 80% lysis was
verified by light microscopy. After addition of 0.4 ml of lysis
buffer, cell lysates were spun at 390.times. g for 1 min at
4.degree. C. The supernatant was then spun at 436,000.times. g for
20 min at 4.degree. C., and the pellets were resuspended in the
same volume of lysis buffer. To assess the relative amount of Rho1p
and Cdc42p in each fraction, equal volumes of each fraction were
loaded onto a sodium dodecyl sulfate-12.5% polyacrylamide gel for
immunoblot analysis. Guinea pig polyclonal antibody against Rho1p
and mouse monoclonal antibody against HA (12CA5, Boeringer
Mannheim, Germany) were used at 1:500 and 1:100, respectively.
Alkaline phosphatase-conjugated goat anti-guinea pig IgG and
anti-mouse IgG were used at 1:5000. Antibody-antigen complexes were
detected with 5-bromo-4-chloro-3-indoryl-phosphate and nitro blue
tetrazolium.
[0270] C. Results and Discussion
[0271] Mutation points of cdc43-2.about.cdc43-7 were determined
after DNA fragments containing the cdc43-2.about.cdc43-7 mutations
were cloned by the gap repair method.(17) to yield
YCpT-cdc43-2.about.YCpT-cdc43-7. Based on the subcloning analysis
(see Materials and Methods), we concluded that all of the cdc43
mutations resided within the 1.0-kb region between the Nsp(7524)V
and EcoT22I, nearly corresponding to the entire coding region of
CAL1/CDC43. Nucleotide sequencing of the 1.0-kb Nsp(7524)V-EcoT22I
fragment from the YCpT-cdc43-2.about.YCpT-cdc43-7 revealed that
each of the cdc43 mutants possessed a single base pair change
within the ORF. FIG. 10 shows the amino acid changes in the cdc43
sequences. cdc43-4 and cdc43-6 resulted from an identical
nucleotide change, and hereafter are referred to as cdc43-6.
cdc43-5 had a amino acid change at the same position as cdc43-4 and
cdc43-6, but resulted in a different amino acid change. FIG. 10
shows that the four cdc43/cal1 mutations (cdc43-5 cdc43-6, cdc43-7,
cal1-1) were mapped within the domain homologous to the b-subunits
of other protein isoprenyltransferases (a.a. position 159-350).
Interestingly enough, these mutations affect the conserved amino
acid residues among the subunits of GGPTase I from four different
species (3, 18, 19). The other two cdc43 mutations (cdc43-2 and
cdc43-3) were mapped outside of the homologous domain.
[0272] We have previously shown the functional interaction between
RHO1 and CAL1 based on the observation that overproduction of Rho1p
suppressed the temperature sensitivity of cal1-1 (See reference of
Example 3). In order to know whether the suppression by
overproduction of Rho1p was seen only with the cal1-1 allele, we
examined the ability of overproduction of Rho1p to suppress the
cdc43 mutations. Since the restrictive temperatures of the cdc43
mutants were different, effects of the Rho1p overexpression were
examined at five different temperatures (23.degree. C., 28.degree.
C., 30.degree. C., 33.degree. C. and 37.degree. C.). We found that
the cdc43 mutations were not suppressed effectively by
overproduction of Rho1p (Table 1). None of the mutations was
suppressed at 37.degree. C., while cal1-1 was suppressed at this
temperature. cdc43-2 and cdc43-7 with multicopy RHO1 grew slightly
faster than those with vector alone at 30.degree. C., while cal1-1
was suppressed completely at this temperature. Slight growth
improvement of cdc43-5 by overproduction of Rho1p was observed only
at 23.degree. C. These results indicate that among the cal1/cdc43
mutations so far isolated, cal1-1 is a unique mutation that is
effectively suppressed by overproduction of Rho1p.
1TABLE 1 Effect of overproduction of Rho1p and Cdc42p in the
cal1/cdc43 mutants growth on YPD YPD +Ca strain plasmid 23.degree.
C. 28.degree. C. 30.degree. C. 33.degree. C. 37.degree. C.
33.degree. C. 37.degree. C. cal1-1 pYO324 + + .+-. -- -- ++ +
YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++ ++ ++ ++ + ++ +
YEpT-CDC42 n.d. n.d. n.d. n.d. n.d. n.d. n.d. cdc43-2 pYO324 ++ +
.+-. -- -- -- -- YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++ + + --
-- -- -- YEpT-CDC42 ++ + .+-. -- -- -- -- cdc43-3 pYO324 ++ ++ ++
.+-. -- -- -- YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++ ++ ++
.+-. -- -- -- YEpT-CDC42 ++ ++ ++ .+-. -- + -- cdc43-5 pYO324 +
.+-. -- -- -- -- -- YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++
.+-. -- -- -- -- -- YEpT-CDC42 ++ ++ ++ ++ + ++ cdc43-6 pYO324 ++ +
.+-. -- -- -- -- YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++ + .+-.
.+-. -- -- -- YEpT-CDC42 ++ + .+-. -- -- -- -- cdc43-7 pYO324 ++ +
.+-. -- -- .+-. -- YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++ + +
-- -- .+-. -- YEpT-CDC42 ++ + .+-. -- -- + --
[0273] Since overproduction of Rho1p suppressed a mutation of the
CAL1/CDC43 gene, we next attempted to examine multicopy suppression
of the cdc43 mutations by overproduction of another essential
substrate of GGPTase I, Cdc42p. We found that overproduction of
Cdc42p suppressed the temperature-sensitive phenotype of cdc43-5
(Table 1); the cdc43-5 mutant with multiple copies of CDC42 grew
well at 37.degree. C. Among the cdc43 mutations, cdc43-5 was most
effectively suppressed by overproduction of Cdc42p; cdc43-3,
cdc43-6 and cdc43-7 were suppressed slightly by overproduction of
Cdc42p at the intermediate temperature, and cdc43-2 was not
suppressed at all at any temperature examined.
[0274] Several trials to introduce multiple copies of CDC42 into
the cal1-1 strain were unsuccessful. Reasoning that overexpression
of Cdc42p might be a lethal event in the cal1-1 strain, we
attempted to increase the levels of Cdc42p by placing its
expression under the control of the GAL1 promoter that was induced
by galactose in the medium. The cal1-1 strain with pGAL-CDC42 could
grow on solid media containing glucose but did not grow on media
containing galactose (FIG. 11). This growth inhibition was observed
at any temperature examined (23.degree. C., 30.degree. C. and
37.degree. C.). Since pGAL-CDC42 was not toxic in the wild-type
strain and many of the other cdc43 mutants (FIG. 11), we concluded
that lethality caused by the overexpression of Cdc42p is specific
to the cal1-1 mutant. Although CDC42 on a multicopy plasmid is not
toxic in cdc43-7, pGAL-CDC42 is dereterious in cdc43-7 (FIG. 11).
This may be due to the fact that the expression level of Cdc42p by
pGAL-CDC42 is more than that expressed by multiple copies of
CDC42.
[0275] cal1-1 was suppressed most effectively by overexpression of
Rho1p, while cdc43-5 was suppressed by overexpression of Cdc42p. To
test the possibility that the allele-specific suppression is due to
substrate specificity of the mutant GGPTase I, we examined the
partitioning of Rho1p and Cdc42p in the cal1-1 and cdc43-5 mutant
strains. It was already shown that soluble Cdc42p increases in the
cdc43-2 strain grown at the restrictive temperature (15),
suggesting that membrane localization of small GTPases is dependent
on geranylgeranyl modification. We found that the proportion of
Rho1p found in the soluble fraction of cal1-1 dramatically
increases after the temperature shift (FIG. 12). Rho1p from cdc43-5
strain grown at 37.degree. C. for 2 hr was almost exclusively in
the particulate fraction, indicating that increase of soluble Rho1p
is specific to cal1-1. The proportion of HA-tagged Cdc42p found in
the soluble fraction of cdc43-5 increased after 2 hr incubation at
37.degree. C., while cal1-1 did not affect partitioning of
HA-tagged Cdc42p (FIG. 12). Temperature-shift itself did not affect
the partitioning of these GTPases in the wild-type control strain.
These results suggested that cal1-1 and cdc43-5 specifically impair
geranylgeranylation of Rho1p and Cdc42p, respectively.
[0276] We have previously shown that Rho1p is a regulatory subunit
of 1,3-.beta.-glucan synthase (see Example 2 above). To directly
examine involvement of GGPTase I in the Rho1 function, we measured
GS activity in membrane fractions of the cal1-1 and cdc43-5 mutant
cells grown at permissive temperature (FIG. 13). We found that
cal1-1 displayed dramatically reduced activity relative to
wild-type. cdc43-5 mutant instead displayed only slightly reduced
activity, probably due to the fact that cdc43-5 impairs
geranylgeranylation of Cdc42p more than geranylgeranylation of
Rho1. We tested whether purified, recombinant GST-Rho1 restored GS
activity to the membrane fraction of the cal1-1 mutant. GS activity
was restored by the addition of constitutively activated Rho1 .
These results indicate that the GS-deficient cal1-1 mutant membrane
lack the Rho1 function.
[0277] Multiple copies of either Rho1p or Cdc42p suppressed
specific alleles of cal1/cdc43 (Table 2): cal1-1 was suppressed
effectively by multicopy RHO1, while cdc43-5 was suppressed
effectively by multicopy CDC42. Given both Rho1p from the cal1-1
strain and Cdc42p from the cdc43-5 strain accumulate in the soluble
fraction, substrate specificipy of the mutant GGPTase I likely
accounts for the allele-specific suppression. In our current model,
cal1-1 and cdc43-5 selectively impair the in vivo
geranylgeranylation of Rho1p and Cdc42p, respectively. This is
consistent with observation of the mutant phenotypes; terminal
phenotypes of cdc43-5 and cdc42 are undistinguished, and those of
cal1-1 and temperature-sensitive rho1 strains are somewhat similar.
This is also consistent with our observation that overexpression of
Cdc42p is lethal specifically in the cal1-1 strain, because
overexpression of Cdc42p likely sequesters the cal1-1 GGPTase I to
further impair geranylgeranylatikn of Rho1p. GS activity was
dramatically reduced in cal1-1 but not in cdc43-5. Taken together,
our genetic and biochemical results suggest that the CAL1/CDC43
GGPTase I has an ability to prenylate the substrate GTPases by some
domain-specific, substrate-specific recognition mechanisms.
2TABLE 2 Summary of the effect of the GTPases in the cal1/cdc43
mutants overproduction Phenotype Cdc42p Rho1p suppression cdc43-5
cal1-1 (cdc43-3, -4, -7) (cdc43-2, -5, -7) deleterious cal1-1
(cdc43-7)
[0278] C. References in Example 3
[0279] 1. W. R. Schafer and J. Rine (1992) Annu. Rev. Genet.
26:209; S. Clarke (1992) Annu. Rev. Biochem. 61:355
[0280] 2. C. A. Omer and J. B. Gibbs (1994) Mol. Microbiol.
11:219
[0281] 3. Y. Ohya et al. (1991) J. Biol. Chem. 266:12356
[0282] 4. D. I. Johnson et al. (1991) Gene 98:149
[0283] 5. L. E. Goodman et al. (1988) Yeast 4:271
[0284] 6. B. He et al. (1991) Proc. Natl. Acad. Sci. USA
88:11373
[0285] 7. K. Fujimura et al. (1994) J. Biol. Chem. 269:9205; G.
Rossi et al. (1991) Nature 351:158
[0286] 8. M. S. Boguski et al. (1992) New Biol. 4:408
[0287] 9. Y. Ohya et al. (1984) Mol. Gen. Genet. 193:389
[0288] 10. A. E. M. Adams et al. (1990) J. Cell. Biol. 111:131
[0289] 11. P. Madaule et al. (1987) Proc. Natl. Acad. Sci. USA
84:779
[0290] 12. H. Qadota et al. (1994) Proc. Natl. Acad. Sci. USA
91:9317
[0291] 13. D. I. Johnson and J. R. Pringle (1990) J. Cell. Biol.
111:143
[0292] 14. Y. Ohya et al. (1993) Mol. Biol. Cell 4:1017
[0293] 15. M. Ziman et al. (1993) ibid. 1307
[0294] 16. M. Rose et al. (1990) Methods in yeast genetics. A
laboratory manual. CSH Lab. Press, CSH, NY.
[0295] 17. T. L. Orr-Weaver et al. (1983) Methods in Enzymol.
101:228
[0296] 18. M. Diaz et al. (1993) EMBO J. 12:5245
[0297] 19. F. L. Zhang et al. (1994) J. Biol. Chem. 269:3175
[0298] 20. H. Qadota et al. (1992) Yeast 8:735
[0299] 22. Inoue et al. (1995) Eur. J. Biochem. 231: 845
[0300] All of the above-cited references and publications are
hereby incorporated by reference.
Equivalents
[0301] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific polypeptides, nucleic acids, methods,
assays and reagents described herein. Such equivalents are
considered to be within the scope of this invention and are covered
by the following claims.
Sequence CWU 1
1
* * * * *