Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2002-03-20
2004-07-13
Hutson, Richard (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S219000, C435S212000, C435S252300, C435S320100, C435S325000, C435S410000, C435S006120, C435S348000, C435S254200, C536S023100, C536S023200, C536S023500
Reexamination Certificate
active
06762045
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to caspases and their role in apoptosis, and in particular, to nucleic acids encoding membrane derived caspase-3, the encoded polypeptides, antibodies thereto, and methods of producing and using membrane derived caspase-3 polypeptide.
BACKGROUND OF THE INVENTION
The normal physiological process of programmed cell death, also known as apoptosis, plays a critical role in the maintenance of tissue homeostasis. The apoptotic process in multicellular organisms ensures that the rate of new cell accumulation produced by cell division is offset by a commensurate rate of cell loss due to death. A typical result of apoptosis are certain morphological changes in a cell, including fragmentation of nuclear chromatin, compaction of cytoplasmic organelles, dilatation of the endoplasmic reticulum, decreased cell volume, and alterations in the plasma membrane. The end result of programmed cell death is phagocytosis of apoptotic cells and prevention of an inflammatory response. Disturbances in apoptosis that prevent or delay normal cell turnover can be just as important to the pathogenesis of diseases as are known abnormalities in the regulation of cell proliferation and the cell cycle. Similar to cell division, which is controlled by complex interactions between cell cycle regulatory proteins, apoptosis is regulated under normal circumstances by a complex network of gene products that interact to either induce or inhibit cell death.
The stimuli that regulate the function of these apoptotic gene products include both extracellular and intracellular signals. Either the presence or removal of a particular stimulus can be sufficient to evoke a positive or negative apoptotic signal. Physiological stimuli that inhibit or reduce the likelihood of apoptosis include, for example, growth factors, extracellular matrix, CD40 ligand, viral gene products, neutral amino acids, zinc, estrogen, and androgens. In contrast, stimuli that promote apoptosis include, for example, tumor necrosis factor (TNF), Fas, transforming growth factor &bgr; (TGF
&bgr;
), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment, intracellular calcium, and glucocorticoids. Other stimuli, including those of environmental and pathogenic origin, may also induce or inhibit apoptosis. Although diverse signals and complex interactions of cellular gene products mediate apoptosis, the results of these interactions ultimately lead into a cell death pathway that is evolutionarily conserved between humans and invertebrates.
Several gene families and products that modulate the apoptotic process have now been identified. One family is the Bcl-2 proteins, which can function to modulate apoptosis in a wide variety of cell systems (Oltvai and Korsmeyer,
Cell
79:189-192, 1994; Reed,
Nature
387:773-776, 1997). The over-expression of Bcl-2 has been shown to inhibit the activation of cytoplasmic caspases following apoptotic stimuli in several cell systems (Armstrong et al.,
J. Biol. Chem.
271:16850-16855, 1996; Chinnaiyan et al.,
J. Biol. Chem.
271:4573-4576, 1996; Boulakia et al.,
Oncogene
12:29-36, 1996; Srinivasan et al.,
J. Neurosci.
16:5654-60, 1996). Although Bcl-2 inhibits the onset of apoptosis, it does not impede already initiated apoptosis (McCarthy et al.,
J. Cell Biol.
136:215-217, 1997). Most Bcl-2 family members associate with cellular membranes, such as the mitochondrial outer membrane, the nuclear envelope, and the endoplasmic reticulum (Reed,
Nature
387:773-776, 1997; Krajewski et al.,
Cancer Res.
53:47014714, 1993; Yang et al.,
J. Cell. Biol.
128:1173-1184, 1995; Lithgow et al.,
Cell Growth Differ
3:411-417, 1994); however, it remains unclear how the membrane bound Bcl-2 exerts control over another key set of apoptosis regulators, the soluble cytoplasmic, aspartate-specific cysteine proteases (“caspases”).
The caspase family of cysteine proteases are essential effectors of the apoptotic process (Yuan et al.,
Cell
75:641-652, 1993; Alnemri et al.,
Cell
87:171, 1996; Cohen,
Biochem.
326:1-16, 1997; Miller,
Semin. Immunol
9:35-49, 1997; Salvesen and Dixit,
Cell
91:443-446, 1997). Caspases are synthesized as inactive zymogens, which are activated by proteolytic processing to yield large (~18 kDa) and small (~12 kDa) subunits that associate to form active enzymes (Thornberry et al.,
Nature
396:768-774, 1992; Nicholson et al.,
Nature
376:37-43, 1995; Stennicke and Salvesen,
J. Biol. Chem.
272:25719-25723, 1997). Diverse apoptotic stimuli cause the activation of specific caspases which then initiate a protease cascade by proteolytically processing additional caspases (Srinivasula et al.,
Proc. Natl. Acad Sci USA
93:14486-14491, 1996; Yu et al.,
Cancer Res.
58:402-408, 1998). Once activated, these downstream (executioner) caspases kill cells by cleaving specific molecular targets that are essential for cell viability or by activating additional pro-apoptotic factors (Liu et al.,
Cell
89:175-184, 1997; Enari et al.,
Nature
391:43-50, 1998; Salvesen and Dixit,
Cell
91:443446, 1997).
Caspase-3 is an example of a downstream “executioner” caspase thought to cleave a number of important cellular proteins involved in DNA replication, DNA repair, RNA splicing, protein phosphorylation, and chromosomal fragmentation during apoptosis (Cohen et al.,
Biochem. J.
326:1-16, 1997, Enari et al.,
Nature
391:43-50, 1998, Liu et al.,
Cell
89:175-84, 1997). This enzyme is synthesized as a 32 kDa procaspase that is processed into mature 20/17 kDa (large) and 12 kDa (small) subunits by cleavage at Asp 9, Asp 28, and Asp 175 (Fernandes-Alnemri et al.,
J. Biol. Chem.
269:30761-64, 1994; Tewari et al.,
Cell,
81:801-9, 1995; Fernandes-Alnemri et al.,
Proc.Natl.Acad.Sci. USA,
93:7464-69, 1996, Nicholson et al.,
Nature
376:37-43, 1995). Procaspase-3 can be activated by a number of proteases involved in apoptosis, including caspases-1, -8, -9, and -10, as well as the serine protease Granzyme B (Stennicke et al.,
J. Biol. Chem.
273:27084-90, 1998, Fernandes-Alnemri et al.,
Proc.Natl.Acad.Sci. USA,
93:7464-69, 1996; Quan et al.,
Proc Natl Acad Sci USA,
93:1972-76, 1996, Krebs et al.,
J. Cell Biol.
144:915-26, 1999). Immunocytochemical experiments indicate that procaspase-3 is primarily a cytoplasmic protein (Krajewski et al.,
Cancer Res.
57:1605-13, 1997; Posmantur et al.,
J. Neurochem.
68:2328-37, 1997; Chandler et al.,
J. Biol. Chem.
273:10815-18, 1998), while activated caspase-3-like enzyme is found in the cytoplasm and the nucleus (Martins et al.,
J. Biol. Chem.
272:7421-30, 1997). Additionally, it was reported that procaspase-3 may localize to the mitochondrial intermembrane space (Mancini et al.,
J. Cell Biol.
140:1485-95, 1998).
The dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states because apoptosis maintains tissue homeostasis in a range of physiological processes, including embryonic development, immune cell regulation and normal cellular turnover. For example, the loss of apoptosis can lead to the accumulation of self-reactive lymphocytes associated with many autoimmune diseases. Additionally, abnormal loss or inhibition of apoptosis can also lead to the accumulation of virally infected cells and hyperproliferative cells, such as neoplastic or tumor cells. Similarly, the irregular activation of apoptosis can contribute to a variety of pathological disease states, including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases, and ischemic injury. Treatments that are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can change the natural progression of many of these diseases.
Thus, there exists a need to identify apoptotic genes and their gene products and methods of modulating apoptosis for the therapeutic treatment of human diseases. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides in
Fritz Lawrence C.
Krebs Joseph F.
Srinivasan Anupama
Wu Joe C.
Hutson Richard
Idun Pharmaceuticals, Inc.
Seed Intellectual Property Law Group PLLC
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