Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-06-16
2002-06-11
Caputa, Anthony C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000, C530S324000
Reexamination Certificate
active
06403765
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally the regulation of apoptosis, and more particularly, to truncated Apaf-1 and methods of using truncated Apaf-1 and self-oligomerizing caspases to identify modulators of apoptosis.
BACKGROUND OF THE INVENTION
Apoptosis is the normal physiological process of programmed cell death that maintains tissue homeostasis. Changes to the apoptotic pathway that prevent or delay normal cell turnover can be just as important in the pathogenesis of diseases as are abnormalities in the regulation of the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either prevent or induce cell death.
Since apoptosis functions in maintaining tissue homeostasis in a range of physiological processes such as embryonic development, immune cell regulation and normal cellular turnover, the dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes that occurs with many autoimmune diseases. Inappropriate loss or inhibition of apoptosis can also lead to the accumulation of virally infected cells and of hyperproliferative cells such as neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can also contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. Treatments which are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can alter the natural progression of many of these diseases.
Although apoptosis is mediated by diverse signals and complex interactions of cellular gene products, the results of these interactions ultimately feed into a cell death pathway that is evolutionarily conserved between humans and invertebrates. The pathway, itself, is a cascade of proteolytic events analogous to that of the blood coagulation cascade.
Several gene families and products that modulate the apoptotic process have now been identified. One family is the aspartate-specific cysteine proteases (“caspases”). The human caspase family includes, for example, Ced-3, human ICE (interleukin-1-&bgr; converting enzyme) (caspase-1), ICH-1 (caspase-2), CPP32 (caspase-3), ICE
rel
II (caspase-4), ICE
rel
III (caspase-5), Mch2 (caspase-6), ICE-LAP3 (casepase-7), Mch5 (caspase-8), ICE-LAP6 (caspase-9), Mch4 (caspase-10), and others.
The caspase proteins share several common features. In this regard, caspases are cysteine proteases (named for a cysteine residue in the active site) that cleave substrates at Asp-X bonds. Furthermore, caspases are primarily produced as inactive zymogens that require proteolytic cleavage at specific internal aspartate residues for activation. The primary gene product is arranged such that the N-terminal peptide (prodomain) precedes a large subunit domain, which precedes a small subunit domain. The large subunit contains the conserved active site pentapeptide QACXG (X=R, Q, G) which contains the nucleophilic cysteine residue. The small subunit contains residues that bind the Asp carboxylate side chain and others that determine substrate specificity. Cleavage of a caspase yields the two subunits, the large (generally approximately 20 kD) and the small (generally approximately 10 kD) subunit that associate non-covalently: to form a heterodimer, and, in some caspases, an N-terminal peptide of varying length. The heterodimer may combine non-covalently to form a tetramer.
Caspase zymogens are themselves substrates for caspases. Inspection of the interdomain linkages in each zymogen reveals target sites (i.e. protease sites) that indicate a hierarchical relationship of caspase activation. By analyzing such pathways, it has been demonstrated that caspases are required for apoptosis to occur. Moreover, caspases appear to be necessary for the accurate and limited proteolytic events which are the hallmark of classic apoptosis (see Salvesen and Dixit,
Cell
91:443-446, 1997). Once activated, most caspases can process and activate their own and other inactive procaspases in vitro (Fernandes-Alnemri et al.,
Proc. Natl. Acad. Sci. USA
93:7464-7469, 1996; Srinivasula et al.,
Proc. Natl. Acad. Sci. USA
93:13706-13711, 1996. This characteristic suggests that caspases implicated in apoptosis may execute the apoptotic program through a cascade of sequential activation of initiators and executioner procaspases (Salvesen and Dixit,
Cell
91:443-446, 1997). The initiators are responsible for processing and activation of the executioners. The executioners are responsible for proteolytic cleavage of a number of cellular proteins leading to the characteristic morphological changes and DNA fragmentation that are often associated with apoptosis (reviewed by (Cohen,
Biochem. J
. 326:1-16, 1997; Henkart,
Immunity
4:195-201, 1996; Martin and Green,
Cell
82:349-352, 1995; Nicholson and Thomberry,
TIBS
257:299-306, 1997; Porter et al.,
BioEssays
19:501-507, 1997; Salvesen and Dixit,
Cell
91:443-446, 1997. The first evidence for an apoptotic caspase cascade was obtained from studies on death receptor signaling (reviewed by (Fraser and Evan,
Cell
85:781-784, 1996; Nagata,
Cell
88:355-365, 1997) which indicated that the death signal is transmitted in part by sequential activation of the initiator procaspase-8 and the executioner procaspase-3 (Boldin et al.,
Cell
85:803-815, 1996; Fernandes-Alnemri et al.,
Proc. Natl. Acad. Sci. USA
93:7464-7469, 1996; Muzio et al.,
Cell
85:817-827, 1996; Srinivasula et al.,
Proc. Natl. Acad. Sci. USA
93:13706-13711, 1996). More direct evidence was provided, recently, when it was demonstrated that the cytochrome c death signal is transmitted through activation of a cascade involving procaspase-9 and -3 (Li et al.,
Cell
91:479-489, 1997).
However, it remains unclear how the initiator caspases, like procaspase-8 and -9 are activated. While Apaf-1 is known to play a role in the activation of procaspase-9 the exact mechanism has yet to be determined.
Therefore, there exists a need in the art for methods of assaying compounds for their ability to affect Apaf-1 mediated caspase activity as well as for methods of modulating apoptosis in order to treat diseases and syndromes. The present invention fulfills this need, while further providing other related advantages.
SUMMARY OF THE INVENTION
The present invention generally provides truncated Apaf-1. In one aspect, the invention provides an isolated nucleic acid molecule encoding a truncated Apaf-1 or a variant thereof. In one embodiment, the encoded truncated Apaf-1 is a human truncated Apaf-1. In another embodiment, the human truncated Apaf-1 has the amino acid sequence of SEQ ID NO:2 or a variant thereof. In another embodiment, the nucleic acid molecule encoding a truncated Apaf-1 or variant thereof has the nucleic acid sequence of SEQ ID NO:1 or a variant thereof. In another embodiment, the nucleic acid molecule encodes a truncated Apaf-1 or fragment thereof that oligomerizes with a caspase. In yet another embodiment, the nucleic acid molecule encodes a human truncated Apaf-1 having the amino acid sequence of SEQ ID NO:2 or variant thereof that oligomerizes with a caspase.
It is another aspect of the invention to provide an expression vector comprising any of the nucleic acid molecules encoding a truncated Apaf-1 or a variant thereof referred to above, wherein the nucleic acid molecule encoding the truncated Apaf-1 is operatively linked to a promoter. In one embodiment, the promoter is inducible. In another aspect, the invention provides a host cell transfected with such expression vectors. In certain embodiments, the host cell may be a bacterium, an insect cell or a mammalian cell.
Another aspect of the invention pertains to an isolated truncated Apaf-1 polypeptide or fragment thereof.
Caputa Anthony C.
Holleran Anne L.
SEED Intellectual Property Law Group PLLC
Thomas Jefferson University
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