Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-09-01
2002-11-26
Chan, Christina Y. (Department: 1644)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C530S350000
Reexamination Certificate
active
06485955
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a novel cytoplasmic post-prolyl dipeptidase, and its role in regulating apoptosis, or programmed cell death (PCD).
Apoptosis is a physiological form of cell death, and it shapes a number of diverse biological processes, such as development and homeostasis. It occurs in response to diverse stimuli which fall into two categories. The first is activation induced cell death following specific stimulation; and the second is death by neglect after withdrawal of a life-promoting stimulation, such as a growth factor, serum or cell-cell contact. While these two types of PCD take place under very different circumstances, both depend on the activation of caspases, a family of cysteine proteases which are present in the cytoplasm of cells as inactive proenzymes.
Apoptotic stimuli lead to the activation of certain caspases by specific proteolytic cleavage, enabling them to activate other caspases through a proteolytic cascade, which eventually leads to cell death. Studies of activation-induced cell death through the Fas/TNF receptors have implicated the death effector domain containing protease FLICE (caspase 8) in the initiation of the caspase cascade. However, while most cells contain all the components of the apoptotic machinery, and are susceptible to PCD by neglect, no caspase-cascade regulator induced under these conditions, has been identified.
With respect to apoptosis in the nervous system, recent studies have shown that neuronal cell death resulting from degeneration or trauma occurs by the process of apoptosis, the biochemical hallmarks of which include cytolemmal membrane blebbing, cell soma shrinkage, chromatin condensation, and DNA laddering. Thus, given the role of apoptosis in neural degeneration, it would be highly desirable to have a process for identifying compounds that modulate this apoptosis. Such compounds could be effective as therapeutic agents in the treatment or prevention of neural conditions associated with inappropriate neural degeneration. However, one of the fundamental technological problems associated with the identification of these compounds is the lack of suitable targets to use in screening for regulators of apoptosis. Thus, there is a need to identify new enzymatic targets as a first step in developing new therapeutic compounds.
SUMMARY OF THE INVENTION
We have discovered a novel, cytoplasmic post-prolyl dipeptidase, which has similarities to, but is distinct from, the membrane-bound T-cell serine protease CD26. This enzyme is expressed in quiescent, non-cycling T-cells and in neurons, and is called quiescent cell post-prolyl dipeptidase (QPP; previously termed DPIVb).
In a first aspect, the invention features a substantially pure, recombinant quiescent cell post-prolyl dipeptidase polypeptide (QPP), wherein the polypeptide inhibits quiescent cells from undergoing apoptosis. Preferably, the polypeptide comprises the amino acid sequence of SEQ ID NO 6.
In a second and related aspect, the invention features a substantially pure nucleic acid molecule encoding a QPP polypeptide, wherein the nucleic acid sequence comprises SEQ ID NO 5, or a sequence that is complementary to a sequence that hybridizes to SEQ ID NO 5 under stringent hybridization conditions encoding a QPP polypeptide, or encodes a polypeptide of SEQ ID NO 6.
The invention also features a host cell, for example, yeast,
E. coli
, Chinese hamster ovary (CHO), or fibroblast, transformed or transfected with a transgene that includes a nucleic acid molecule encoding a QPP polypeptide, wherein the nucleic acid molecule is operably linked to a regulatory sequence, and wherein the nucleic acid sequence comprises SEQ ID NO 5, or a sequence that is complementary to a sequence that hybridizes to SEQ ID NO 5 under stringent hybridization conditions, or encodes a polypeptide of SEQ ID NO 6.
In a related aspect, the invention features a method of producing a QPP polypeptide including transforming or transfecting a cell with a nucleic acid molecule encoding a QPP polypeptide, wherein the nucleic acid molecule is operably linked to a regulatory sequence, and further wherein the nucleic acid sequence comprises SEQ ID NO 5, or a sequence that is complementary to a sequence that hybridizes to SEQ ID NO 5 under stringent hybridization conditions, or encodes a polypeptide of SEQ ID NO 6; culturing the cell under conditions for expressing the nucleic acid; and isolating the QPP polypeptide.
The purified QPP sequences of the invention have a number of utilities. The invention features methods for: 1) identifying a therapeutic candidate compound that modulates a QPP effect in quiescent cells, which includes contacting a recombinant QPP polypeptide, preferably, the QPP polypeptide of SEQ ID NO 6, or a transformed or transfected cell encoding and expressing a recombinant QPP polypeptide, with the compound and measuring QPP biological activity; 2) inhibiting apoptosis in a quiescent cell, which includes contacting the cell with a recombinant QPP polypeptide, preferably, the QPP polypeptide of SEQ ID NO 6, in an amount sufficient to protect the cell from death when an apoptosis stimulus is administered; and 3) treating a mammal for a condition related to quiescent cell death, that includes administering to the mammal a recombinant QPP polypeptide, preferably, the QPP polypeptide of SEQ ID NO 6, in an amount sufficient to inhibit the cell death. Preferably, the condition is a neurodegenerative disorder or an immune system disorder, and the QPP polypeptide may be administered by transplanting a host cell into the mammal, wherein the host cell is ex vivo transfected with a transgene comprising a nucleic acid molecule encoding a QPP polypeptide, wherein the nucleic acid molecule is operably linked to a regulatory sequence and is expressed in the transplanted host cell.
The recombinant QPP sequences can also be used to make antibodies (polyclonal, monoclonal, or recombinant) using conventional methods, involving immunization of, e.g., rabbits, mice, or human volunteers. The antibodies can be used in standard ELISA assays to measure QPP levels in patients being tested for diseases which potentially involve increased or decreased QPP levels; for example, HIV patients, who have lost QPP-containing T-cells, will exhibit decreased QPP levels, with the QPP concentration being diagnostic of the stage of the disease. Generally, because QPP is a cytoplasmic enzyme, the assay is carried out on peripheral blood lymphocyte samples which have first been treated to lyse T-cells to release the enzyme.
By “substantially pure” is meant a polypeptide or nucleic acid that has been separated from components which normally accompany it, or which accompany it upon recumbent expression. An enzyme is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably, at least 90%, and most preferably, at least 99%, pure QPP by weight. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “apoptosis” is meant the process of programmed cell death wherein a dying cell displays a set of well-characterized biochemical hallmarks which include cytolemmal membrane blebbing, cell soma shrinkage, chromatin condensation, and DNA laddering.
By “stringent hybridization conditions” is meant incubation conditions that destabilize mismatched heteroduplexes. Conditions for high stringency hybridization, such as for PCR, Northern, Southern, in situ hybridization, or DNA sequencing, are described, for example, in F. Ausubel et al.,
Current Protocols in Molecular Biology
, John Wiley & Sons, New York, N.Y., 1994.
By “transformed” or “transfected” cell is meant a cell in which foreign nucleic acid molecules have been introduced. Lipofection, DEAE-dextranmediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, retroviral delivery, electroporation,
Huber Brigitte T.
Underwood Robert H.
Chan Christina Y.
Huynh Phu N.
The Trustees of Tufts University
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