U.S. patent application number 11/598864 was filed with the patent office on 2007-03-15 for phosphorylated glyoxalase i and its use.
This patent application is currently assigned to Vlaams Interuniversitair Instituut Voor Biotechnologie VZW. Invention is credited to Katia Vancompernolle.
Application Number | 20070059772 11/598864 |
Document ID | / |
Family ID | 8179836 |
Filed Date | 2007-03-15 |
United States Patent
Application |
20070059772 |
Kind Code |
A1 |
Vancompernolle; Katia |
March 15, 2007 |
Phosphorylated glyoxalase I and its use
Abstract
The present invention relates to a phosphorylated form of
mammalian glyoxalase I. The present invention relates further to
the use of phosphorylated mammalian glyoxalase I to modulate
MG-modification of proteins (AGE formation) and consequent cell
death, especially upon stress such as oxidative stress, or upon TNF
treatment.
Inventors: |
Vancompernolle; Katia;
(Heusden, BE) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Assignee: |
Vlaams Interuniversitair Instituut
Voor Biotechnologie VZW
Zwijnaarde
BE
|
Family ID: |
8179836 |
Appl. No.: |
11/598864 |
Filed: |
November 14, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10630451 |
Jul 30, 2003 |
|
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11598864 |
Nov 14, 2006 |
|
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PCT/EP02/01118 |
Jan 30, 2002 |
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10630451 |
Jul 30, 2003 |
|
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Current U.S.
Class: |
435/7.1 ;
424/85.1; 435/189; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/88 20130101 |
Class at
Publication: |
435/007.1 ;
435/189; 435/069.1; 435/320.1; 435/325; 536/023.2; 424/085.1 |
International
Class: |
A61K 38/19 20060101
A61K038/19; G01N 33/53 20060101 G01N033/53; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C12N 9/02 20060101
C12N009/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 31, 2001 |
EP |
01200353.9 |
Claims
1.-11. (canceled)
12. A process for modulating methylglyoxal-modification of proteins
in a cell, said process comprising: contacting the cell with a
means for phosphorylating a glyoxalase I associated with the
cell.
13. The process of claim 12, wherein the means for phosphorylating
a glyoxalase I is selected from the group consisting of TNF, PKA, a
phosphorylation mutant of glyoxalase I, and combinations of any
thereof.
14. The process of claim 12, wherein the means for phosphorylating
a glyoxalase I is TNF.
15. The process of claim 12, wherein the glyoxalase I is mammalian
glyoxalase I.
16. The process of claim 12, wherein the glyoxalase I comprises an
amino acid sequence of SEQ ID NO: 1.
17. The process of claim 12, wherein the modulation of the
methylglyoxal-modification of proteins occurs in a cell.
18. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of co-pending application
Ser. No. 10/630,451, filed Jul. 30, 2003, which is a continuation
of co-pending International Patent Application No. PCT/EP02/01118
filed on Jan. 30, 2002 designating the United States of America
(International Publication No. WO 02/061065 published in English on
Aug. 8, 2002), which claims priority to European Patent Application
No. 01200353.9, filed Jan. 31, 2001, the contents of the entirety
of each of which are incorporated herein by this reference in their
entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to biotechnology,
and, more particularly, to a phosphorylated form of mammalian
glyoxalase I. The present invention relates further to the use of
phosphorylated mammalian glyoxalase I to modulate methylglyoxal
(MG)-modification of proteins and consequent cell death, especially
upon stress such as oxidative stress, or upon TNF treatment.
BACKGROUND
[0003] Tumor Necrosis Factor (TNF) is a pleiotropic cytokine,
originally described for its ability to cause hemorrhagic necrosis
of certain tumors in vivo (Carswell et al., 1975). In addition to
its anti-tumor and anti-malignant cell effects, TNF has been
reported to influence mitogenesis, differentiation, and
immunoregulation of various cell types.
[0004] The activities of TNF are mediated through two cell-surface
receptors, namely TNF-R55 (CD120a) and TNF-R75 (CD120b), which are
expressed by most cell types. TNF's effects are mediated primarily
through TNF-R55. Upon activation of the receptor, adaptor proteins
such as TRADD and TRAF are recruited and bind to the intracellular
part of the clustered receptor (for review, see Wallach et al.,
1999). Those receptor-associated molecules that initiate signaling
events are largely specific to the TNF/nerve growth factor receptor
family. However, the downstream signaling molecules are not unique
to the TNF system, but also mediate effects of other inducers.
Downstream signaling molecules in the TNF system identified so far
include: caspases, phospholipases, the three mitogen-activated
protein (MAP) kinases, and the NF-.kappa.B activation cascade.
[0005] TNF-induced cell death in L929 cells is characterized by a
necrosis-like phenotype and does not involve DNA fragmentation
(reviewed by Fiers et al., 1999). It is independent of caspase
activation and cytochrome c release, but is dependent on
mitochondria and is accompanied by increased production of reactive
oxygen intermediates (ROI) in the mitochondria that are essential
to the death process (Goossens et al., 1995; Goossens et al.,
1999). The latter was demonstrated by the fact that lipophylic
anti-oxidantia, when added three hours after TNF treatment, could
not only arrest the ongoing increased ROI production, but could
also arrest cell death (Goossens et al., 1995). Furthermore, the
mitochondria translocate from a dispersed distribution to a
perinuclear cluster (De Vos et al., 2000); functional implications
of this mitochondrial translocation remain unclear.
[0006] Glyoxalase I, together with glyoxalase II, constitutes the
glyoxalase system that is an integral component of the cellular
metabolism of .alpha.-ketoaldehydes and is responsible for the
detoxification of the latter. The prime physiological substrate of
the glyoxalase system is methylglyoxal (MG), which is cytotoxic.
The major source of intracellular MG is the glycolysis namely,
nonenzymatic and enzymatic elimination of phosphate from
dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The
glyoxalase system, using glutathione (GSH) as cofactor, catalyzes
the conversion of methylglyoxal to D-lactate in two consecutive
steps. Glyoxalase I catalyzes the isomerization of the
hemithioacetal, produced by the nonenzymatic conjugation of
methylglyoxal with glutathione (GSH), to S-D-lactoylglutathione
which is then hydrolyzed by glyoxalase II to D-lactate and GSH.
D-lactate is then further metabolized to pyruvate by 2-hydroxy-acid
dehydrogenase localized in the mitochondria. In addition to its
role as a detoxification system, it has been suggested that
glyoxalase I, together with its substrate MG, is involved in the
regulation of cellular growth (for a review, see Kalapos, 1999),
but until now this role has not been found. Increased expression of
glyoxalase I occurs in diabetic patients and in some types of
tumors such as colon carcinoma (Ranganathan et al., 1993), breast
cancer (Rulli et al., 2001), prostate cancer (Davidson et al.,
1999). It is also uniquely overexpressed in invasive human ovarian
cancer compared to the low malignant potential form of this cancer
(Jones et al., 2002). Also, hypoxia can lead to increased
expression of glyoxalase I (Principato et al., 1990). Recently, it
has been shown that glyoxalase I is involved in resistance of human
leukemia cells to anti-tumor agent-induced apoptosis (Sakamoto et
al., 2000).
DISCLOSURE OF THE INVENTION
[0007] While much effort has been directed at the molecular
mechanism of the caspase-dependent cell death pathway, relatively
little is known about the TNF-induced ROI-dependent cell death
pathway. To identify molecules involved in the latter, we performed
a comparative study of the phosphoproteins from TNF-treated and
control cells by two-dimensional (2-D) gel electrophoresis. It is
known that upon activation of the TNF receptor, several
kinases/phosphatases are activated (Guy et al., 1992; Guy et al.,
1991). However, most of the changes in phosphorylation occur very
rapidly (2 to 15 minutes) upon binding of TNF to its receptors and
most of them are transient and related to the gene-inductive
activities of TNF.
[0008] To identify molecules that are involved in the cytotoxic
process downstream of the receptor-proximal events, lysates from
cells that had been stimulated with TNF for 1.5 hours are studied.
Previously, oncoprotein 18 (Op18, stathmin) has been identified as
a protein with reproducible and large increases in phosphorylation
upon TNF treatment. Op18 is responsible for TNF-induced microtubule
stabilization that promotes cell death (Vancompenolle et al.,
2000). Unexpectedly, we were able to demonstrate that glyoxalase I
is also phosphorylated upon TNF treatment. Phosphorylation of
mammalian glyoxalase I has not yet been described, although the
sequence does contain several potential phosphorylation sites
(Ranganathan et al., 1993). Interestingly, phosphorylation of yeast
GLO1 has been observed during the sexual response of S.
cerevisiae--specifically, during the arrest of cell division at the
G1 phase, which occurs when haploid cells of one sex are exposed to
the mating factor of the opposite type of cells (Inoue et al.,
1990). However, none of these observations suggest that
phosphorylated mammalian glyoxalase I does exist, nor do these
observations suggest which potential phosphorylation sites may be
used.
[0009] It is a first aspect of the invention to provide
phosphorylated mammalian glyoxalase. The phosphorylation may be a
single or a multiple phosphorylation. An exemplary embodiment is a
phosphorylated mammalian glyoxalase I comprising SEQ ID NO:1 of the
incorporated herein SEQUENCE LISTING. Preferably, phosphorylated
mammalian glyoxalase I essentially consists of SEQ ID NO:1. Even
more preferentially, phosphorylated mammalian glyoxalase I consists
of SEQ ID NO:1. Preferably, phosphorylation is carried out at
position Ser 8 and/or Ser 21 and/or Ser 26 and/or Thr 107. Even
more preferably, the phosphorylation is carried out at the PKA
phosphorylation sites Ser 45 and/or Thr 98 (numbering as for human
glyoxalase, including the N-terminal Met residue).
[0010] Another aspect of the invention is the use of a
phosphorylated glyoxalase I to modulate MG-modification of
proteins. Phosphorylated glyoxalase I may be any glyoxalase I,
known to the person skilled in the art, such as a fungal glyoxalase
I or a plant glyoxalase I. Preferably, glyoxalase I is a mammalian
glyoxalase I. MG-modified proteins or advanced glycation end
products (AGEs) are known to be synthesized in response to a number
of pathophysiological conditions in vivo, such as cataract
formation (Shamsi 2000), vascular complications associated with
chronic diabetes (Shinohara et al., 1998), tissue damage after
ischemia/reperfusion (Oya et al., 1999) and aging (Corman et al.,
1998). The term "AGE," as used here, is used for any
MG-modification of a protein, irrespective of the way it is formed.
The term "MG-modification of proteins" is considered as being
equivalent with the term AGE formation.
[0011] Still another aspect of the invention is the use of
phosphorylated glyoxalase I, or an inhibitor of the phosphorylation
of glyoxalase I, preferably mammalian glyoxalase I to modulated
TNF-induced cell death. This inhibitor can be any inhibitor that
inhibits the phosphorylation of glyoxalase I. Preferably, the
inhibitor is an inhibitor of the PKA activity.
[0012] Alternatively, a mutant form of glyoxalase I may be used
that affects phosphorylation in it ("phosphorylation mutant"),
i.e., it can no longer be phosphorylated at one or more
phosphorylation sites and/or it becomes phosphorylated at other
sites. On the basis of the knowledge of the phosphorylation sites,
such mutants can be easily constructed by the person skilled in the
art and include, as a nonlimiting example, glyoxalase I forms where
the Ser 45 and/or the Thr 98 have been replaced by another amino
acid or any other mutant that affects phosphorylation on these or
other sites. Therefore, another aspect of the invention is the use
of a phosphorylation mutant of glyoxalase I, preferably mammalian
glyoxalase I, to modulate TNF-induced cell death. This modulation
can be realized by replacing the endogenous glyoxalase I by the
mutant form, or by expressing the mutant glyoxalase I form beside
the endogenous glyoxalase I.
[0013] A further aspect of the invention is the use of
phosphorylated glyoxalase I, or an inhibitor of the phosphorylation
of glyoxalase I, or a phosphorylation mutant of glyoxalase I, to
modulate stress-induced cell death. Preferably, the stress is
oxidative stress. Oxidative stress, followed by ROI induction and
AGE formation is known to occur in several organisms, including
plants, yeast, fungi and mammalians. An exemplary embodiment is the
use of mammalian phosphorylated glyoxalase I to modulate oxidative
stress-induced cell death.
[0014] Still another aspect of the invention is the use of PKA to
phosphorylate glyoxalase I. By modulating the phosphorylation of
glyoxalase I, TNF-induced cell death and stress-induced cell death,
preferably oxidative stress, can be modulated.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1: phosphorylation of glyoxalase I in control cells
(left panel) and after 1.5 hours of TNF treatment (right
panel).
[0016] FIG. 2: effect of the glyoxalase I inhibitor
S-p-bromobenzylglutathione diester on TNF-induced cytotoxicity in
L929s cells, in function of the incubation time. TNF is added at a
concentration of 1000 units/ml; the inhibitor is added 1 hour 10
minutes prior to TNF at a concentration of 10 .mu.M (GI10) or 20
.mu.M (GI20). The time scale is calculated from the moment of TNF
addition.
[0017] FIG. 3: western blots, developed with anti-human glyoxalase
I polyclonal antibody, of 2-dimensional gels (pH 3-10) from total
cell lysates derived from control cells (C), glyoxalase I inhibitor
S-p-bromobenzylglutathione diester treated cells (I), TNF-treated
cells (TNF) and cells treated with TNF and glyoxalase I inhibitor
(TNF+I).
[0018] FIG. 4: effect of different concentrations exogeneously
added methylglyoxal on TNF-induced cell death. Measurement after
5.5 hours of incubation with TNF and methylglyoxal at a
concentration as indicated.
[0019] FIG. 5: effect of different concentrations of the AGE
formation inhibitor, aminoguanidine, on the TNF-induced cell death.
Measurement after 16 hours of incubation with TNF and
aminoguanidine at a concentration as indicated.
[0020] FIG. 6: inhibition of TNF-induced cell death by the PKA
inhibitor H89, in function of the incubation time. The
concentration of the inhibitor H89 is as indicated on the
graph.
[0021] FIG. 7: western blot of a 2-dimensional gel (IEF pH 4-7)
developed with a polyclonal antibody against human GLO1. C:
control; TNF: TNF-treated sample; H89: control sample with addition
of the PKA inhibitor H89; TNF+H89: TNF-treated sample, with
addition of the PKA inhibitor H89. Ac and Bs indicate the acidic
(Ac) and basic (Bs) side of the gel. Upon TNF treatment of the
cells, a more acidic isoform of GLO1 becomes apparent; the arrow
indicates the more acidic phosphorylated form. This new isoform is
derived from the most basic isoform, present in the control cells,
and its formation is inhibited by the presence of the PKA inhibitor
H89. Note that the two-dimensional pattern of the TNF-treated
sample in the presence of H89 is identical to the control sample C
and the control sample H89.
[0022] FIGS. 8A and 8B: Formation of a specific MG-derived AGE
during TNF-induced cell death and the inhibition by several agents.
FIG. 8A: Western blot with the anti-AGE antibody mAb6B that was
developed against MG-modified keyhole limpet hemocyanin (recognizes
also MG-modified BSA). Immunocomplexes were visualized by enhanced
chemiluminescence (ECL) and evaluated by scanning densitometry. To
analyze AGE formation in TNF-induced cell death, all TNF treatments
(1000 U/ml, 2.5 hours) were done in the presence of cycloheximide
(CHX) to synchronize cell death. Equal amounts of total cytosolic
protein from cells incubated under different conditions were loaded
in each lane. a: control cells; b: TNF-treated cells; C: control
anti-oxidant butylated hydroxyanisole (BHA; 100 .mu.M); d:
TNF-treated cells in the presence of BHA (100 .mu.M), the
antioxidant agent BHA was administered 0.5 hour after TNF
administration to allow initiation of TNF signaling; e: control
2-deoxyglucose; f: TNF-treated cells in the presence of
2-deoxyglucose (2:1 ratio to glucose), 2-deoxyglucose was
administered at the same time as TNF. Note the appearance of the
specific MG-derived AGE exclusively in TNF-treated cells (indicated
by arrowheads), while no significant changes in the other AGEs can
be observed. The formation of this MG-derived AGE is strongly
inhibited (65%) in the presence of BHA or 2-deoxyglucose. FIG. 8B:
Formation of the specific MG-derived AGE is inhibited by the
inhibitors that also inhibit the phosphorylation of GLO1. This
figure represents the percentage relative increase of the ECL
signal (as evaluated by scanning densitometry) of this specific
MG-derived AGE in TNF-treated cells (1000 U/ml in the presence of
CHX, 1.5 hours) over the background in control cells.
[0023] FIG. 9: The anti-glucose metabolite 2-deoxyglucose strongly
inhibits TNF-induced cell death in L929 cells. TNF-induced cell
death was measured as a function of time by flow cytometry using
propidium iodide (PI) uptake as a parameter for the percentage of
death cells. 2-Deoxyglucose was used in a 2:1 ratio to glucose.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The invention is further explained with the aid of the
following illustrative Examples.
EXAMPLES
Materials and Methods to the Examples
Cell Lines and Cultures
[0025] All L929 cells were cultured in Dulbecco's modified Eagle's
medium supplemented with heat-inactivated fetal calf serum (5%
v/v), heat-inactivated newborn calf serum (5% v/v), penicillin (100
units/ml), streptomycin (0.1 mg/ml), and L-glutamine (2 mM), at
37.degree. C. in a humidified incubator under a 5% CO.sub.2
atmosphere.
Reagents
[0026] Murine TNF (mTNF) was obtained from Roche Diagnostics and
was used at 1000 IU/ml unless indicated otherwise. Propidium iodide
(PI) and cycloheximide (CHX) (all from Sigma) were used at
concentrations of 30 .mu.M and 50 .mu.g/ml, respectively.
Measurement of TNF-Induced Cell Death by Flow Cytometry
[0027] Cell death in L929 was induced by addition of TNF to the
cell suspension. Cell death was measured by quantifying PI-positive
cells by FACS (FACSCalibur, Becton Dickinson, San Jose, Calif.).
The PI dye was excited with an argon-ion laser at 488 nm; PI
fluorescence was measured above 590 nm using a long-pass filter.
Routinely, 3,000 cells were analyzed. Cell death is expressed as
the percentage of PI-positive cells in the total cell
population.
Radiolabeling of Cells and Preparation of the Sub-Cellular Protein
Fractions
[0028] L929 cells were plated 48 hours prior to the experiment.
.sup.32P labeling was carried out as described in (Guy et al.,
1992). TNF treatments (1000 IU/ml, 1.5 hours) were done in the
presence of cycloheximide (CHX), to synchronize cell death. To
simplify the 2-D phosphoprotein pattern and subsequent
computer-assisted analysis, we prepared two subcellular protein
fractions. The cytosolic protein fraction, containing soluble
cytoplasmic molecules and molecules derived from single-membrane
organelles, was obtained as the supernatant from digitonin
(0.03%)-permeabilized cells. After rinsing once with excess PBS
buffer, the remaining cell fraction was lysed in a CHAPS
(2%)-containing buffer as described in (Guy et al., 1992). This
lysate was then centrifuged (20,000 g) and the supernatant was used
as the organelle fraction; it is enriched for mitochondrial and
cytoskeleton-derived proteins.
Two-Dimensional (2D) Gel Electrophoresis
[0029] Isoelectric focusing. Isoelectric focusing was carried out
on 18 cm IPG strips, pH 4-7 (Amersham Pharmacia Biotech), according
to the manufacturer's instructions. Protein samples were
precipitated with ethanol and redissolved in lysis buffer.
[0030] SDS-PAGE. The second dimension (SDS-PAGE) was run on large
vertical gels (12.5% acrylamide, Biorad).
Western Blotting
[0031] Proteins were separated by SDS-PAGE (12.5%) and transferred
to a PVDF membrane (Hybond-P, Amersham Pharmacia Biotech). The
blots were incubated with an anti-human glyoxalase I antibody
(kindly provided by Dr. P. Thornalley, University of Essex, UK),
followed by ECL-based detection (reagents of Amersham Pharmacia
Biotech; software for analysis by Totallab).
Amino Acid Sequence Analysis by MALDI-Mass Spectrometry
[0032] Following in-gel digestion of the excised protein with
endoproteinase Lys-C (sequencing grade; Boehringer, Mannheim,
Germany), a 10% aliquot of the generated peptide mixture was
purified and concentrated on Poros.RTM. 50 R2 beads (Gevaert et
al., 1998; Gevaert et al., 1997) and used for MALDI-MS peptide mass
fingerprint analysis. However, partly due to contamination with
human keratin peptides, the obtained peptide mass map did not lead
to any unambiguous protein identification in a nonredundant protein
database. Therefore, the remainder of the peptide mixture was
separated by RP-HPLC, a total of 20 fractions containing eluting
peptides were obtained, which were all analyzed by MALDI-MS.
Adequate peptide ions were further selected for post-source decay
(PSD) analysis (Spengler et al., 1992). A PSD-spectrum obtained
from a peptide ion with a mass of 902.42 Da (measured in linear
mode) present in the first RP-HPLC fraction, could be unambiguously
assigned to the peptide NH.sub.2--SLDFYTR--COOH (SEQ ID NO: 2)
present in human glyoxalase I (database entry number 417246) using
the SEQUEST algorithm and a nonredundant protein database.
Following a search in an EST-database, the same peptide sequence
was identified in many different mouse EST-clones. The identified
peptide contains an arginine residue at its C-terminus instead of a
lysine, an observation which we made several times when
endoproteinase Lys-C was used as the protease.
[0033] In order to confirm our initial finding, PSD-analysis was
conducted on a peptide with an apparent mass of 1396.53 Da present
in RP-HPLC fraction 11. Based on the partially .sup.18O-labeled
y-type fragment ions, a peptide sequence tag (391.24)YAI/LF(885.67)
could easily be obtained. Furthermore, a SEQUEST search in a
nonredundant protein database lead to the identification of the
peptide NH.sub.2--FSLYFLAYEDK--COOH (SEQ ID NO: 3) also belonging
to human glyoxalase I. Again, the same peptide sequence was found
in different mouse EST clones using the PSD data and a SEQUEST
search in an EST database. Based upon the amino acid sequence of
human glyoxalase I, masses of peptide ions observed in the
different RP-HPLC fractions could be assigned to the identified
protein. Hereby, a total of 38% of the amino acid sequence of the
protein was covered, again confirming the identification of
glyoxalase I.
Assay of Glyoxalase I Activity
[0034] The glyoxalase I assay was performed according to a
spectrophotometric method monitoring the increase in absorbance at
240 nm due to the formation of S-D-lactoylglutathione for 4 minutes
at 20.degree. C. The standard assay mixture contained 2 mM MG and 2
mM GSH in a sodium phosphate buffer (50 mM, pH 6.6). Before
initiating the reaction by adding the total cytosolic protein
fraction to the assay mixture, the mixture was allowed to stand for
10 minutes to ensure the equilibration of hemithioacetal
formation.
D-Lactate Measurements
[0035] D-Lactate measurements were performed by a fluorometric
assay using an endpoint enzymatic assay with D-lactate
dehydrogenase (McLellan et al. 1992).
Intracellular Methylglyoxal Measurements
[0036] Intracellular free methylglyoxal is detected as the
2-methylquinoxaline (2-MQ) derivative of methylglyoxal formed with
o-phenylenediamine (o-PD) using the general approach of (Chaplen et
al., 1996). Samples arrived frozen on dry ice and were stored at
20.degree. C. until assayed. Samples were thawed at room
temperature and maintained on ice during the assay procedure.
Sample volume was increased to 2.5 ml with MilliQ water and the
samples were sonicated (5 s, 30 W). 5 M HClO.sub.4 (PCA; 0.25 ml)
was added to precipitate macromolecules and the resulting mixture
was incubated on ice for 20 minutes. Samples were then centrifuged
(12,000.times.g, 10 minutes) to remove precipitated materials. The
supernatant was passed through a C-18 SPE cartridge (Waters Sep-Pak
tC18 plus cartridge, Millipore Corp., Marlborough, Mass.) that had
been prepared by flushing with 6-8 ml of acetonitrile followed by
6-8 ml of 10 mM KH.sub.2PO.sub.4 (pH 2.5, adjusted with
concentrated H.sub.3PO.sub.4). The pre-derivatization SPE step
removes phenol red and other interfering compounds. Samples were
supplemented with 12.5 nmol 5-methylquinoxaline (5-MQ; internal
standard) and 250 nmol o-PD (derivatizing agent) and reacted at
20.degree. C. for 3.5 to 4 hours.
Sample Concentration
[0037] All samples are concentrated after derivatization. For
concentration, the samples are passed through a C-18 SPE cartridge,
prepared as described above, at a rate of 1-2 ml/minutes. The
cartridges are then rinsed with 1-2 ml 10 mM KH.sub.2PO.sub.4 (pH
2.5) and the retentate eluted with 2 ml of acetonitrile. Eluates
were evaporated to a volume of 200 ml using a Savant Speed-Vac
Concentrator vacuum centrifugation unit (Savant Instruments,
Farmingdale, N.Y.) and filtered through 0.2 mm Gelman PVDF filters
(Fisher Scientific, Chicago, Ill.) into sample vials.
HPLC of Quinoxalines
[0038] HPLC was performed as described previously (Chaplen et al.,
1996) but with a mobile phase consisting of 35% acetonitrile/0.1%
trifluoroacetic acid, pH 2.4 and 65% 10 mM phosphate/0.1%
trifluoroacetic acetic acid in HPLC grade water, pH 2.4. Under
these modified conditions, 2-MQ eluted after 7.5 minutes and 5-MQ
eluted after 11.2 minutes.
Detection of MG-Derived AGEs
[0039] L929 cells were seeded 48 hours prior to the experiment. TNF
incubations (1000 U/ml) were done in the presence of CHX to
synchronize cell death. After TNF incubations (1.5 hours or 2.5
hours), the cells were rinsed three times with ice-cold PBS buffer
and cell lysates were prepared in a CHAPS-containing cytosol
extraction buffer (Guy et al., 1992). MG-derived AGEs were detected
by Western blotting using the mAb6B (Oya et al., 1999). To use the
antibody sparingly, SDS-PAGE gels were only run over a distance of
5 cm.
Example 1
TNF Induces Increased Phosphorylation of Glyoxalase I
[0040] FIG. 1 shows the autoradiogram of the two-dimensional gels
from TNF-treated and control samples that were derived from cells
labeled with .sup.32P-orthophosphate. The protein spot with
increased phosphorylation identified as glyoxalase I is indicated
by an arrow. It was identified by mass spectrometry analysis of a
peptide mixture derived from an in-gel digestion of the excised
protein spot. The increased phosphorylation of glyoxalase I is
already observed after 15 minutes of TNF treatment, but is much
more pronounced after 1.5 hours of TNF treatment (FIG. 1). This
indicates that the TNF-induced phosphorylation of GLO1 is an early
but lasting event.
Example 2
The Glyoxalase I Inhibitor S-p-bromobenzylglutathione Cyclopentyl
Diester Inhibits TNF-Induced Cell Death
[0041] To examine the role of glyoxalase I in TNF-induced cell
death, we tested the effect of the cell permeable competitive
inhibitor of glyoxalase I S-p-bromobenzylglutathione diester on
cell death. Preincubation (1 hour 10 minutes) of L929 cells with
this inhibitor strongly inhibits TNF-induced cell death in a
concentration-dependent manner (FIG. 2). An inhibition of 60% was
obtained at a concentration of 20 .mu.M of the inhibitor. However,
when the inhibitor and TNF were added at the same time, a
synergistic effect on TNF-induced cell death was obtained (50%
increase in cell death at a concentration of 20 .mu.M of the
inhibitor). This synergistic effect is more pronounced at lower
doses of TNF; that is, when the cells die more slowly.
Example 3
The Glyoxalase I Inhibitor S-p-bromobenzylglutathione Cyclopentyl
Diester Inhibits the TNF-Induced Phosphorylation of Glyoxalase
I
[0042] This differential effect of the inhibitor on TNF-induced
cell death prompted us to investigate whether the binding of the
inhibitor to GLO1 competes and thus inhibits the TNF-induced
phosphorylation of GLO1. FIG. 3 shows the Western blots, developed
with an anti-human glyoxalase I polyclonal antibody, of
2-dimensional gels (pH 3-10) from total cell lysates derived from
TNF-treated and control cells and from TNF-treated and control
cells that were first preincubated with the glyoxalase I inhibitor
for 1 hour 10 minutes. In the upper panels, you can see that TNF
induces a more acidic phosphoisoform of glyoxalase I which is not
well separated from the non-phosphorylated form (it fills the space
between the left-most isoform and the main non-phosphorylated
form). In the lower panels, you can see that in the presence of the
glyoxalase I inhibitor, TNF cannot induce the more acidic
phosphoisoform of glyoxalase I (identical 2-D patterns as in the
control). These data show that the competitive inhibitor of
glyoxalase I S-p-bromobenzylglutathione inhibits the TNF-induced
phosphorylation of GLO1 and that phosphorylated glyoxalase I is
essential for cell death. These data also suggest that
phosphorylation of glyoxalase I modulates the active site of the
enzyme.
[0043] Thus, the differential effect of the GLO1 inhibitor on
TNF-induced cell death can be explained as follows: [0044] when the
cells are pretreated with the inhibitor, the inhibitor is already
bound to GLO1 and thus hinders the TNF-induced phosphorylation of
GLO1 which then leads to inhibition of phosphorylated GLO1-mediated
MG-modification of proteins and consequent cell death. [0045]
however, when the cells are treated with the inhibitor and TNF
together, the TNF-induced phosphorylation of GLO1 occurs first (via
a receptor-activated kinase cascade) and the inhibitor can only
bind to nonphosphorylated GLO1 (and not to phosphorylated GLO1)
leading to inhibition of GLO1 and thus to accumulation of MG,
resulting in phosphorylated GLO1-mediated MG-modification of
proteins and consequent cell death.
Example 4
TNF-Induced Phosphorylation of Glyoxalase I does not Inhibit
Methylglyoxal Detoxification
[0046] For many years, .alpha.-ketoaldehydes, exemplified by
methylglyoxal, have been known to be carcinostatic, but their
direct use as anti-cancer drugs is prevented by their rapid
detoxification in vivo by the glyoxalase system. Therefore,
glyoxalase I inhibitors have been developed as potential
anti-cancer agents (Vince and Wadd, 1969; Thornalley et al., 1996).
Bearing this in mind, one would expect that TNF-induced
phosphorylation of GLO1 would result in inhibition of the enzyme
and thus in accumulation of MG with cytotoxicity as a consequence.
However, our experiments with the GLO1 inhibitor do not support
this expectation, because we would then expect a synergistic effect
of the preincubated inhibitor on TNF-induced cell death. Indeed,
measurements of GLO1 activity in lysates derived from TNF-treated
and control cells showed no inhibition, but even a limited increase
in GLO1 activity in TNF-treated cells. These experiments were
repeated several times and each time gave the same results, with an
average increase of 8% after 1 hour of TNF treatment and 12% (from
0.086.+-.0.003 to 0.106.+-.0.001 units per 8.5 .mu.g of total
protein) after 1.5 hours of TNF treatment. Measurement of the
concentration of the end product of the glyoxalase system D-lactate
showed an increase of 60% after 1.5 hours of TNF treatment compared
to control cells. This further confirmed that TNF did not inhibit
GLO1 activity and that an increased flux of MG is converted through
the glyoxalase system in TNF-treated cells.
Example 5
TNF Increases the Intracellular Concentrations of Methylglyoxal
[0047] As we consider it very unlikely that TNF would cause an
increased detoxification of MG through the glyoxalase system, a
more plausible explanation is that TNF induces an increase in the
intracellular concentrations of MG via a pathway other than
inhibition of glyoxalase I. An increase in the intracellular
concentration of MG would then also automatically result in an
increased flux of MG through the glyoxalase system and an increased
GLO1 activity. Therefore, intracellular concentrations of MG were
measured with a method that not only measures free MG, but also MG
bound to biological molecules (majority of the MG), mainly proteins
(Chaplen et al., 1998). Two independent experiments were performed
in which intracellular concentrations of MG were measured in
TNF-treated (1.5 hours) L929 cells compared to control cells. Each
sample was measured in triplicate and each time gave very
reproducible results. These results showed that TNF strongly
increased the intracellular concentrations of MG, with an increase
of 32% (from 0.91 .mu.Mole in control cells to 1.20 .mu.Mole in
TNF-treated cells) in the one experiment and 94% (from 1.24
.mu.Mole in control cells to 2.39 .mu.Mole in TNF-treated cells) in
the other experiment.
[0048] Also, exogenously added MG is strongly synergistic with
TNF-induced cell death in a concentration-dependent manner (FIG.
4), while MG alone and used at the same concentrations is not
cytotoxic for L929 cells. The synergistic effect of exogenously
added MG is more pronounced at lower doses of TNF (100 U/ml) and
also earlier in TNF treatment. This result can be explained by the
fact that the TNF-induced increase of endogenous MG is more drastic
at higher doses of TNF (1000 U/ml) and later in TNF treatment.
Example 6
Inhibition of AGE Formation Inhibits TNF-Induced Cell Death
[0049] Increased endogenously produced levels of dicarbonyls,
especially methylglyoxal, are involved in numerous pathogenic
processes in vivo, including the formation of advanced glycation
end-products (AGEs) which contribute to the pathophysiology of
aging and to complications associated with chronic diabetes. They
have been detected in several pathophysiological conditions in
vivo, such as cataract formation, vascular complications in
diabetes, and tissue damage after ischemia/reperfusion. All these
conditions are characterized by increased oxidative stress, and
recently it was shown that mitochondrial ROS are the direct cause
of increased concentrations of MG and thus AGEs formation in
diabetic hyperglycemia (Nishikawa et al., 2000). Since TNF-induced
cell death in L929 cells is characterized by increased production
of mitochondrial ROS (Goossens et al., 1995; Goossens et al., 1999)
which are essential for cell death and increased levels of MG, we
tested whether irreversible protein modification by MG plays a role
in TNF-induced cell death. For this we used aminoguanidine, a
nucleophilic hydrazine compound and inhibitor of advanced
nonenzymatic glycosylation product formation (Brownlee et al.,
1986). The percentage of cell death in L929 cells after 16 hours of
TNF treatment (20 U/ml) with and without aminoguanidine is shown in
FIG. 5. A maximum inhibition of cell death of 25% was obtained in
the presence of 600 or 800 .mu.M of aminoguanidine and 15%
inhibition in the presence of 400 .mu.M. This inhibition was less
pronounced (average of 15% to 20%) when the cells died more rapidly
by giving higher doses of TNF (500-1000 U/ml). This could be due to
the fact that the reaction of aminoguanidine with MG and
MG-modified proteins is rather slow and that the MG protein
modifications that occur during TNF-induced cell death are more
rapid at higher doses of TNF and could even be enzymatically
catalyzed. These data indicate that irreversible protein
modification by methylglyoxal might play a role in TNF-induced cell
death.
Example 7
The PKA Inhibitor Inhibits TNF-Induced Cell Death and TNF-Induced
Phosphorylation of Glyoxalase I
[0050] As it has already been shown that PKA is activated by TNF
(Zhang et al., 1988), we examined whether pretreatment (2 hours) of
L929 cells with the PKA inhibitor H89 had an effect on TNF-induced
cell death. As shown in FIG. 6, the PKA inhibitor inhibits
TNF-induced cell death in a concentration-dependent fashion and to
a similar extent as the glyoxalase I inhibitor. Even an inhibitory
effect was already obtained at relatively low concentrations of the
inhibitor (1 .mu.M), while at the highest concentration (5 .mu.M)
an inhibition of more then 50% was obtained. These data thus
indicate that PKA plays a role in TNF-induced cell death. Next, we
examined whether PKA was also responsible for the TNF-induced
phosphorylation of endogenous glyoxalase I in L929 cells. As shown
in FIG. 3 and FIG. 7, pretreatment of the cells with the PKA
inhibitor (5 .mu.M) completely abolished the induction of the more
acidic isoform by TNF. This suggests that the inhibitory effect of
the PKA inhibitor on TNF-induced cell death could be largely due to
the inhibition of phosphorylation of glyoxalase I.
Example 8
Formation of a Specific Methylglyoxal-Derived AGE During
TNF-Induced Cell Death
[0051] Given the demonstrated role of MG in AGE formation and the
accumulation of MG noted in response to TNF treatment, we next
sought to determine whether irreversible protein modification by MG
is a critical step in TNF-induced cell death. Immunoblots of L929
protein extracts were performed with a monoclonal antibody raised
against in vitro MG-modified keyhole limpet hemocyanin. This
antibody (mAb6B) recognizes epitopes in arterial walls of diabetic
kidneys and of tissue injured by ischemia/reperfusion (Oya et al.,
1999). The immunoblots showed a distinct differential protein band
specifically present in TNF-treated (2.5 hours) cells along with
several protein bands that were present in both the control and
TNF-treated cells (FIG. 2A). The band was already present, although
very weakly, after 1.5 hours of TNF treatment. These data indicate
that protein modification by MG is not a random process during
TNF-induced cell death, but rather involves specific target
molecules for MG.
[0052] To demonstrate that the TNF-induced MG-derived AGE
identified here by the antibody was formed as a consequence of
oxidative stress, as in the case of diabetic hyperglycemia
(Nishikawa et al., 2000), and only under cytotoxic conditions, L929
cells were treated with the anti-oxidant BHA. BHA arrests
TNF-induced ROS production and cell death (Goossens et al., 1995).
The formation of this specific MG-derived AGE in TNF-treated cells,
as measured by densitometric analysis of the ECL signal from
immunoblots, was reduced by 65% in the presence of BHA (FIG. 8A).
The anti-glucose metabolite 2-deoxyglucose was then used to
determine whether the TNF-induced increase in MG concentration was
derived from glycolytic intermediates, which are usually considered
to be the main intracellular source of MG. In the presence of
2-deoxyglucose (2:1 ratio to glucose), TNF-induced cell death was
inhibited by 65% (FIG. 9), as was the formation of the specific
MG-derived AGE noted in FIG. 8A. The possibility exists that
inhibition of AGE formation in the presence of 2-deoxyglucose is an
indirect result of the inhibition of mitochondrial ROS, which would
mean the latter are derived from increased glycolysis. Indeed, it
has been reported that TNF highly increases glycolysis and glucose
uptake in L929 cells (Matthews, 1983; Kim and Kim, 2001).
[0053] Taken together, our results clearly indicate that glycolysis
plays an important role in TNF-induced necrosis and that
TNF-induced mitochondrial ROS, as in diabetic hyperglycemia, can
lead to accumulation of MG and subsequent formation of a specific
MG-derived AGE.
Example 9
The TNF-Induced Phosphorylation of GLO1 is Involved in Formation of
the Specific MG-Derived AGE
[0054] Until now, AGE formation has been described as nonenzymatic,
irreversible modifications of Lys and Arg residues slowly formed
through long-term exposure to high concentrations of sugars and
reactive compounds such as MG. Yet in TNF-induced cell death,
MG-modification of proteins occurs very rapidly (within 1.5 to 2.5
hours of initiating TNF treatment). This suggests that
MG-modification of specific target molecules could be enzymatically
catalyzed by phosphorylated GLO1. To test this hypothesis, L929
cells were pretreated with s-p-bromobenzylglutathione cyclopentyl
diester (BBGD 20 .mu.M; kindly provided by Dr. P. Thornalley) and
the PKA inhibitor H89 (5 .mu.M), respectively, to determine whether
these inhibitors of GLO1 phosphorylation interfere with the
formation of MG-derived protein modifications during TNF-induced
cell death. In cell cultures pretreated with BBGD and the PKA
inhibitor, the formation of the specific MG-derived AGE, which is
recognized by mAb6B, as measured by densitometric analysis of the
ECL signal from immunoblots, was reduced by 50% and 70%,
respectively, after 1.5 hours of TNF treatment (FIG. 8B).
Inhibition was less pronounced (10% and 20% respectively) after 2.5
hours of TNF treatment, which could be due to the fact that, at
later time points of TNF treatment, MG accumulation is dominant
over the low basal levels of the phosphorylated form of GLO1 which
are always present in L929 cells. The inhibitory effects with
pretreatment of BBGD on TNF-mediated AGE formation and cell death
is less pronounced during longer TNF treatments, possibly as a
consequence of MG accumulation as described above. In any event,
these data indicate that formation of the specific MG-derived AGE
during cell death requires the TNF-induced phosphorylation of
GLO1.
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Sequence CWU 1
1
3 1 184 PRT Homo sapiens Lactoylglutathione lyase; Glyoxalase I
Accession Q04760 1 Met Ala Glu Pro Gln Pro Pro Ser Gly Gly Leu Thr
Asp Glu Ala Ala 1 5 10 15 Leu Ser Cys Cys Ser Asp Ala Asp Pro Ser
Thr Lys Asp Phe Leu Leu 20 25 30 Gln Gln Thr Met Leu Arg Val Lys
Asp Pro Lys Lys Ser Leu Asp Phe 35 40 45 Tyr Thr Arg Val Leu Gly
Met Thr Leu Ile Gln Lys Cys Asp Phe Pro 50 55 60 Ile Met Lys Phe
Ser Leu Tyr Phe Leu Ala Tyr Glu Asp Lys Asn Asp 65 70 75 80 Ile Pro
Lys Glu Lys Asp Glu Lys Ile Ala Trp Ala Leu Ser Arg Lys 85 90 95
Ala Thr Leu Glu Leu Thr His Asn Trp Gly Thr Glu Asp Asp Ala Thr 100
105 110 Gln Ser Tyr His Asn Gly Asn Ser Asp Pro Arg Gly Phe Gly His
Ile 115 120 125 Gly Ile Ala Val Pro Asp Val Tyr Ser Ala Cys Lys Arg
Phe Glu Glu 130 135 140 Leu Gly Val Lys Phe Val Lys Lys Pro Asp Asp
Gly Lys Met Lys Gly 145 150 155 160 Leu Ala Phe Ile Gln Asp Pro Asp
Gly Tyr Trp Ile Glu Ile Leu Asn 165 170 175 Pro Asn Lys Met Ala Thr
Leu Met 180 2 7 PRT Homo sapiens 2 Ser Leu Asp Phe Tyr Thr Arg 1 5
3 11 PRT Homo sapiens 3 Phe Asp Leu Tyr Phe Leu Ala Tyr Glu Asp Lys
1 5 10
* * * * *