U.S. patent application number 13/190869 was filed with the patent office on 2012-01-05 for methods and kits for detecting mastitis.
This patent application is currently assigned to University of Medicine and Dentistry of New Jersey. Invention is credited to Kiran Madura.
Application Number | 20120003662 13/190869 |
Document ID | / |
Family ID | 47601533 |
Filed Date | 2012-01-05 |
United States Patent
Application |
20120003662 |
Kind Code |
A1 |
Madura; Kiran |
January 5, 2012 |
Methods and Kits for Detecting Mastitis
Abstract
Methods and kits for determining if one or more animals have
mastitis and for monitoring animals and the quality of the milk
they produce are disclosed. Kits and test assays disclosed are used
to determine the quantity of proteasomes and proteins thereof, the
activity of proteasome enzymes, the quantity of proteasome bound
and regulating proteins, and the quantity of ubiquinated protein.
Components and reagents for use in the kits and assays are also
disclosed.
Inventors: |
Madura; Kiran; (Bridgewater,
NJ) |
Assignee: |
University of Medicine and
Dentistry of New Jersey
Somerset
NJ
|
Family ID: |
47601533 |
Appl. No.: |
13/190869 |
Filed: |
July 26, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12738340 |
Apr 16, 2010 |
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PCT/US08/80514 |
Oct 20, 2008 |
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13190869 |
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60999495 |
Oct 18, 2007 |
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Current U.S.
Class: |
435/6.18 ;
435/24; 435/7.4 |
Current CPC
Class: |
G01N 33/573 20130101;
C12Q 1/6888 20130101; G01N 2800/365 20130101; C12Q 1/37
20130101 |
Class at
Publication: |
435/6.18 ;
435/24; 435/7.4 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/573 20060101 G01N033/573; C12Q 1/37 20060101
C12Q001/37 |
Goverment Interests
[0002] This invention was made with government support under grant
number CA83875 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method for determining if one or more animals has mastitis
comprising (a) performing a test assay for determining the level of
proteasomes present in a test sample of milk collected from one or
more animals, and (b) comparing the level of proteasomes present in
the test sample with a standard level of proteasomes in a control
sample of milk, wherein an increase in the level of proteasomes
present in the test sample relative to the standard level of
proteasomes present in the control sample of milk indicates one or
more animals has mastitis.
2. The method of claim 1, wherein the test assay for determining
the level of proteasomes present in the test sample detects
proteasome activity in the sample.
3. The method of claim 2, wherein the proteasome activity is
measured by measuring the level of conversion of a proteasome
enzyme substrate to a product.
4. The method of claim 3, wherein the proteasome enzyme substrate
comprises Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr (SEQ ID NO:8);
Z-Gly-Gly-Leu; Suc-Leu-Leu-Val-Tyr (SEQ ID NO:9); Suc-Leu-Tyr;
Ac-Arg-Leu-Arg; Boc-Leu-Arg-Arg; Bz-Val-Gly-Arg; Ac-Gly-Pro-Leu-Asp
(SEQ ID NO:7); Z-Leu-Leu-Glu; or Ac-nLeu-Pro-nLeu-Asp (SEQ ID
NO:6).
5. The method of claim 4, wherein the substrate comprises a
fluorogenic label, luminescent label or a chromogenic label.
6. The method of claim 2, wherein the proteasome enzyme substrate
is an ATPase substrate.
7. The method of claim 2, wherein the proteasome enzyme substrate
is a de-ubiquitinase substrate.
8. The method of claim 1, wherein the test assay for determining
the level of proteasomes present in the test sample is an assay
comprising protein detection or detection of nucleic acid molecules
encoding proteins.
9. The method of claim 8, wherein the test assay detects one or
more molecules comprising one or more proteasome proteins, one or
more proteasome binding proteins, one or more proteasome regulating
proteins, a nucleic acid molecule that encodes a proteasome
protein, a proteasome regulating protein or a proteasome binding
protein.
10. The method of claim 8, wherein the test assay for determining
the level of proteasomes present in the test sample is an assay to
determine the level of immunoproteasomes present in the test
sample.
11. The method of claim 8, wherein the test assay for determining
the level of proteasomes present in the test sample is an assay to
detect the level of ubiquinated proteins in a sample or copper
complex binding to proteasomes.
12. A kit for detecting proteasome enzyme activity in a sample of
fluid comprising: a) a ubiquitin-like domain affinity matrix, and
b) a container comprising a cap, wherein said container or cap
comprises a proteasome enzyme substrate.
13. The kit of claim 12, wherein the proteasome enzyme substrate
comprises Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr (SEQ ID NO:8);
Z-Gly-Gly-Leu; Suc-Leu-Leu-Val-Tyr (SEQ ID NO:9); Suc-Leu-Tyr;
Ac-Arg-Leu-Arg; Boc-Leu-Arg-Arg; Bz-Val-Gly-Arg; Ac-Gly-Pro-Leu-Asp
(SEQ ID NO:7); Z-Leu-Leu-Glu; or Ac-nLeu-Pro-nLeu-Asp (SEQ ID
NO:6).
14. The kit of claim 13, wherein the substrate comprises a
fluorogenic label, luminescent label or a chromogenic label.
15. The kit of claim 12, wherein the proteasome enzyme substrate is
an ATPase substrate.
16. The kit of claim 12, wherein the proteasome enzyme substrate is
a de-ubiquitinase substrate.
17. The kit of claim 12, further comprising a reagent for a test
assay for determining the level of proteasomes present in the test
sample, wherein said assay comprises protein detection or detection
of nucleic acid molecules encoding proteins.
18. The kit of claim 17, wherein the test assay detects one or more
molecules comprising one or more proteasome proteins, one or more
proteasome binding proteins, one or more proteasome regulating
proteins, a nucleic acid molecule that encodes a proteasome
protein, a proteasome regulating protein or a proteasome binding
protein.
19. The kit of claim 17, wherein the test assay is an assay to
determine the level of immunoproteasomes present in the test
sample.
20. The kit of claim 17, wherein the test assay is an assay to
detect the level of ubiquinated proteins in a sample or copper
complex binding to proteasomes.
21. A method for determining milk quality comprising (a) collecting
a test sample of milk, (b) performing a test assay for determining
the level of proteasomes present in the test sample, and (c)
comparing the level of proteasomes present in the test sample with
a standard level of proteasomes in a control sample of high quality
milk or comparing the level of proteasomes present in the test
sample with a standard level of proteasomes in a control sample of
low quality milk; wherein an increase in the level of proteasomes
present in the test sample relative to the standard level of
proteasomes present in the control sample of high quality milk
indicates the milk is of lower quality; an increase in the level of
proteasomes present in the test sample relative to the standard
level of proteasomes present in the control sample of low quality
milk indicates the milk is of higher quality; a substantially
similar level of proteasomes present in the test sample relative to
the standard level of proteasomes present in the control sample of
high quality milk indicates the milk is of high quality; and a
substantially similar level of proteasomes present in the test
sample relative to the standard level of proteasomes present in the
control sample of low quality milk indicates the milk is of lower
quality.
22. A kit for determining the level of proteasomes present in a
test sample comprising a first antibody and second antibody that
each specifically bind one or more proteasomal subunits.
23. The kit of claim 22, wherein the first and second antibodies
bind the same proteasomal subunit at distinct epitopes.
24. The kit of claim 22, wherein the first and second antibodies
bind two distinct proteasomal subunits.
25. The kit of claim 22, wherein the first antibody is bound to a
solid support and the second antibody comprises a detectable label.
Description
[0001] This application is a continuation-in-part application of
U.S. Ser. No. 12/738,340, filed Apr. 16, 2010, which claims
priority to PCT/US08/80514, filed Oct. 20, 2008, and U.S.
Provisional Application No. 60/999,495, filed Oct. 18, 2007, which
are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Mastitis is a general term that describes inflammation in
the milk glands and tissue lining the mammary gland/udder.
Inflammation is a cellular- and organ-specific response to injury
that can arise from a number of environmental events and pathogenic
agents. Mastitis can be induced by physical injury (such as that
caused by the milking apparatus), and from bacterial and fungal
infections. Infections of the nipples, ducts and udders might be
caused by mycoplasma, pseudomonas, staphylococcus and
streptococcus, as well as coliforms such Escherichia coli. Probably
more than a dozen different bacterial species, in addition to yeast
and fungi, can cause mastitis in cow, sheep and goat. Yeast and
fungal infections yield a sub-clinical form of infection and
therefore may go undetected. However, it is not known if
yeast/fungi can aggravate infection caused by coincident bacterial
infection.
[0004] The living environment of dairy cows is conducive to
infection, since the soil and manure are rich sources of bacteria.
The animals are in frequent contact with the soil, and this is
evident when one compares natural fluctuations in the sub-clinical
infections during wet and dry seasons; wet environments cause
higher levels of somatic cell count (SCC) values. Furthermore, the
milking process is itself highly conducive to cross contamination.
Therefore, animal management procedures play a key role in
controlling the incidence of mastitis. Separation of infected
animals, clean handling techniques, and well fitting vacuum suction
cups can play an important role in minimizing the incidence of
infection. Treatment of the affected animal includes antibiotics to
eliminate the bacteria, and anti-inflammatory agents to reduce
swelling.
[0005] In cows, the four teats drain separate compartments
(quarters) in the udder, and the infection can migrate from one
quarter to another in a single animal. Additionally,
animal-to-animal and human contact can also extend the infection to
other animals in the milk line. These issues are of considerable
concern, since the milking process typically results in the
collection of milk from many animals (50-200) in a common reservoir
(bulk tank). Not surprisingly, a single heavily infected animal can
compromise the quality of the entire milk output from a dairy
facility. Culling chronically infected, unresponsive animals is a
prudent measure. However, reduced milk output, poor milk quality,
veterinary and health issues have a significant negative impact on
the viability and productivity in dairy farms. In this regard, the
technologies developed and described herein are expected to
contribute to improving early diagnostics, minimizing
cross-contamination, sequestering problematic animals, and
monitoring efficacy of treatment.
[0006] Mastitis is characterized as clinical or pre-clinical
mastitis. Sub-clinical mastitis is not visually detected (e.g.,
redness, soreness, swelling), and milk quality is not severely
diminished. However, some changes may be observed, including clots
and runny consistency of the milk. Redness, soreness and swelling
of the teats are occasionally observed. Clinical mastitis can be
mild or severe, based on the level of SCC in the milk. A heavily
infected animal will display significant redness, soreness, and a
hard teat, which will necessitate antibiotic treatment. The onset
of mastitis can be rapid, and cause impaired breathing, failure to
eat, and significantly increased body temperature. A systemic
infection is considered grave, and the animal is removed from the
milk line, and treated with antibiotics for approximately 3 days.
Following the treatment, the animal is kept off the milk line until
antibiotic levels in the milk are reduced. Some animals may
tolerate chronic sub-clinical mastitis, although the extent of this
occurrence and its effect of milk output and quality have not been
carefully determined, because there is no straightforward way to
measure it. Therefore, there is a need for detecting the early
stage of infection to permit prompt removal of affected individuals
before the bulk reservoir is affected. Because chronically affected
individually oscillate between high baseline of sub-clinical
infection and full-blown infection, these candidates require
constant monitoring, which is not possible with conventional
methods, but can be easily achieved with the Invention described
here.
[0007] The standard assay for measuring mastitis in the dairy
industry is to count the number of cells (originating from the
cow), in the milk. This estimate is termed the somatic cell count
(SCC). Specifically, the SCC reflects the levels of cells,
including immune cells, such as leukocytes that are released from
the lining and tissues of the udder of the infected animal, into
the udder cavity. Although these cells are present in the milk, it
is difficult to estimate their levels due to the difficulty of
visualizing clear cells in the turbid milk suspension. The number
of somatic cells in a given volume of milk (typically 1 ml)
provides a semi-quantitative estimate of the degree of infection,
because unaffected animals typically have low SCC levels. The SCC
level may also be confounded by high levels of a pathogen,
including bacteria, yeast and fungi, each of which is a causative
agent in mastitis. There are a number of limitations of the SCC
type assay. Due to turbidity, milk cell counts cannot be made, and
instead the samples are tested at remote laboratories using
independent measurements. The time lapse between the collection of
a milk sample and the transmittal of the cell count information to
the dairy farmer can exceed one month. This extraordinary delay in
providing key information to the field agent or dairy farmer
severely limits, prompt action that might otherwise curtail the
transmission of infection to unaffected animals. High level of SCC
in the bulk tank deteriorates milk quality, and garners a lower
price for the farmer. Furthermore, mastitis reduces milk output in
the affected animal, and increases the likelihood of more frequent
infections. Surprisingly, infected animals are only treated for a
fixed term with antibiotics, and SCC levels are not typically
monitored to confirm successful outcome. Consequently, an animal
might be treated excessively, or not sufficiently. In the latter
case, animals that respond poorly, or slowly to the antibiotic
regimen, are likely to become chronic offenders that become drug
resistant, yield poor quality of milk, incur large veterinarian
expenses, eventually leading to the culling of the animal.
[0008] Federal and State guidelines allow up to about
7.5.times.10.sup.5 somatic cells/ml of raw milk. This is the upper
limit, and milk quality is negatively, affected at such high SCC
levels. Typical values in the bulk tank may range from
2-4.times.10.sup.5 SCC. The level permitted by the European dairy
industry is more stringent (4.times.10.sup.5 somatic cells/ml).
[0009] There are currently two broadly available mastitis tests for
monitoring milk quality. One assay, called the Somatic Cell Count
(SCC) determines the level of somatic cells in the milk. The
weaknesses of the SCC assay include inaccuracy, since it is
negatively influenced by the presence of pathogens (the primary
cause of mastitis) in the milk; insensitivity as it only provides a
threshold value of the levels of somatic cells; and failure to
provide early-diagnostic information, because the results are
provided to the farm up to a month after the initial acquisition of
milk samples. Moreover, an individual infected animal is not
identified because SCC levels are typically measured in the bulk
milk reservoir, which can contain milk from 50-100 cows. Therefore,
this compromises the quality of milk in the bulk reservoir and
delays detection of the affected animal. Moreover, advanced
mastitis necessitates more aggressive treatment, prolonged
withdrawal of the animal from the milk line, and a higher
probability of generating a chronically affected individual, all of
which represent significant economic liabilities to the farm. The
SCC assay is hindered by the expense and delay of testing samples
at a remote laboratory. Significantly, the delay prevents the
implementation of prompt remedial action. Generally, the
farmer/field agent recognizes symptoms in an affected animal and
removes it from the milk line. However, this represents an action
after the infection has occurred.
[0010] The second assay is termed the California Mastitis Test
(CMT). In this method, milk from each quadrant of the udder is
deposited into each of four shallow receptacles, to which a
proprietary solution is added. Gentle mixing results in clumping of
mastitis-positive samples. This is an imprecise assay that does not
give any quantitative measurement of the level of infection.
Moreover, the assay is quite insensitive, and does not detect lower
levels of persistent mastitis.
[0011] While other assays have been suggested, these assays have
not been developed for commercial use. For example, Pyorala ((2003)
Vet. Res. 34:565-578) suggests that ATP has a strong positive
correlation with SCC and has been considered as an alternative to
SCC as an indicator of mastitis. Similarly, GB 2001434 indicates
that ATP levels correlate with somatic cell count numbers and can
be used to determine the hygiene of milk or the health condition of
cows. However, neither of these assays detects the level of
proteasome activity via a proteasomal substrate or hydrolysis of
ATP.
[0012] Clinical mastitis causes greater than $2 billion in directly
attributable losses for the dairy industry. However, this is an
underestimate, because the financial loss caused by low quality
milk and poor yield from sub-clinical cows, treatment of affected
animals, withdrawal from the milk line, and occasional culling of
ill animals is not estimated. Accordingly, there is a need in the
art for rapid, reliable and accurate tests for detecting
mastitis.
SUMMARY OF THE INVENTION
[0013] The present invention features methods and kits for
detecting and monitoring mastitis. In one embodiment, the method of
the invention involves the steps of (a) performing a test assay for
determining the level of proteasomes present in a test sample of
milk collected from one or more animals, and (b) comparing the
level of proteasomes present in the test sample with a standard
level of proteasomes in a control sample of milk, wherein an
increase in the level of proteasomes present in the test sample
relative to the standard level of proteasomes present in the
control sample of milk indicates one or more animals has mastitis.
In certain embodiments of this method, the test assay for
determining the level of proteasomes present in the test sample
detects proteasome activity in the sample by, e.g., measuring the
level of conversion of a proteasome enzyme substrate to a product.
Examples of such substrates include an ATPase substrate, a
de-ubiquitinase substrate, or a substrate such as
Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr (SEQ ID NO:8); Z-Gly-Gly-Leu;
Suc-Leu-Leu-Val-Tyr (SEQ ID NO:9); Suc-Leu-Tyr; Ac-Arg-Leu-Arg;
Boc-Leu-Arg-Arg; Bz-Val-Gly-Arg; Ac-Gly-Pro-Leu-Asp (SEQ ID NO:7);
Z-Leu-Leu-Glu; and Ac-nLeu-Pro-nLeu-Asp (SEQ ID NO:6), which may be
labeled with a fluorogenic label, luminescent label or a
chromogenic label. In other embodiments of this method, the test
assay for determining the level of proteasomes present in the test
sample is an assay that detects nucleic acid molecules encoding
proteins or an assay that detects proteins such as one or more
proteasome proteins, one or more proteasome binding proteins, one
or more proteasome regulating proteins, a nucleic acid molecule
that encodes a proteasome protein, a proteasome regulating protein
or a proteasome binding protein. In certain embodiments, the test
assay for determining the level of proteasomes present in the test
sample is an assay to determine the level of immunoproteasomes
present in the test sample. In other embodiment, the test assay for
determining the level of proteasomes present in the test sample is
an assay to detect the level of ubiquinated proteins in a sample or
copper complex binding to proteasomes. A kit for carrying out this
assay is also provided.
[0014] As another embodiment, the method of the invention involves
the steps of (a) collecting a test sample of milk, (b) performing a
test assay for determining the level of proteasomes present in the
test sample, and (c) comparing the level of proteasomes present in
the test sample with a standard level of proteasomes in a control
sample of high quality milk or comparing the level of proteasomes
present in the test sample with a standard level of proteasomes in
a control sample of low quality milk; wherein an increase in the
level of proteasomes present in the test sample relative to the
standard level of proteasomes present in the control sample of high
quality milk indicates the milk is of lower quality; an increase in
the level of proteasomes present in the test sample relative to the
standard level of proteasomes present in the control sample of low
quality milk indicates the milk is of higher quality; a
substantially similar level of proteasomes present in the test
sample relative to the standard level of proteasomes present in the
control sample of high quality milk indicates the milk is of high
quality; and a substantially similar level of proteasomes present
in the test sample relative to the standard level of proteasomes
present in the control sample of low quality milk indicates the
milk is of lower quality. A kit for carrying out this assay is also
provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the fold increase in proteasome activity in
affected cow milk, as compared to normal healthy cow milk. Sunshine
and R-Dale were young and healthy. COW1 was chronically infected.
COW2 was injured. COW3 and COW4 were suspicious and under
observation.
[0016] FIG. 2 shows the altered abundance of proteasome
subunits.
[0017] FIG. 3 shows the altered abundance of immunoproteasome
subunits.
[0018] FIG. 4 shows proteasome activity in a chronically infected
cow over the course of a month.
[0019] FIG. 5 shows a depiction of examples of luminescent assays,
wherein Suc-LLVY (SEQ ID NO:9), Z-LRR or Z-nLPnLD (SEQ ID NO:6) are
attached to luciferin.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The Ub/proteasome proteolytic pathway is required for
efficient cell-cycle control, stress response, DNA repair, and
differentiation (Glickman & Ciechanover (2002) Physiol. Rev.
82:373-428; Pickart (1997) FASEB J. 11:1055-1066; Varshaysky (1997)
Trends Biochem. Sci. 22:383-387). Mutations in this pathway can
cause pleiotropic defects because of its involvement in virtually
all aspects of cell function. Consequently, the characterization of
the Ub/proteasome pathway for the development of treatment for
cancer and other malignancies is an area of active investigation
(Voorhees, et al. (2003) Clin. Cancer Res. 9:6316-6325; Yang, et
al. (2004) Clin. Cancer Res. 10:2570-7; Yang, et al. (2004) Clin.
Cancer Res. 10:2220-2221; Rossi & Loda (2003) Breast Cancer
Res. 5:16-22; Ohta & Fukuda (2004) Oncogene 23:2079-2088).
[0021] The 26S proteasome is composed of a catalytic (20S) particle
and a regulatory (19S) particle. The structure and function of the
20S catalytic particle is conserved in evolution, and its
compartmentalized organization ensures that the proteolytic
activities are sequestered within the interior of the proteasome
(Baumeister, et al. (1998) Cell 92:367-380). The large 19S
regulatory particle interacts with the 20S particle to facilitate
recognition, unfolding and degradation of ubiquitinated substrates
(Glickman, et al. (1998) Mol. Cell. Biol. 18:3149-3162; Groll, et
al. (2000) Nat. Struct. Biol. 7:1062-1067). Ubiquitin (Ub) is
covalently attached to lysine side-chains in cellular proteins
(Pickart (2000) Trends Biochem. Sci. 25:544-548). The ligation of
Ub to proteins requires the action of three enzymes termed
Ub-activating (E1), Ub-conjugating (E2), and Ub-ligases (E3)
(Glickman & Ciechanover (2002) supra). The sequential addition
of Ub moieties results in the formation of a multi-Ub chain, which
facilitates protein degradation by promoting translocation of
substrates to the proteasome (Gregori, et al. (1990) J. Biol. Chem.
265:8354-8357; Thrower, et al. (2000) EMBO J. 19:94-102).
[0022] Malignant conditions are frequently associated with altered
abundance and stability of regulatory proteins. It is therefore
likely that the expression of a unique repertoire of proteins
underlies the transition from normal to abnormal growth. Proteasome
activity has been found to be elevated in esophageal cancer and
cancer cachexia (Wyke, et al. (2004) Br. J. Cancer 91:1742-1750;
Zhang, et al. (2004) World J. Gastroenterol. 10:2779-2784).
[0023] It has been shown that the co-translational degradation of
newly synthesized misfolded proteins requires the Ub/proteasome
system (Schubert, et al. (2000) Nature 404:770-774; Reits, et al.
(2000) Nature 404:774-778; Turner & Varshaysky (2000) Science
289:2117-2220). Moreover, translation elongation factor 1-alpha
(eEF1A) is required for the efficient degradation of nascent
polypeptide chains, especially in ATP-depleting conditions, and in
the presence of protein synthesis inhibitors (Chuang, et al. (2005)
Mol. Cell. Biol. 25:403-413). eEF1A expression is increased in
certain cancers, e.g., T-lymphoblastic cancer (Lamberti, et al.
(2004) Amino Acids 26:443-448; Dapas, et al. (2003) Eur. J.
Biochem. 270:3251-3262), a result that reflects a more general
response to aberrant growth (Ejiri (2002) Biosci. Biotechnol.
Biochem. 66:1-21).
[0024] It has now been shown that the detection of proteasomes in
milk can be used as an indicator of mastitis in a milk-producing
animal as well as a measure useful to assess the quality of the
milk. Proteasomes are found in the somatic cells found in milk
including the immune cells such as leukocytes which are present in
increasing numbers as the level of infection progresses, as an
animal goes from uninfected to severe mastitis. Additionally, the
infectious organism responsible for mastitis also has proteasomes
that can also be detected using distinct proteasome assays and
antibodies.
[0025] Proteasomes are very robust by their nature and remain
enzymatically active in milk. Thus, measuring the level of enzyme
activity in a sample as compared to standard levels or ranges
typical of uninfected, preclinical-mastitis, mild clinical
mastitis, severe clinical mastitis and the like indicates not only
a diagnosis of mastitis but also the severity of the condition.
Proteasome enzyme activity assays are fast and simple and can be
carried out with very little equipment or expense at the site where
the milk is collected. When used in the context of dairy livestock,
management and treatment of animals can be undertaken to maximize
yield, minimize time off the line, and to improve the overall
health and well being of the animals through early diagnosis and
monitoring during treatment.
[0026] Likewise, because proteasomes are present in milk, they
provide a useful target for detection by immunoassay, or as targets
for detection of mRNA encoding proteasome protein components. In
the case of immunoassay, the level of proteasome protein in a
sample may be compared to standard levels or ranges typical of
uninfected, preclinical-mastitis, mild clinical mastitis, severe
clinical mastitis and the like to diagnosis mastitis and assess its
severity. Similarly, the level of mRNA that encodes a proteasome
protein in a sample may be quantified and compared to standard
levels or ranges typical of uninfected, preclinical-mastitis, mild
clinical mastitis, severe clinical mastitis and the like to
diagnosis mastitis and assess its severity. These methods may be
used to improve the productivity and health of animals.
[0027] Other methods of detecting proteasome levels include, for
example, detection of levels of proteasome bound factors or mRNA
encoding the same, detection of levels of proteasome regulatory
factors or mRNA encoding the same, and levels based on interaction
with organic copper complexes.
[0028] For the purposes of the present invention, the term "level
of proteasomes present" is meant to refer to the quantity of
proteasomes present. The proteasomes may be from the animal's
somatic cells or from a bacteria, fungus and/or yeast organism
infecting the animal. The level of proteasomes may be determined by
direct quantification or by determining the level of proteasome
enzyme activity or activity of enzymes associated with proteasome.
The term "standard level of proteasomes in a control sample of milk
from an uninfected animal" is meant to refer to a typical quantity
or range of quantities of proteasomes found in a sample of milk
from an uninfected animal, wherein the sample is of a known sample
volume, preferably the same volume as being used in a test assay.
The term "proteasome enzyme activity" is used herein to refer to
enzymatic activity by one of the several proteasome enzymes present
as part of the proteasome complex whereby a substrate is processed
by the enzyme into one or more products which are distinguishable
from the substrate. Proteasome enzymes include the
chymotrypsin-like or chymotryptic protease, the trypsin-like or
tryptic protease and the caspase-like or post-acidic protease. The
term "level of conversion of a proteasome enzyme substrate to a
product" is meant to refer to the quantity of substrate converted
to a product by an enzyme in a test period.
[0029] The proteasome contains three enzyme activities: 1) protease
activity, 2) ATPase activity and 3) de-ubiquitination activity.
Three different proteases perform the protease activity. These
include the trypsin-like enzyme, the chymotrypsin like enzyme, and
the caspase-like enzyme. Each of these enzymes cleaves peptide
bonds at the C-terminus of different types of amino acids.
[0030] In the enzyme assays disclosed herein, the level of enzyme
activity is determined by measuring the cleavage or processing of a
substrate into products. Typically, enzyme assays use labeled
substrate in which the label becomes detectable after processing.
In such case, the level of activity is indicated by measuring the
amount of product produced. It is also possible to measure enzyme
activity by using a labeled substrate in which the label is
detectable when the substrate is unprocessed and becomes
undetectable after processing. In such case, the level of activity
is indicated by measuring the amount of substrate processed. As an
alternative, Verma, et al. ((2000) Mol. Biol. Cell. 11(10):3425-39)
refer to the identification of nucleotide-sensitive
proteasome-interacting proteins by mass spectrometric analysis of
affinity-purified proteasomes.
[0031] Protease Assays. As noted above, there are three different
proteases, the trypsin-like enzyme, the chymotrypsin like enzyme,
and the caspase-like enzyme, and each cleaves peptide bonds at the
C-terminus of different types of amino acids. In its usual
functioning in the cells, proteins are labeled for destruction by
attachment to ubiquitin and delivered to the proteasome where the
proteins are de-ubiquitinated and unfolded so that they can be
moved into the proteasome and processed by the proteases. The three
proteases of the proteasome are located internally in a proteasome
structure and so the proteins to be cleaved are unfolded and
translocated into the proteasome. The proteases recognize and
cleave by hydrolysis peptide bonds which are at the C terminus of
different amino acid residues. The trypsin-like enzyme cleaves
after a basic amino acid residue. The chymotrypsin like enzyme
cleaves after a hydrophobic amino acid residue. The caspase-like
enzyme cleaves after an acidic amino acid residue. A detailed
analysis of the amino acid sequence preferred by the hydrolytic
activities present in the proteasome is described in the art (see,
e.g., Dick, et al. (1998) J. Biol. Chem. 273:25637-25646).
[0032] In assays provided herein, substrates are used that are
spontaneously processed by the proteasomes. Specifically, these
substrates do not need to be attached to ubiquitin, or unfolded. To
be spontaneously processed by a protease, the substrate must be of
a size that it can enter the proteasome without the need for
delivery proteins, de-ubiquitination, or unfolding. Thus,
substrates that can be spontaneously processed are typically no
longer in length than the length of a 3-6 amino acid peptide, and
are no more than 13 Angstroms cross-wise so that it can freely
enter the proteasome. The structure of the 20S proteasome from
yeast at 2.4 .ANG. resolution is known (Groll, et al. (1997) Nature
386:463-471). The crystal structure of the 20S proteasome from the
yeast Saccharomyces cerevisiae was reported to show that its 28
protein subunits are arranged as a complex in four stacked rings
and occupy unique locations. The interior of the particle, which
harbors the active sites of the three proteases, is only accessible
by two 13 Angstrom diameter axial pores.
[0033] To be processed, the substrate must include a peptide bond
at the C-terminus of an amino acid residue. Accordingly, the
general formulae for the substrates are X-Y-A=Z for trypsin-like
substrate, X-Y-B=Z for chymotrypsin-like substrate and X-Y-C=Z for
caspase-like substrate, wherein X is a blocking moiety which
prevents N-terminal proteases from processing the substrate, Y is
absent or moiety which links X to A, A is a basic amino acid or
moiety which forms a peptide bond with Z that is recognized and
cleaved by the trypsin-like protease, B is a hydrophobic amino acid
or moiety which forms a peptide bond with Z that is recognized and
cleaved by the chymotrypsin-like protease, C is an acidic amino
acid or moiety which forms a peptide bond with Z that is recognized
and cleaved by the caspase-like protease, = is a peptide bond that
links A, B or C to Z and that is recognized and cleaved by the
protease, and Z is a label that is either undetectable when linked
to A, B or C and becomes detectable when = is hydrolyzed, or is
detectable when linked to A, B or C and becomes undetectable when =
is hydrolyzed.
[0034] Typically, the substrates are N-terminally blocked linear
peptides that are not folded and have a secondary structure and
linked at their C-terminus to detectable labels by a peptide bond.
Thus, according to the formulae above, the peptides are Y-A, Y-B or
Y-C in which Y is 1-4 amino acids, A is an amino acid which is
recognized by trypsin-like protease, B is an amino acid which is
recognized by chymotrypsin-like protease, and C is an amino acid
which is recognized by caspase-like protease. N-terminal blocking
moieties are well-known in the art and include, but are not limited
to, succinimide (Suc), (Boc), benzoyloxycarbonyl (Z) and benzoyl
(Bz).
[0035] Detectable labels useful in the methods of the invention are
well-known and include fluorescent, colorimetric and luminescent
labels. Examples of fluorescent labels include
7-amino-4-methylcoumarylamide (AMC). Examples of luminescent labels
include luciferin. Examples of colorimetric labels include copper
complex reagents. Fluoresence may be measured using standard
commercially available fluorometer equipment. Examples of
fluoresence detectors include Turner 7000 (Sunnyvale, Calif.),
Turner AQUAFLUOR (Sunnyvale, Calif.), and TECAN Genesis plate
reader. Fluorescent, luminescent and colorimetric labels and
methods of detecting and measuring quantities with them are
well-known and readily understood by those skilled in the art.
[0036] As used herein, the terms "trypsin-like substrate" and
"tryptic substrate" are used interchangeably and meant to refer to
substrates which can be spontaneously processed by the trypsin-like
protease which is also referred to as tryptic protease. Examples of
amino acids recognized by trypsin-like protease, i.e. A amino
acids, include the basic amino acids Arginine; Lysine; and in some
cases Histidine. Examples of trypsin-like substrates include
Ac-Arg-Leu-Arg-AMC; Boc-Leu-Arg-Arg-AMC; and Bz-Val-Gly-Arg-AMC. In
some embodiments the substrate includes the amino acid sequence as
one of these substrates but a different blocking moiety and or
label.
[0037] The terms "chymotrypsin-like substrate" and "chymotryptic
substrate" are used interchangeably and meant to refer to
substrates which can be spontaneously processed by the
chymotrypsin-like protease which is also referred to as
chymotryptic protease. Examples of amino acids recognized by
chymotrypsin-like protease, i.e. B amino acids, include the
hydrophobic amino acids Tryptophan; Tyrosine; Phenylalanine;
Methionine; Leucine; and Isoleucine. Examples of chymotrypsin-like
substrates include Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-AMC (SEQ ID
NO:1); Z-Gly-Gly-Leu-.beta.NA; Z-Gly-Gly-Leu-AMC;
Suc-Leu-Leu-Val-Tyr-Luciferin (SEQ ID NO:2); and Suc-Leu-Tyr-AMC.
In some embodiments, the substrate includes the amino acid sequence
as one of these substrates but a different blocking moiety and or
label.
[0038] As used herein, the terms "caspase-like substrate" and
"post-acidic protease substrate" are used interchangeably and meant
to refer to substrates which can be spontaneously processed by the
caspase-like protease which is also referred to as post-acidic
protease. Examples of amino acids recognized by caspase-like
protease, i.e. C amino acids, include the acidic amino acids
Glutamic acid and Aspartic acid. Examples of caspase-like
substrates include Ac-Gly-Pro-Leu-Asp-AMC (SEQ ID NO:3);
Z-Leu-Leu-Glu-AMC; Z-Leu-Leu-Glu-.beta.NA; and
Ac-nLeu-Pro-nLeu-Asp-AMC (SEQ ID NO:4). In some embodiments the
substrate comprises the amino acid sequence as one of these
substrates but a different blocking moiety and or label.
[0039] The hydrolysis of test substrates by chymotryptic, tryptic
and post-acidic proteases is well-described in the literature.
These substrates are typically small peptide derivatives that are
linked to a fluorogenic reagent that upon release by cleavage by a
proteasome protease can be excited by near-UV light to generate a
highly sensitive and specific fluorescent signal. The structure,
sequence, hydrophobicity and other biophysical properties of these
synthetic substrates have been described, and the compounds that
are commercially available represent versions that can be generated
in bulk and yield good signal. For example, Sato, et al. ((1991)
Eye Res. 10:485-489) refer to the use of labeled diisopropyl
fluorophosphates to measure chymotrypsin-like activity. The
degradation of beta-galactosidase-based substrates can be monitored
using labeled proteins, and by measuring enzymatic activity using
ONPG (o-Nitrophenyl-beta-galactopyranoside) or CPRG (chlorophenol
red-beta-D-galactopyranoside). A plating and chromogenic assay can
also be performed using IPTG (isopropyl
beta-D-thiogalactopyranoside) and X-gal (5-
bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Other enzymes
have also been developed into proteasome substrates (including
glucuronidase, DHFR, GST and GFP). Moreover, Bachmair &
Varshaysky ((1989) Cell 56:1019-1032) report that the degradation
of DHFR and beta-galactosidase-type substrates show a loss of
signal, in the presence of productive degradation by the
proteasome. In contrast, degradation of fluorogenic peptide
substrates yields a time-dependent increase in fluorescence signal,
as the substrate is hydrolyzed by the proteasome.
[0040] In carrying out a method of the invention, some embodiments
provide that a test sample is added to a container such as a
cuvette or test tube. Generally, 0.5-2 ml, typically 1 ml samples
are tested. In some embodiments, the container is preloaded with
substrate. In some embodiments, the container is preloaded with
substrate that is dried and adhered to an inner surface of the
container. In other embodiments, the container has a cap with a
reservoir that includes substrate. After adding the milk, the cap
is used to close the container upon depressing it, the substrate is
displaced from the reservoir into the container, typically by
rupturing the reservoir. In some embodiments, the substrate is
provided in a container such as a dropper or metered dispensers and
the substrate is added to the container. The test sample may be
mixed and maintained for a period of time sufficient to allow
detection of an amount of detectable label that corresponds to the
assessment standard being used. Generally, the time of the reaction
is 30 seconds to about 5 minutes. After sufficient time has
elapsed, if the detectable label is a fluorescent label, the
container may be inserted in the fluorometer or luminometer, and
the level of fluorescence or luminescence detected. After
sufficient time has elapsed, if the detectable label is a
luminescent label, the light emitted from the container may be
measured and the level of luminescence detected. After sufficient
time has elapsed, if the detectable label is a colorimetric label,
the container may be inserted in the colorimeter and the level of
light of a predetermined wavelength is detected or, if the change
in color can be detected visually in the presence of a level of
proteasomes indicative of some level of infection, the visual
inspection of the container may be undertaken to determine the
results. In each case, the results may be compared to standards
known to correspond to one or more levels of infection.
[0041] In other embodiments of the invention, a test sample is
contacted with a solid surface, such as a stick of plastic or
paper. In accordance with this embodiment, the solid surface can be
preloaded with substrate such that contacting the test sample with
the solid surface places the test sample in contact with the
substrate disposed thereon. Alternatively, for example, substrate
is contacted with the solid surface after or with the test sample
so that the sample is mixed with the substrate. The solid surface
with the test sample is maintained for a specified period of time
sufficient to allow enzyme present in the sample to process the
substrate and produce a detectable label that manifests itself as a
color change on the solid surface. The color of the solid surface
may be compared to a standard color chart that includes the colors
for one or more levels of proteasomes corresponding to one or more
levels of infection. Alternatively, the amount of substrate on the
surface may be measured with sufficient precision such that when
contacted for a specified period of time with a sample containing
proteasomes above a threshold amount that corresponds to a level of
infection, the solid surface changes color such that it can be
visually detected. That is, the amount of reagent disposed on a
test strip is calibrated so that a detectable reaction only occurs
if the amount of enzyme present is greater than a threshold level.
Thus, a test strip is contacted with a milk sample for a specified
time and if it undergoes a detectable reaction in that time period,
such as changing color, the assay indicates protease levels above a
known threshold. For example, the test strip may be manufactured so
that it will only change color after a specified time if the
protease levels are consistent with a pre-clinical mastitis level,
or clinical mastitis level.
[0042] ATPase Assays. The intact 26S proteasome contains two 19S
regulatory particles. Each 19S particle contains a ring of six
ATPases. Therefore, an intact 26S proteasome will contain 12 ATP
hydrolyzing enzymes. In this respect, the term "ATPase subunit" is
meant to refer to proteasome subunits which can hydrolyze ATP. For
example, DeMartino, et al. ((1994) J. Biol. Chem. 269:20878-20884)
report that PA700, an ATP-dependent activator of the 20S
proteasome, is an ATPase containing multiple members of a
nucleotide-binding protein family.
[0043] Purified proteasomes have robust ATPase activity that can be
measured by a broad range of assays that are well-known and
routinely performed by those skilled in the art. A luciferase-based
ATPase activity is one of the most sensitive. In some embodiments,
the assay determines ATP hydrolyzing activities present in raw
milk. In some embodiments, the assay includes first isolating
proteasomes from a sample and then measuring proteasome-specific
ATPase activity. Proteasome isolation may be accomplished, for
example, by the method that is disclosed in U.S. Pat. No.
6,294,363, which is incorporated herein by reference. Typical
detection methods include a luciferase/luciferin-based approach
which generates a robust luminescent signal of high specificity,
and very broad dynamic range (linearity over 5-orders of magnitude)
however, other systems using different types of detectable signals
may also be routine performed. These methods provide another way to
monitor proteasome abundance and activity.
[0044] De-Ubiquitization Assays. Ubiquitination, the conjugation of
proteins to ubiquitin that occurs in a number of cellular processes
including endocytosis, DNA repair and degradation by the 26S
proteasome is reversible as a number of deubiquitinating enzymes
mediate the disassembly of ubiquitin-protein conjugates. Some
deubiquitinating enzymes are associated with the 26S proteasome
contributing to and regulating the particle's activity. At least
three different deubiquitinating enzymes have been detected in
proteasomes.
[0045] For example, Stone, et al. ((2004) J. Mol. Biol.
344(3):697-706) refer to Uch2/Uch37 as the major deubiquitinating
enzyme associated with the 26S proteasome in fission yeast. Fission
yeast Uch2 and Ubp6, two proteasome associated deubiquitinating
enzymes is reported to be characterized. The human orthologues of
these enzymes are known as Uch37 and Usp14, respectively. The
subunit Uch2/Uch37 is reported to be the major deubiquitinating
enzyme associated with the fission yeast 26S proteasome while the
activity of Ubp6 appears to play a more regulatory and/or
structural role involving the proteasome subunits Mts1/Rpn9,
Mts2/Rpt2 and Mts3/Rpn12. Ubp6 is reported to become essential when
activity of these subunits is compromised by conditional
mutations.
[0046] Proteasome activity can be measured by monitoring associated
de-ubiquitinating activity. The vast majority of substrates that
are degraded by the proteasome are attached to a chain of
ubiquitins. However, these ubiquitins are not degraded along with
the substrate, but are released, recycled and used again to target
additional proteins to the proteasome. Significantly, the
de-ubiquitination of the substrate is required for successful
degradation.
[0047] Deubiquitinating activity can be measured using well-known
routine assays and commercially available fluorogenic or
luminescent substrates. In this respect, the term
"de-ubiquitinating substrate" is meant to refer to substrates which
can be processed by the proteasome de-ubiquitinating enzyme. The
method to measure proteasome-associated de-ubiquitination involves
the addition of a substrate such as Ubiquitin-AMC,
Ubiquitin-Luciferin, Arg-Gly-Gly-AMC, Arg-Leu-Arg-Gly-Gly-AMC (SEQ
ID NO:10), Arg-Leu-Gly-Gly-Luciferin (SEQ ID NO:10), to other
peptide derivatives, to a reaction that contains the
de-ubiquitinating activity. Cleavage of the label from the
ubiquitin, or from the peptide (e.g., Arg-Gly-Gly-AMC) generates a
highly specific signal that can be readily detected with a
fluorometer or luminometer. Therefore, the key aspect is that
measurement of de-ubiquitinating activity of the proteasome
(irrespective of the substrate used), provides an alternate way to
measure the level of proteasome and proteasome activity.
[0048] Immunoassays. A simple way to gauge the level of the
proteasome is by using immunologic methods. Both monoclonal and
polyclonal antibodies are available to all subunits of the
proteasome. Antibodies against human proteasome subunits can
cross-react with the bovine counterparts. The intact 26S proteasome
contains approximately 32 subunits, while the immunoproteasome
contains approximately 16 subunits (e.g., LMP2, LMP7, and
MECL1/LMP10), as well as the proteasome activator, PA28. For
example, Kopp, et al. ((1997) PNAS 94:2939-2944) refer to subunit
arrangement in the human 20S proteasome and report that in human
20S proteasomes two copies of each of seven different .alpha.-type
and seven different (.beta.-type subunits are assembled to form a
stack of four seven-membered rings, giving the general structure
.alpha.1-7, .beta.-7, .beta.-7, .alpha.-7. By means of
immunoelectron microscopy and chemical crosslinking of neighboring
subunits, the positions of the individual subunits in the
proteasome were determined. The topography shows that for the
trypsin-like, the chymotrypsin-like, and the post-glutamyl cleaving
activities, the pairs of .beta.-type subunits, which are thought to
form active sites, are nearest neighbors.
[0049] Tanaka & Tsurumi ((1997) Mol. Biol. Reports
24:(1-2)3-11) refer to the subunits and functions of the 26S
proteasome and report that the 26S proteasome is an eukaryotic
ATP-dependent, dumbbell-shaped protease complex with a molecular
mass of approximately 2000 kDa. It is composed of a central 20S
proteasome, functioning as a catalytic machine, and two large
V-shaped terminal modules, having possible regulatory roles,
composed of multiple subunits of 25-110 kDa attached to the central
portion in opposite orientations. The regulatory subunits are
classified into two subgroups, a subgroup of at least 6 ATPases
that constitute a unique multi-gene family encoding homologous
polypeptides conserved during evolution and a subgroup of
approximately 15 non-ATPase subunits, most of which are
structurally unrelated to each other.
[0050] Coret, et al. ((1994) Biochemistry 33:12229-12237) refer to
PRES and PRE6, as genes encoding 20S proteasome subunits from yeast
and report that the 20S proteasome of eukaryotes is an abundant
multicatalytic/multifunctional proteinase complex composed of an
array of nonidentical subunits which are encoded by a- or O-type
members of the proteasomal gene family. In budding yeast, 14
subunits had been detected but only 12 proteasomal genes had been
cloned. The authors cloned two additional proteasomal genes, PRES
and PRE6, which both encode essential .alpha.-type subunits.
Sequence comparison of all known eukaryotic proteasomal proteins
show the presence of a total of 14 subgroups, which can be divided
into seven a- and seven O-type groups. Including the Pre5 and Pre6
proteins, every subgroup contains a single yeast member.
[0051] In accordance with the instant invention, the level or
amount any subunit, associated protein or activator of the
proteasome or immunoproteasome can be measured to determine whether
an animal has mastitis. One or more of these proteins can be
measured using antibodies that are purchased or generated by
methods routinely used in the art. Methods of immuno-detection
include ELISA, immunoblotting, and antibody-based interference
assays. Antibodies conjugated to biotin, magnetic beads, or enzymes
(such as alkaline phosphatase) represent other ways to monitor the
levels of proteasome subunits.
[0052] Diagnostic marker proteins in a sample of raw milk can be
detected via binding assays, wherein a binding moiety which
specifically recognizes the marker protein (for instance an
antibody or naturally occurring binding partner), is introduced
into a sample suspected of containing the marker protein. In such
an assay, the binding partner is generally labeled as, for example,
with a radioisotopic or fluorescent marker. Labeled antibodies can
be used in a similar manner in order to isolate selected marker
proteins.
[0053] Using the instant marker proteins or antibodies, the skilled
artisan can use any one of a variety of detection methods for
detecting mastitis. The methods typically employ the steps of
detecting, by some means, the level of one or more Ub/proteasome
pathway marker protein in a sample and comparing said level to that
of the marker protein in a control sample to determine whether
there is a detectable increase (e.g., 1.5-fold or more) in the
level of the marker protein. In general, the test sample can be
obtained from young healthy cows, and confirmed independently using
SCC analysis.
[0054] In a protein-based assay, the marker protein in a sample is
detected, for example, by combining the marker protein with an
antibody-based binding moiety capable of specifically binding the
marker protein. An antibody-based binding moiety of the present
invention is intended to include an antibody, antibody fragment or
antibody derivative. Binding proteins can also be designed which
have enhanced affinity for a target protein. Optionally, the
binding moiety can be linked with a detectable label, such as an
enzymatic, fluorescent, radioactive, phosphorescent or colored
particle label. The labeled complex is detected, e.g., visually or
with the aid of a spectrophotometer or other detector.
[0055] A Ub/proteasome pathway or ancillary marker protein can also
be detected using any of a wide range of immunoassay techniques
with qualitative or quantitative results. For example, the skilled
artisan can employ the sandwich immunoassay format to detect
mastitis in a milk sample. Alternatively, the skilled artisan can
use conventional immunohistochemical procedures for detecting the
presence of the proteasome or ancillary protein using one or more
labeled binding moieties. It is contemplated that either an
absolute, semi-quantitative, or relative level of protein
expression can be detected using the immunoassays disclosed
herein.
[0056] In a sandwich immunoassay, two antibodies capable of binding
the marker protein generally is used, e.g., one immobilized onto a
solid support and one free in solution and labeled with a
detectable chemical compound. Examples of chemical labels that are
useful for the second antibody include radioisotopes, fluorescent
compounds, and enzymes or other molecules that generate colored or
electrochemically active products when exposed to a reactant or
enzyme substrate. When a sample containing the marker protein is
placed in this system, the marker protein binds to both the
immobilized antibody and the labeled antibody, to form a "sandwich"
immune complex on the support's surface. The complexed protein is
detected by washing away non-bound sample components and excess
labeled antibody, and measuring the amount of labeled antibody
complexed to protein on the support's surface. Alternatively, the
antibody free in solution can be detected by a third antibody
labeled with a detectable moiety which binds the free antibody or,
for example, a hapten coupled thereto.
[0057] Both the sandwich immunoassay and tissue immunohistochemical
procedures are highly specific and very sensitive, provided that
labels with good limits of detection are used. A detailed review of
immunological assay design, theory and protocols can be found in
numerous texts in the art, including Practical Immunology, Butt,
ed. (1984) Marcel Dekker, New York; and Antibodies, A Laboratory
Approach, Harlow, et al., eds. (1988) Cold Spring Harbor
Laboratory.
[0058] In general, immunoassay design considerations include
preparation of antibodies (e.g., monoclonal or polyclonal
antibodies), antibody fragments, or antibody derivatives having
sufficiently high binding specificity for the target protein to
form a complex that can be distinguished reliably from products of
nonspecific interactions. As used herein, the term "antibody" is
understood to mean binding proteins, for example, antibodies or
other proteins comprising an immunoglobulin variable region-like
binding domain, having the appropriate binding affinities and
specificities for the target protein. Higher antibody binding
specificity will permit detection of lower amounts of the target
protein. As used herein, the terms "specific binding" or
"specifically binds" are understood to mean that the binding
moiety, for example, an antibody, has a binding affinity for the
target protein of greater than 10.sup.5 M.sup.-1 or greater than
about 10.sup.7 M.sup.-1.
[0059] Antibody binding domains also can be produced
biosynthetically and the amino acid sequence of the binding domain
manipulated to enhance binding affinity with a preferred epitope on
the target protein. Specific antibody methodologies are
well-understood and described in the literature. A more detailed
description of their preparation can be found, for example, in
Practical Immunology (1984) supra.
[0060] Chimeric antibodies are also contemplated. Techniques
developed for the production of chimeric antibodies (Morrison, et
al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, et
al. (1984) Nature 312:604-608; Takeda, et al. (1985) Nature
314:452-454) by splicing the genes from a mouse antibody molecule
of appropriate antigen specificity together with genes from a human
antibody molecule of appropriate biological activity can be used. A
chimeric antibody is a molecule in which different portions are
derived from different animal species, such as those having a
variable region derived from a murine monoclonal antibody and a
human immunoglobulin constant region.
[0061] Antibodies can also be modified, e.g., to produce a number
of well-characterized fragments generated by digestion with various
peptidases. For example, pepsin digestion of an antibody produces
F(ab)'.sub.2. The F(ab)'.sub.2 can further be reduced under mild
conditions to break the disulfide linkage in the hinge region,
thereby converting the F(ab)'.sub.2 dimer into an Fab' monomer. The
Fab' monomer is essentially a Fab with part of the hinge region
(see, Fundamental Immunology, Third Edition (1993) Paul, ed., Raven
Press, N.Y.). While various antibody fragments are defined in terms
of the digestion of an intact antibody, one of skill will
appreciate that such fragments can be synthesized de novo either
chemically or by utilizing recombinant DNA methodology.
Accordingly, the term antibody fragment also includes fragments
either produced by the modification of whole antibodies or those
synthesized de novo using recombinant DNA methodologies. Thus, an
antibody fragment includes, but is not limited to, single chain
antibodies, Fab fragments, F(ab').sub.2 fragments, diabodies
(Holliger, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444; Poljak
(1994) Structure 2:1121-1123), fragments produced by a Fab
expression library (Huse, et al. (1989) Science 246:1275-1281), and
epitope-binding fragments of any of the above.
[0062] Antibody derivatives such as peptide aptamers, which are
selected for specifically binding to a proteasome subunit or
ancillary protein in mastitis, are also provided in the instant
invention. Peptide aptamers can be rationally designed or screened
for in a library of aptamers (e.g., provided by Aptanomics SA,
Lyon, France). In general, peptide aptamers are synthetic
recognition molecules whose design is based on the structure of
antibodies. Peptide aptamers consist of a variable peptide loop
attached at both ends to a protein scaffold. This double structural
constraint greatly increases the binding affinity of the peptide
aptamer to levels comparable to that of an antibody (nanomolar
range).
[0063] Binding moieties to the instant protein markers are used in
the diagnostic assays disclosed herein as well as in kits to detect
the level of the proteins in a milk sample. For example, a kit of
the invention can include one or more binding moieties (e.g.,
antibodies, antibody fragments, or antibody derivatives) which bind
specifically to one or more Ub/proteasome pathway or ancillary
proteins and which permit the relative level and/or specific
concentration of the Ub/proteasome pathway or ancillary proteins to
be detected and/or quantitated in a milk sample.
[0064] Suitable kits for detecting Ub/proteasome pathway or
ancillary proteins are contemplated to include, e.g., a receptacle
or other means for capturing a sample to be evaluated, and means
for detecting the presence and/or quantity in the sample of one or
more of the Ub/proteasome pathway or ancillary proteins described
herein. As used herein, "means for detecting" in one embodiment
includes one or more antibodies specific for these proteins and
means for detecting the binding of the antibodies to these proteins
by, e.g., a standard sandwich immunoassay as described herein.
Where the presence of a protein within a cell is to be detected,
e.g., as from a milk sample, the kit also includes a means for
separating the proteasomes from fat globules and casein present in
the milk. The kit can further contain written information, such as
procedures for carrying out the method of the present invention or
analytical information, such as representative values for
proteasome activity as a function of SCC levels, as well as with
low and high expression levels of a specific reference protein.
Such a kit, is developed, validated and commercially available (see
U.S. Pat. No. 6,294,363).
[0065] Antibodies against ubiquitin and its derivatives also
provide a method for monitoring the abundance of this pathway,
since the majority of proteasome-substrates are attached to
multi-ubiquitin chains. A kit to achieve this is developed,
validated and commercially available (see US Application No.
2005/0287608). An ubiquitin binding matrix may be used to collect
ubiquitinated proteins from raw milk. The level of such proteins
may be measured and compared to the range found in milk from
uninfected individuals.
[0066] Nucleic Acid Based Assays. In other embodiments, the
occurrence of mastitis can also be determined by detecting, in a
milk sample, one or more nucleic acid molecules encoding one or
more Ub/proteasome pathway or ancillary proteins. Using methods
well-known to those of ordinary skill in the art, one or more
oligonucleotide probes are designed to specifically hybridize with
a nucleic acid molecule encoding a Ub/proteasome pathway or
ancillary protein, e.g., nucleic acid molecules disclosed in the
GENBANK Accession Nos. disclosed herein.
[0067] The expression of proteasome subunits and
proteasome-associated factors can be readily determined, in real
time, using quantitative PCR. The sequence of the human genome is
available, and the design of effective PCR oligonucleotides can be
generated using both public and proprietary programming software.
(Bovine-specific RNA sequences can be based on homology to other
mammalian species, and further refined as nucleic acid sequences
become available). The 26S proteasome contains approximately 32
distinct subunits, whose sequence is highly conserved across
evolution. The immunoproteasome contains fewer than 32, but does
contain approximately 5 novel subunits. Additionally, hundreds of
regulatory factors form transient interactions with the proteasome.
Antibodies against human, mouse, rat, dog, worm, fly, yeast and
other species can be tested for cross-reactivity against the bovine
proteins. In tests conducted thus far, all the human specific
antibodies examined have reacted against the bovine proteins.
[0068] A target nucleic acid molecule encoding an Ub/proteasome
pathway or ancillary protein marker can be detected using a labeled
binding moiety capable of specifically binding the target nucleic
acid. The binding moiety can be, for example, a protein, a nucleic
acid or a peptide nucleic acid. Additionally, a target nucleic
acid, such as an mRNA encoding a Ub/proteasome pathway or ancillary
protein, can be detected by conducting, for example, a northern
blot analysis using labeled oligonucleotides, e.g., nucleic acid
fragments complementary to and capable of hybridizing specifically
with at least a portion of a target nucleic acid.
[0069] More specifically, gene probes composed of complementary RNA
or, preferably, DNA to the Ub/proteasome pathway or ancillary
protein nucleotide sequences or mRNA sequences encoding
Ub/proteasome pathway or ancillary proteins can be produced using
established recombinant techniques or oligonucleotide synthesis.
The probes hybridize with complementary nucleic acid sequences
presented in the test specimen, and provide exquisite specificity.
A short, well-defined probe, coding for a single unique sequence is
most precise and preferred. Larger probes are generally less
specific. While an oligonucleotide of any length can hybridize to
an mRNA transcript, oligonucleotides typically within the range of
8-100 nucleotides, preferably within the range of 15-50
nucleotides, are envisioned to be most useful in standard
hybridization assays. Choices of probe length and sequence allow
one to choose the degree of specificity desired. Hybridization is
generally carried out at from 50.degree. C. to 65.degree. C. in a
high salt buffer solution, formamide or other agents to set the
degree of complementarity required. Furthermore, the state of the
art is such that probes can be manufactured to recognize
essentially any DNA or RNA sequence. For additional methodologies,
see, for example, Guide to Molecular Techniques, Berger, et al.
(1987) Methods of Enzymology, Vol. 152. A wide variety of different
labels coupled to the probes or antibodies can be employed in the
instant assays. The labeled reagents can be provided in solution or
coupled to an insoluble support, depending on the design of the
assay. The various conjugates can be joined covalently or
non-covalently, directly or indirectly. When bonded covalently, the
particular linkage group will depend upon the nature of the two
moieties to be bonded. A large number of linking groups and methods
for linking are taught in the literature. Broadly, the labels can
be divided into the following categories: chromogens; catalyzed
reactions; chemiluminescence; radioactive labels; and
colloidal-sized colored particles. The chromogens include compounds
which absorb light in a distinctive range so that a color is
observed, or emit light when irradiated with light of a particular
wavelength or wavelength range, e.g., fluorescers. Both enzymatic
and nonenzymatic catalysts can be employed. In choosing a
proteasome substrate, there will be many considerations including
the stability of the substrate. A chemiluminescent label involves a
compound that becomes electronically excited by a chemical reaction
and emits light that serves as a detectable signal or donates
energy to a fluorescent acceptor. Radioactive labels include
various radioisotopes found in common use such as the unstable
forms of hydrogen, iodine, phosphorus, sulphur, or the like.
Colloidal-sized colored particles involve material such as
colloidal gold that, in aggregate, form a visually detectable
distinctive spot corresponding to the site of a substance to be
detected. Additional information on labeling technology is
disclosed, for example, in U.S. Pat. No. 4,366,241.
[0070] Once the level or activity of Ub/proteasome pathway or
ancillary protein or nucleic acid encoding such protein is detected
or measured in the test milk sample, it is compared to the level or
activity of Ub/proteasome pathway or ancillary protein or nucleic
acid encoding such protein in one or more controls in order to
determine whether the subject from which the sample was obtained
has mastitis.
[0071] As exemplified herein, a control can be a milk sample
obtained from a healthy cow. By using one or more control samples
(e.g., in a panel), the skilled technician can compare the level of
proteasome activity, or abundance of ancillary proteins in an
immunological or immunohistochemical assay, or absolute level of
expression in an ELISA for a semi-quantitative or quantitative
result. Although the present methodology emphasizes the
characterization of proteasome activity as a key measure of
infection, measuring the abundance of the proteasome and ancillary
components, and/or the levels of specific mRNAs or DNA, also serves
as useful indicators of mastitis.
[0072] Interaction with Organic Copper Complexes. The abundance of
the proteasome can be determined by examining its interaction with
organic copper complexes (including NCI-109268 and
bis-8-hydroxyquinoline copper(II) [Cu(8-OHQ).sub.2], and
5,7-dichloro-8-hydroxyquinoline-copper(II)). These reagents inhibit
the chymotryptic activity of the proteasome, and generate a green
stain upon interaction with the proteasome. The ability of specific
proteasome subunits and/or regulatory factors to bind copper can be
used in the instant detection system to measure proteasome
levels.
[0073] A variety of copper complexes can bind and inhibit the
proteasome. Furthermore, the interaction results in a green stain
that can be detected. Copper-binding compounds have been reported
as proteasome inhibitors and apoptosis inducers in human cancer.
Several copper-binding compounds have been reported to
spontaneously complex with copper and form active proteasome
inhibitors and apoptosis inducers. For example, compounds in the
quinoline and dithiocarbamate families may bind with copper and
inhibit the proteasome activity. Compounds, such as clioquinol and
pyrrolidinedithiocarbamate as examples, form complexes with copper
and bind to proteasomes.
[0074] Detection of Proteasome Regulatory Factors. The proteasome
can be rapidly purified, for example, as disclosed above. For
example, U.S. Pat. No. 6,294,363 provides for one-step, rapid
purification of proteasomes. The presence of key regulatory factors
can be established using immunologic methods, such as antibodies.
The presence of these factors in raw milk provides a key indicator
for the presence of proteasomes, and therefore a gauge of the level
of inflammatory cells in the milk. As noted above, this provides a
way to assess the level of infection, which is strongly related to
the level of pathogen/infection.
[0075] Detection of Proteasome Bound Factors. An important, and
additional application of the instant methods relates to the fact
that some proteasome-bound factors have well-described biochemical
properties. It has been shown that ubiquitin-conjugating enzymes
(Ubc4, Ubc5), and translation elongation factor (eEF1A), can be
detected in association with the proteasome. In addition, the
association of ubiquitin-E3 ligases (Ubr1, Scf), de-glycosylases
(Png1) and kinases (Cdc2) with the proteasome has been described.
Proteasomes have also been reported to have RNase activity.
Notably, all these factors have well-described biochemical
properties that can be readily measured. Therefore, measuring these
and other biochemical activities in purified proteasomes provides a
method for detecting the levels of proteasomes, and the likelihood
of mastitis.
[0076] For example, Gautier-Bert, et al. ((2003) Mol. Biol. Rep.
30:1-7) refer to and report partially reconstituted 20S
proteasome/RNA complexes using oligonucleotides corresponding to
ARE (adenosine- and uridine-rich element) (AUUUA).sub.4 (SEQ ID
NO:5) and HIV-TAR (human immunodeficiency virus-Tat transactivation
response element), a stem-loop structure in the 5' UTR
(untranslated region) of HIV-mRNAs. RNAs which associate with
proteasomes are degraded by proteasomal endonuclease activity. The
formation of these 20S proteasome/RNA substrate complexes is rather
specific since 20S proteasomes do not interfere with truncated TAR
that is not cleaved by proteasomal endonuclease. In addition,
affinity of proteasomes for (AUUUA).sub.4 (SEQ ID NO:5) is much
stronger than it is for HIV-TAR.
[0077] In addition, Jarrousse, et al. ((1999) J. Biol. Chem.
274:5925-5930) refer to possible involvement of proteasomes
(Prosomes) in mRNA decay and report on a cellular target for
proteasomal endonuclease activity, indicating that 20S proteasomes
interact with the 3'-untranslated region of certain cytoplasmic
mRNAs in vivo, and 20S proteasomes isolated from Friend leukemia
virus-infected mouse spleen cells associate with an mRNA fragment
showing great homology to the 3'-untranslated region of tumor
necrosis factor .beta. (TNF.beta.) mRNA that contains AUUUA
sequences. Destabilization of oligoribonucleotides corresponding to
the 3'-untranslated region of TNR.beta. by 20S proteasomes and the
creation a specific cleavage pattern are reported. The cleavage
reaction is accelerated with increasing number of AUUUA motifs, and
major cleavage sites are localized at the 5' side of the A
residues.
[0078] Detectably labeled RNA substrates can be used in assays to
detect proteasome RNase activity in a milk sample. The level of
RNase activity relative to the level of RNase activity in milk from
an uninfected animal and/or infected animal indicates whether the
animal is infected or not.
[0079] Proteasome associated Peptide N-glycanase (PNGase;
glycoamidase; N-glycanase) activity can also be measured as an
indication of proteasome levels. Png1 is a deglycosylating enzyme
that removes asparagine-linked (N-linked) glycans from
glycoproteins/glycopeptides that have been targeted for degradation
by the proteasome. The interaction between Pngl and the
proteasome-docking (shuttle) factor Rad23 is significant, because
this interaction promotes the localization of PNGase activity with
the proteasome. Suzuki, et al. ((2002) FASEB J. 16:635-641) refer
to the occurrence, primary structure, and potential functions of
cytoplasmic peptide:N-glycanase (PNGase) in eykaryotic cells.
Suzuki & Lennarz ((2003) Biochem. Biophys. Res. Commun.
302:1-5) hypothesize that a glycoprotein-degradation complex is
formed by protein-protein interaction involves cytoplasmic
peptide:N-glycanase.
[0080] Assays measuring removal of asparagine-linked (N-linked)
glycans from glycoprotein/glycopeptides substrates by Pngl are
useful in measuring proteasome levels in a sample. In such assays,
the substrate includes a detectable label that is either detectable
before processing by Pngl but not after, or not detectable before
processing by Pngl but detectable after the enzyme reaction.
[0081] Proteasome-associated lipase activity may also be used as an
enzyme-based assay which correlates to proteasome levels. In this
respect, Ohsak, et al. ((2006) MBC 17:2674-2683) refer to
cytoplasmic lipid droplets as being sites of convergence of
proteasomal and autophagic degradation of Apolipoprotein B. Lipid
esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes
are reported to be used to synthesize very low-density lipoproteins
(VLDLs), into which apolipoprotein B (ApoB) is integrated
cotranslationally. Using Huh7 cells derived from human hepatoma and
competent for VLDL secretion, ApoB was found to be is highly
concentrated around CLDs to make "ApoB-crescents." ApoB-crescents
were reportedly seen in <10% of Huh7 cells under normal
conditions, but the ratio increased to nearly 50% after 12 hours of
proteasomal inhibition by
N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy
reportedly showed ApoB to be localized to a cluster of
electron-lucent particles 50-100 nm in diameter adhering to CLDs.
ApoB, proteasome subunits, and ubiquitinated proteins were detected
in the CLD fraction, and this ApoB was ubiquitinated. Proteasome
inhibition reportedly caused increases in autophagic vacuoles and
ApoB in lysosomes in which ApoB-crescents began to decrease after
12-24 hours of proteasomal inhibition, but the decrease was blocked
by an autophagy inhibitor, 3-methyladenine inhibition of autophagy
alone reportedly caused an increase in ApoB-crescents. Proteasomal
and autophagy/lysosomal degradation of ApoB may occur around CLDs
and that the CLD surface functions as a unique platform for
convergence of the two pathways.
[0082] Accordingly, detectably labeled lipase substrates can be
used to determine proteasome-associated lipase activity as an
indication of proteasome levels in a sample. The reagents referred
to herein may be adapted to detectably label lipase substrates.
Enzyme activity can be measured and used as an indicator of
proteasome levels.
[0083] The invention is described in greater detail by the
following non-limiting examples.
EXAMPLE 1
Application of Proteasome Assays to Detecting Mastitis
[0084] The methods and systems provided are particularly useful for
the early detection of mastitis and monitoring or treatment for
various milk producing animals such as the dairy cow. However, the
methods are broadly applicable to other organisms, including human,
and to other conditions such as malignant growth and pathology. The
method involves detecting the abundance and activity of the
ubiquitin/proteasome pathway in a sample of unprocessed (raw) milk,
wherein an increase in proteasome activity in the sample, as
compared to a control, is indicative of mastitis. The invention
also includes detecting the level of ancillary proteins of the
ubiquitin/proteasome pathway and of the immunoproteasome. In a
different embodiment of this invention the mRNA expression of
components of the ubiquitin/proteasome pathway are measured using
variations of polymerase chain reaction-based amplification.
Oligonucleotide-based detection of released nuclear DNA from
somatic cells is also envisioned. There has been no prior
description of a measurement of the activity of the
ubiquitin/proteasome pathway in milk or utility of such an approach
for detecting mastitis.
[0085] A kit that will permit early detection, routine monitoring
of milk quality, and surveillance of animals that are recovering
following illness is also embraced by this invention. The ability
to monitor milk quality and animal health can have a direct
positive effect on animal welfare and husbandry. Furthermore, a
diagnostic kit developed from the invention will be applied on-site
at milking facilities. While this technology and methodology can
monitor milk quality and animal welfare, its applicability is not
restricted to the dairy industry. The invention is highly novel,
and broadly applicable to all mammals that are of commercial
interest and personal value. For instance, other farm animals
(including goat, sheep and horse), and animals of non-commercial
value (pets) can also suffer from mastitis. Mastitis is a medical
concern in the lactating woman, and the invention can be readily
adapted to the nursing mother. The applicability of this invention
was verified for monitoring the quality of dairy milk, and
following the drug response of infected dairy cows. The invention
provides a mechanism to monitor efficacy of drug regimen, and
recovery of the animal.
[0086] Mastitis is an inflammatory response, which can cause the
release of somatic cells into the milk, possibly through lesions in
the lining of the udder. Because systemic inflammation involves
activation of white blood cells, it was envisioned that a
significant fraction of the somatic cells in the milk represent cow
white blood cells. In earlier studies proteasome activity had been
detected in blood cells. However, detection of white blood cells or
proteasome activity in raw whole milk has not been envisaged or
demonstrated by direct or indirect experiments. Also, prior to the
instant invention, it was not clear whether the milk could be used
in its native raw state or if it needed to be purified to remove
other interfering components so that a reliable and accurate
determination of the proteasome and related assays could be carried
out. It was recognized that milk contains high levels of lipid
micelles, and proteins including casein. High sensitive enzymatic
assays for analyzing raw milk have not been described, since the
turbidity caused by scattering of light by the colloidal particles
in the milk emulsion precludes most biochemical measurements.
Therefore, it was determined whether proteasome activity could be
detectable in bulk milk. The experimental findings herein
demonstrate that proteasome activity can be measured in whole raw
milk, without a prior need for purifying somatic cells.
[0087] In contrast, performing a somatic cell count (SCC) requires
specialized instruments, and cannot be performed by straightforward
microscopy, because turbidity and light scattering by the milk
suspension interferes with the visual inspection of the somatic
cells. Furthermore, large aggregates of casein and lipid vesicles
prevent clear visualization of the somatic cells. Consequently, the
SCC is determined by external laboratories and the dairy farm is
not informed of the results for several weeks or months.
[0088] This delay provides no benefit to the dairy farmer for
monitoring, identifying, quarantining and restoring an infected
animal to the milk line. Moreover, the presence of a single
infected animal greatly increases the probability of
cross-contamination and infection of other animals. Note that the
same vacuum tubes are used for collecting milk from many cows, and
inadequate treatment with disinfectant can contaminate the next cow
that is tethered to the lines.
[0089] In the experiments conducted, high levels of a proteasome
variant, called the `Immunoproteasome,` were detected in
mastitis-affected milk. This form of the proteasome contains
distinct subunits, and is specifically required for the immune
response, consistent with the inflammation that accompanies
mastitis.
[0090] Affinity purification and immunological experiments were
also carried out to define the abundance and activity of various
proteasome isoforms. It was determined that the higher proteasome
activity in mastitis milk was due to increased expression of
proteasomes, and proteasome-isoforms. As noted above, high levels
of the `immunoproteasome` were detected. This finding provides a
powerful means to monitor the onset, progression and remission of
inflammation, using a straightforward antibody-based assay, which
will complement the activity measurement.
[0091] In total, the detection of proteasome level may be achieved
by measuring both proteasome activity and abundance. The 26S
proteasome and its component 19S and 20S particles can be detected.
The `immunoproteasome` and the associated PA28/13S complex, and
novel 20S catalytic particle can also be the subject of a test
assay. Regulatory factors that form transient interactions with the
proteasome including, but not restricted to, eEF1A, Hsp70, E2 and
E3 enzymes, Sts1, Centrin and the family of UbL/UBA shuttle factors
(e.g., Rad23, Dsk2, Dd11), and multi-ubiquitinated proteins that
are degraded by the proteasome can also be used in such assays.
[0092] These assays provide measurements for mastitis that are both
sensitive and quantitative. Moreover, these assays can be performed
in the field and can be provided in a simple kit format. These key
advances address the critical deficiencies of the current methods,
because the instant invention detects inflammation before physical
manifestation of mastitis is observed, and the assay can be
performed on-site. Therefore, an early detection method is
envisioned that overcomes the deficiencies in the current methods.
In dairy farms, milk from a large number of cows is collected in a
common reservoir. It is therefore critical to detect infected
animals and to remove them from the milk line, before the quality
of the milk in the common reservoir has deteriorated. This is not
possible with either SCC or the CMT assay. CMT is non-quantitative,
and SCC results are provided weeks after sampling. In contrast, the
present invention can detect infected milk originating from fewer
than 1/100 infected animals, and the results can be provided in
minutes. Thus, the presence of a single infected animal can be
initially detected by testing the bulk reservoir. Based on the
outcome of the test, the offending animal can be identified,
withdrawn and treated. The outstanding sensitivity of the instant
assay stands in contrast to the high SCC level that is permitted in
unprocessed whole milk (Maximum about 750,000 SCC/ml; typical bulk
reservoir averages >200,000 SCC/ml, whereas high quality farms
may achieve levels below about 40,000 SCC/ml). Thus, the instant
invention provides extraordinary sensitivity, wherein, high
proteasome activity in milk, caused by a single infected animal,
can be readily detected in the bulk reservoir. Once the affected
animal is identified, the invention allows quantitatively
monitoring treatment and recovery, and assisting the farmer in
returning the cow to the milk line.
[0093] While some embodiments embrace detecting the level of
Ub/proteasome pathway or ancillary protein, other embodiments
embrace detecting the activity of such proteins. As exemplified
herein, an increase in Ub/proteasome pathway proteins is correlated
with an increase in proteasome activity. Accordingly, proteasome
activity, e.g., as determined using a substrate such as
Suc-LLVY-Luciferin (SEQ ID NO:2) or detection of multi-ubiquinated
proteins, can also be used for pre-clinical detection of
mastitis.
EXAMPLE 2
Proteasome Activity in Raw Milk
[0094] Raw milk samples were obtained from a local farm once a week
for 16 consecutive weeks. Samples (approximately 30 ml) were
collected from the daily first milking (about 5:00 am) and then
stored on ice. All samples were collected by 8:30 am, and processed
for proteasome activity by 10:30 am. Proteasome activity was
determined at 37.degree. C. with the fluorogenic substrate
Suc-LLVY-AMC (SEQ ID NO:2), and quantified using a TURNER 7000
fluorometer. Overall protein concentration was highly uniform, and
reflected the very high levels of casein and other abundant milk
proteins. Therefore, a volume-based assay was most reliable. The
results of this analysis are presented in FIG. 1.
[0095] In the first set of seven columns of FIG. 1, the level of
proteasome activity was measured in triplicate from samples taken
from the bulk reservoir (maintained at 10.degree. C.). Milk in this
reservoir represented the collective output from approximately 50
cows. Typically, the reservoir contained the output of several days
of milking. Note the very low levels of proteasome activity in the
bulk reservoir (BULK), and relatively consistent values in seven
independent samplings. A specific young healthy female (named
Sunshine) was sampled twice, on independent occasions, and low
proteasome activity was detected. Another individual cow (Dale) was
examined one day after calving and it showed high activity that was
restored to lower levels over the next two weeks. It is well known
that calving results in high SCC levels in the milk. However, this
is a transient phenomenon that can be recapitulated. Two
independent cows, suffering from mastitis, were examined.
[0096] COW1 had very high levels that required antibiotic
treatment. Following a single treatment regimen, proteasome
activity was examined and a precipitous drop to near normal levels
was detected. However, treatment was insufficient and proteasome
activity increased dramatically over the course of the next week.
Treatment was restored, and lower proteasome activity was observed.
Note that baseline levels remained elevated. It was significant
that failure to monitor treatment response in a quantitative manner
results in inadequate treatment, premature return to the milk line,
degradation of bulk tank quality, and recurrence of the infection.
Neither the SCC nor the CMT assays currently in use can provide
these key measurements. In contrast, the present invention permits
rapid, real-time quantitative analysis of milk quality and animal
health.
[0097] In COW2, rapid onset of mastitis, following injury of one
teat, required antibiotic treatment. Proteasome activity in this
animal was followed over the next two weeks and a dramatic decline
was observed as the injury/infection receded and the animal was
returned to the milk line. Note that baseline levels were elevated
compared to Sunshine, and unaffected bulk reservoir levels.
[0098] COW3 was termed a `chronic offender` by the farmer. This
animal was examined over the course of several weeks, and as noted
by the dairy farmer, this individual had chronic elevated
proteasome activity that fluctuated quite widely. This would
represent a highly troublesome animal that would need constant
monitoring.
[0099] COW4 was under watch by the farmer, as an animal with
potential pre-clinical mastitis. As suspected, elevated levels of
proteasome activity were detected, which were restored to the
normal range without treatment. These two samples (COW3 and COW4)
illustrate the significant difficult of relying on subjective
criterion for identifying troublesome animals. The present
invention provides rapid and quantitative assessment of milk
quality and animal health, and removes intuition and guess-work
from the decision.
[0100] To determine proteasome levels, raw milk samples were
separated in SDS-polyacrylamide gels, and the separated proteins
were transferred to nitrocellulose. The filters were treated with
antibodies against the proteasome subunits Rpn2 and Rpt1, as well
as Ub (ubiquitin). Milk samples from the Bulk Reservoir, and Cow1,
Cow2, Cow3 and Cow4 were analyzed. The samples were acquired on the
dates indicated (for example; S19=Sep. 16, 2006). Note that low
levels of proteasome subunits and ubiquitin were detected in the
Bulk Reservoir, consistent with low proteasome activity (see FIG.
2A). In contrast, Cow1 and Cow2 had significantly elevated amounts
of both proteasome subunits and high molecular weight
multi-Ubiquitinated proteins, consistent with dramatic increase in
proteasome activity (FIG. 2A). The levels of proteasome subunits
and ubiquitin cross-reacting material were elevated in Cow3 and
Cow4, but not to the same degree as Cow1 and Cow2, again in
agreement with the activity measurements (FIG. 2A).
[0101] To further verify these results, proteasomes were purified
from raw milk using a UbL affinity matrix. Highly purified
proteasomes were separated in SDS-polyacrylamide gels and examined
by immunoblotting. Consistent with the previous panel, higher
proteasome levels were detected in Cow1 and Cow2, with a more
moderate increase in Cow3 and Cow4, entirely consistent with the
activity measurements (FIG. 2B).
[0102] In an independent measurement of proteasome abundance, the
levels of the Rpt1 proteasome subunit in raw milk were retested on
specific dates for a number of candidate animals. As shown in FIG.
3A, a sampling from Oct. 9, 2006, when Cow1 and Cow2 showed
dramatically elevated proteasome activity, detected a corresponding
elevation in the levels of the Rpt1 proteasome subunit. In
contrast, Rpt1 was not detected in Bulk tank, Dale, SunShine, or
Cow3 and Cow4. Note that on October 9, both Cow3 and Cow4 had low
proteasome activity.
[0103] The same immunofilters were reacted with antibodies against
the immunoproteasome subunit PA28a. High levels were observed in
Cow1 and Cow2 (lanes 5 and 7), consistent with an immune response
that occurs during the inflammatory response following infection
and injury.
[0104] To confirm that intact proteasomes were present in mastitis
milk, a 10-fold higher amount of raw milk was applied to UbL matrix
to purify proteasomes. Immunoblots were examined and Rpt1 was
detected in Cow1 and Cow2 (FIG. 3B). Lower amounts were observed in
Cow3, and very low levels were detected in Cow4. In contrast, Rpt1
was not detected in Bulk tank, Dale or SunShine.
[0105] A careful assessment of an individual cow, chronically
infected with mastitis was followed over the course of
approximately 4 weeks. Proteasome activity was compared to the
averaged value from the bulk tank, taken at each time point (FIG.
4). Note that the average value was units of proteasome activity.
The activity in the infected animal ranged 100-fold, from 21 to
2156. Significantly, the animal responded efficiently to treatment
with antibiotic (reduced from 2156 to 21), but significantly higher
levels were detected within one week following termination of
treatment.
EXAMPLE 3
Assays for Determining Proteasome Activity
[0106] To monitor chymotryptic, tryptic and caspase-like activities
as an indication of proteasome activity, labeled substrates
specific for one or more proteases can be used. To determined
proteasome subunit abundance, high-affinity immunologic methods
were used. To detect de-ubiquitylation activity, the levels of
ubiquitin, multi-ubiquitin, and ubiquitinated substrates can be
determined using immunologic methods. Alternatively, the assembly
of catalytically active 26S proteasomes, from its component 19S and
20S particles, can be measured. The level of the `Immunoproteasome`
and the associated PA28/13S complex, and novel 20S catalytic
particle, can be determined using immunologic methods.
EXAMPLE 4
Use of a Fluorogenic Substrate to Determine Proteasome
(Chymotryptic) Activity
[0107] Raw milk (50 .mu.l) is suspended in 500 .mu.l lysis buffer
(50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM EDTA and 1% TRITON X-100),
lysed by sonication, and total protein quantified using the
Bradford assay (BIORAD). Protein extracts contain a protease
inhibitor cocktail (Roche), to prevent non-proteasomal proteolysis.
Protein extracts (20 .mu.g) are characterized in triplicate in a
96-well microtiter plate. Reactions are incubated for 5 minutes at
room temperature, in the presence or absence of the proteasome
inhibitors epoxomicin and lactacystin. Proteasome activity is
determined by examining the hydrolysis of SUC-LLVY-AMC (SEQ ID
NO:2) (Boston Biochem), in 25 mM HEPES pH 7.5, 0.5 mM EDTA. Each
reaction, containing 40 .mu.M of SUC-LLVY-AMC (SEQ ID NO:2), is
incubated for 1 hour at 30.degree. C., and the fluorescence signal
quantified over time using a TECAN INFINITE F200 plate reader.
Proteasome activity is calculated after subtracting non-specific
activity that is insensitive epoxomicin-sensitive hydrolysis.
Steady-state and kinetic measurements are obtained.
EXAMPLE 5
Peptide Substrates
[0108] The P4 position from the cleavage site is important for the
ability of the proteasome hydrolytic activity to recognize a
substrate. This implied that there is a length constraint that must
be met to generate an ideal proteasome substrate. Many different
residues can be tolerated at the P4 position, although differences
in cleavage efficiency can be detected. Proteasome substrates
include the peptide-based substrate Suc-LLVY-AMC (SEQ ID NO:2), and
the eight-residue long Suc-RPFHLLVY-AMC (SEQ ID NO:1). The
amino-terminus is generally blocked using succinimide (Suc),
acetate (Ac), benzoyl (Bz), BOC (tert-butyloxycarbonyl),
carboxybenzyl (Z), vinyl and phenyl groups. Other peptide
substrates are listed in Table 1.
TABLE-US-00001 TABLE 1 Activity Substrate SEQ ID NO: Caspase-like
Ac-nLPnLD 6 Trypsin-like Ac-RLR Caspase-like Ac-GPLD 7 Trypsin-like
Boc-LRR Trypsin-like Bz-VGR Chymotrypsin-like Suc-RPFHLLVY 8
Chymotrypsin-like Z-GGL Caspase-like Z-LLE Chymotrypsin-like
Suc-LLVY 9
[0109] These substrates can be conjugated (e.g., at the C-terminus)
with a detectable label including, but not limited to, a
fluorogenic label, luminescent label or a chromogenic label.
EXAMPLE 6
Luminescent Assay for Detecting Proteasome Activity
[0110] In addition to the commonly used fluorogenic assay using,
e.g., AMC, a luminescent assay using the firefly luciferase enzyme
is also available using commercially available materials and
well-known techniques. Examples of luminescent assays include the
reaction set forth in FIG. 5. In this assay, cleavage of
LLVY-Luciferin by the proteasome releases Luciferin. In the
presence of luciferase, a catalytic reaction, requiring ATP,
releases a photon of light, the signal of which is detectable with
a hand-held luminometer. As with a fluorogenic assay, this assay
for proteasome activity is sensitive, linear, and quantitative; has
a wide dynamic range; and has a low signal background.
[0111] Protocol for Measuring Proteasome Activity in Raw Milk Using
a Luminescent Readout. A sample of milk (0.2-0.5 ml) is placed in a
clear UV-light transmissible tube. A defined quantity of the
chymotryptic substrate, Suc-Leu-Leu-Val-Tyr-Luciferin (SEQ ID NO:2)
is added. A defined quantity of the enzyme luciferase is added to
generate a bioluminescent readout. In particular embodiments, a
genetically modified Photuris pennsylvanica luciferase enzyme
(LucPpe2.sup.m) is employed, which has improved thermostability
ranging from 22.degree. C. to 60.degree. C., and catalyzes cleavage
over a longer period, allowing much more accurate measurements to
be obtained (see U.S. Pat. Nos. 6,602,677 and 7,241,584,
incorporated herein by reference). In this respect, the assay can
be conducted over a broad dynamic range (>5-orders of
magnitude), and in a convenient time-frame (1-10 minute duration).
Moreover, the use of a genetically modified Luciferase is an
improvement over existing technology for using bioluminescence
measurements, because the release of light is significantly more
controlled, and does not cause the rapid burst in photon release as
observed with native bioluminescence generating enzymes.
[0112] Cleavage of Suc-LLVY-Luciferin (SEQ ID NO:2) by the
proteasome, releases free Luciferin. In a reaction containing
Luciferin and ATP (already present in raw milk), Luciferin
generates light. The release of light is detected by a luminometer.
The reactions are performed at ambient temperature. Experimental
evidence shows approximately a 2-fold increase in signal from
15.degree. C..fwdarw.37.degree. C., within the typical range of
dairy farm housing of cows. The resulting signal is highly
reproducible, rapid, quantitative, and is a function of proteasome
abundance in the raw milk and the availability of free ATP; both
are strong indicators of somatic cell levels.
[0113] Proteasome Activity in Raw Milk. It has been shown that
there is strong correlation between ATP levels and somatic cells,
as well as proteasome activity and somatic cells. Somatic cell
counts ranging from <20,000/ml to greater than 1.times.107/ml
were examined, and corresponding proteasome activity was detected.
At low SCC levels, luminescent signal is detected within 10
minutes. However, at high SCC levels (>1.times.106/ml), signal
can be detected in less than 30 seconds.
[0114] SCC values in the raw milk samples were obtained at a remote
testing facility. The same milk samples were used to generate both
SCC and proteasome activity measurements. Samples representative of
pre-clinical mastitis showed activity in 1-3 minutes. SCC
measurements in these samples ranged from 200,000-400,000/ml.
[0115] Kit for Determining Proteasome Activity in Raw Milk. The
application of a single-use disposable measuring device was
validated. An applicator stick deposits 0.1 ml of raw milk into a
tube. The end of the applicator stick is attached to a small
balloon that contains pre-loaded chemistry. This chemistry is
released by twisting the balloon to break a seal, which combines
with the raw milk. The tube is agitated gently to mix the reagents,
and then placed in a handheld luminometer. Readings are generated
within 15 seconds. These assays were validated with >1,200
individual raw milk samples. The proteasome assay kit is fully
compatible with a rapid cow-side diagnostic.
EXAMPLE 7
Non-Fluorogenic Substrates for Detecting Proteasome Activity
[0116] A set of approximately 12 non-fluorogenic substrates that
required at least two different targeting pathways were described
by Bachmair & Varshaysky ((1989) Cell 56:1019-1032). These
reporter substrates contained the E. coli beta-galactosidase
enzyme, and their degradation could be demonstrated in bacteria,
human and metazoans (including human cultured cells). More recent
derivatives include degradation substrates based on
glutathione-S-transferase, and green fluorescence protein.
Additionally, there are by now dozens, if not hundreds, of
physiological substrates of the Ub/proteasome pathway. Access to
the interior of the proteasome (which contains the three catalytic
activities) is restricted by a narrow (13 angstrom) translocation
channel.
EXAMPLE 8
Use of Antibodies to Measure Proteasome Abundance
[0117] Protein extract prepared from whole milk is incubated with
GST-UbL SEPHAROSE (CELLXPLORE) to isolate catalytically active
proteasomes. The UbL protein domain (isolated from human Rad23
proteins), forms a high-affinity interaction with catalytically
active proteasomes. This efficient affinity matrix provides the
only known way to isolate functional proteasomes within about 15
minutes. The activities of the proteasome can be determined while
bound to the UbL matrix. The activity of the purified proteasomes
is tested as described above. The beads are washed 3 times with 1
ml lysis buffer containing 1% TRITON X-100. Proteasome hydrolytic
activity is measured on the immobilized proteaseome. The
immobilized proteins are separated in 10% SDS-tricine
polyacrylamide gels, transferred to nitrocellulose, and antibodies
used to detect the levels of Rpt1 and Rpn1 in the 19S regulatory
particle, and the alpha-7 subunit in the 20S catalytic
particle.
EXAMPLE 9
Antibodies Against Proteasomal Subunits
[0118] Antibodies against specific subunits are available
commercially. By way of illustration, the antibodies listed in
Table 2 are available from BIOMOL.
TABLE-US-00002 TABLE 2 Antibody Type Species Reactivity Antigen
(clone) H Y Misc. WB IP IHC Rpn2 mAb (112-1) X X X Rpn5 Rabbit pAb
AT X Rnp6 Rabbit pAb AT, CF X Rpn7 Rabbit pAb X X X Rpn8 Sheep pAb
X X X Rpn10 mAb (S5a-18) X X X X Rpn11 Rabbit pAb X X Rpn12 Rabbit
pAb X X X mAb (p31-27) X X X X mAb (31-38) X X X Rabbit pAb AT X H,
human; Y, yeast; AT, Arabidopsis thaliana; CF, cauliflower; WB,
western blot; IP, immunoprecipitation; IHC,
immunohistochemistry.
[0119] Antibodies that may be used in an immunoprecipitation (IP)
reaction are expected to recognize the intact, functionally active
proteasome. Note that Rpn10 and Rpn12 could both be used.
Similarly, numerous alpha- and beta-subunits in the 20S catalytic
particle may be targeted with antibodies for immunoprecipitation
reactions. These antibodies can also be used in ELISA-based
assays.
[0120] Alternatively, antibodies can be generated against one or
more subunits of the proteaosome and used in the assays herein. In
this respect, two human proteasome subunits were cloned and
purified and monoclonal against these subunits (Rpn8 and Alpha-6)
were generated. Approximately 12-15 hybridomas were generated for
each protein and cell lines expressing productive antibodies
against the relevant proteasome subunits were identified. To
facilitate their use in the instant assays, the hybridomas are
screened to identify antibodies that recognize distinct parts in
the target protein. A pair of non-overlapping antibodies is
identified; one to affinity-purify the proteasome from raw milk and
the second for use in determining the amount of proteasome that was
recovered. Alternatively, antibodies against two different
proteasome subunits are used to immunoprecipitate the proteasome
and to detect the level that was recovered. Because antibodies were
generated against two different proteins, multiple assays can be
developed to produce one or more kits for detecting and evaluating
the severity of mastitis. Advantageously, the use of an
antibody-based assay is economical, and no instrumentation is
required.
EXAMPLE 10
Test-Strip for Antibody-Based Assay
[0121] A kit containing a solid support or test-strip can contain
different amounts of an antibody that recognizes the proteasome.
Microfluidics, lateral flow devices and in-line (real-time)
sensors, are all amenable to this approach. Exposure of the strip
to milk containing proteasomes (as in mastitis) results in binding
to the immobilized antibody. The proteasome is then detected using
a second antibody that recognizes the proteasome. However, the
second antibody can be coupled to a label that permits detection.
For example, conjugation of the second antibody to horse radish
peroxidase (HRP) can be used to generate a colorimetric or
chromogenic signal. However, the antibody could be conjugated to
any enzyme whose activity can be measured. Alternatively, the
second antibody can be coupled to a magnetic bead that can be
detected by incubation with metal particles coated with FITC (for a
fluorescence readout). Still further, the second antibody can be
coupled to biotin, and the reaction developed using strepavidin
conjugated to FITC or other easily detected fluorescence
markers.
[0122] More specifically, a strip of nitrocellulose can be
supported on a plastic matrix to provide stability and ease of use.
A first antibody can be precisely deposited using simple robotic
liquid handling devices. The filter is then blocked to prevent
nonspecific interaction between proteins in the milk and the
filter. The filter is then immersed in the milk sample to be
tested. Incubation for a fixed duration is allowed to occur, e.g.,
15 minutes, the filter is rinsed to remove milk, and a second
antibody is incubated with the test strip to detect the presence of
proteasomes. In one embodiment, the first and second antibodies
bind to the same subunit, but to different epitopes. In this
respect, the two antibodies would recognize the same proteasome
subunit, but different regions in the three-dimensional structure.
To facilitate detection, the second antibody can be coupled to a
chromogenic substrate, biotin, or a reactive enzyme. The reaction
is then detected by hydrolysis of a substrate that deposits a
colored pigment, by binding of streptavidin that is conjugated to a
chromogenic reagent, or exploiting the action of the attached
enzyme to hydrolyze a substrate, respectively. Such reactions are
routinely carried out in the art.
[0123] Alternatively, serial dilutions of antibody against one
subunit of the proteasome can be deposited on a nitrocellulose
strip. The filter is then immersed in the milk sample to be tested
and incubated for a fixed duration, e.g., 15 minutes to capture
proteasomes. The filter is then rinsed to remove milk, and
incubated with an antibody that recognizes a different proteasome
subunit. Because proteasomes remain intact in the presence of ATP,
higher mastitis levels will be accompanied by more ATP in the milk,
and more stable proteasomes. Thus, intact proteasomes can be
recovered following the first antibody treatment, and detected
using the second antibody. The two antibodies can recognize the
same proteasome subunit, but different regions in the
three-dimensional structure. To facilitate detection, the second
antibody can be coupled to a chromogenic substrate, biotin, or a
reactive enzyme. The reaction is detected by hydrolysis of the
substrate that deposits a colored pigment, by binding streptavidin
that is bound to a chromogenic reagent, or by exploiting the action
of the enzyme to hydrolyze a substrate, respectively.
[0124] In yet another embodiment, whole (raw) milk could be
directly incubated with a test-strip containing a small patch (or
patches) of nitrocellulose, or other high-affinity protein binding
surfaces. The strip would then be incubated with an antibody
against a specific proteasome subunit, and detected as described
above.
EXAMLE 11
Test Samples
[0125] In some embodiments, the proteasome level is tested from
samples obtained from a bulk storage tank containing milk from a
plurality of animals. In other embodiments, the proteasome level is
tested from samples obtained from a sample of milk from a single
animal. Such samples may be tested as part of a routine testing
program, or when the dairyman suspects that the animal may have an
infection. In such cases, pre-clinical mastitis may be diagnosed
prior to the appearance of any symptoms of inflammation and
monitoring may be increased or the treatment of such animals may
begin. Samples of milk from a single animal may be tested in cases
where a diagnosis of mastitis has been previously made and
treatment undertaken. Testing informs the dairyman if the animals
condition has been resolved or if a low level infection persists.
This ensures that treatment can be discontinued and animals may be
prepared for return to the line as soon as they are ready but not
prematurely when the risk of relapse is great.
EXAMPLE 12
ATP-Based Assay
[0126] As indicated herein, the stability of the proteasome is
sensitive to ATP levels. Accordingly, ATP and ATPase activity are
of use in detecting and monitoring pre-clinical and clinical
mastitis. In accordance with this embodiment, the proteasome is
rapidly affinity purified using GST-UbL. Raw milk is incubated with
GST-UbL to isolate proteasomes. Excess milk is removed and the
amount of proteasomes is determined by measuring enzymatic
activity. The intact proteasome contains 12 subunits with ATPase
activity. Therefore, the level of proteasome can be ascertained by
measuring ATP hydrolysis by the purified complex. Purified
proteasomes can be incubated in buffer containing a
proteasome-specific substrate. These substrates can provide a
chromogenic, fluorogenic or luminescent readout that typically
monitor the release of ADP.
[0127] Alternatively, hydrolysis of ATP can deplete the amount
available for a luciferase/luciferin-based luminescent assay.
Accordingly, using conventional biochemical assays, it was
determined whether ATP levels were detectable in milk. This
analysis indicated that there was a substantial amount of ATP in
milk. Therefore, it was determined whether ATP levels could be used
as a surrogate for detecting mastitis. The assay proved to be
highly successful, and provided a degree of sensitivity vastly in
excess of current methods. As shown in FIG. 5, the enzyme
luciferase can hydrolyse the substrate luciferin in an
ATP-dependent reaction. The resulting reaction releases a photon of
light that can be detected with a luminometer. Using a hand-held
detector, the luminescent signal is readily detected and a lower
signal is generated with increasing amounts of proteasomes in
mastitis.
[0128] A kit for performing the assay can include a disposable
container and a liquid holding spatula for collecting the milk
sample. The milk sample is deposited into the plastic tube, and the
spatula is secured in place. A plastic bulb attached to the spatula
is twisted and squeezed to release the luciferase enzyme and
luciferin substrate. Once combined with the milk sample, ATP is
required for the reaction to proceed. The reaction is linear over
4-orders of magnitude, and the ATP levels are proportional to the
level of somatic cells in the milk. Thus, this assay provides a
very rapid (minutes) evaluation of milk quality.
[0129] Measuring either ATP levels or ATPase activity is sensitive,
linear, and quantitative; has a wide dynamic range; and has a low
background signal.
EXAMPLE 13
Ubiquitin-Like Domain Affinity Matrix
[0130] Ubiquitin-like (UbL) domains from Rad23 proteins are known
to bind the proteasome (Hiyama, et al. (1999) J. Biol. Chem.
274:28019-28025; Schauber, et al. (1998) Nature 391:715-718). The
UbL domains in two human Rad23 proteins form differential binding
to proteasomes (Chen & Madura (2006) FEBS Lett. 580:3401-3408).
To prepare UbL domain affinity matrices, UbL domains are expressed
in E. coli as fusions to glutathione S-transferase (GST) or
conjugated to beads. GST-UbL efficiently purifies catalytically
active proteasomes from tissue extracts. Furthermore, regulatory
factors that formed sub-stoichiometric interactions with the
proteasome are also detected. See, Chen & Madura (2002) Mol.
Cell Biol. 22:4902-4913; Chuang, et al. (2005) Mol. Cell Biol.
25:403-413; Leggett, et al. (2002) Mol. Cell 10:495-507; Verma, et
al. (2004) Cell 118:99-110.
Sequence CWU 1
1
1018PRTArtificial sequenceSynthetic peptide 1Arg Pro Phe His Leu
Leu Val Tyr1 524PRTArtificial sequenceSynthetic peptide 2Leu Leu
Val Tyr134PRTArtificial sequenceSynthetic peptide 3Gly Pro Leu
Asp144PRTArtificial sequenceSynthetic peptide 4Leu Pro Leu
Asp1520RNAArtificial sequenceSynthetic oligonucleotide 5auuuaauuua
auuuaauuua 2064PRTArtificial sequenceSynthetic peptide 6Leu Pro Leu
Asp174PRTArtificial sequenceSynthetic peptide 7Gly Pro Leu
Asp188PRTArtificial sequenceSynthetic peptide 8Arg Pro Phe His Leu
Leu Val Tyr1 594PRTArtificial sequenceSynthetic peptide. 9Leu Leu
Val Tyr1105PRTArtificial sequenceSynthetic peptide 10Arg Leu Arg
Gly Gly1 5
* * * * *