Regulators of apoptosis

Details Regulators of apoptosis Regulators of apoptosis

1997-02-05

2001-06-05

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S324000, C530S300000, C530S329000, C435S226000

Reexamination Certificate

active

06242569

ABSTRACT:

FIELD OF THE INVENTION
The field of this invention is proteins which regulate cell death.
BACKGROUND
Apoptosis is a cell suicide process of sequential biochemical events triggered by a variety of physiological and stress stimuli. Several lines of evidence indicate that a family of cysteine proteases, or caspases (Alnemri et al., 1996) play a crucial role in execution of apoptosis. Several members of the caspase family have been identified (for review, see Henkart, 1996; for nomenclature, see Alnemri et al., 1996) which share certain characteristic features. For example, all the identified caspases contain a conserved motif QAC(R/Q)G, in which the Cys residue is the structural hallmark of a caspase. This cysteine residue, together with two highly conserved residues, corresponding to His237 and Gly238 in ICE, may form the active site of a caspase (Wilson et al., 1994; Walker et al., 1994). In addition, many members of the family are capable of inducing apoptosis when overexpressed in mammalian cells (Henkart, 1996).
Many divergent stimuli can activate the caspase cascades leading to apoptosis. In recent years, apoptosis induced by TNF and FasL have received extensive attention. TNF elicits a broad range of biological effects (Goeddel, 1986; Beutler and Cerami, 1988; Fiers, 1991) through two distinct membrane receptors, TNF-R1 and TNF-R2, which are expressed at low levels on most cell types (Loetscher et al., 1990; Schall et al., 1990; Smith et al., 1990; Fiers, 1991; Tartaglia and Goeddel, 1992). Apoptosis induced by TNF is mediated primarily through TNF-R1. The intracellular domain of TNF-R1 contains a “death domain” of approximately 80 amino acids that is responsible for signaling cell death by the receptor (Tartaglia et al., 1993). A homologous death domain is also found in the cytoplasmic region of Fas and Wsl/DR3/Apo-3, two other members of the TNF receptor family that can potently induce apoptosis (Itoh and Nagata, 1993; Chinnaiyan et al., 1996b; Kitson et al., 1996; Marsters et al., 1996).
TRADD, a cytoplasmic protein containing a C-terminal death domain, interacts with the death domain of TNF-R1 in a ligand dependent process (Hsu et al., 1995; Hsu et al., 1996a). As observed for TNF-R1, overexpression of TRADD causes both apoptosis and activation of NF-&kgr;B (Hsu et al., 1995). The death domain of TRADD also interacts with the cytoplasmic protein FADD (Hsu et al., 1996a) through their respective death domains. Fas and FADD have also been shown to interact directly through their respective death domains (Boldin et al., 1995; Chinnaiyan et al., 1995). Although the death domains of TNF-R1, Fas, and TRADD induce apoptosis following overexpression in mammalian cells (Tartaglia et al., 1993; Hsu et al., 1995; Itoh and Nagata, 1993; Hsu et al., 1996b), overexpression of the C-terminal death domain of FADD inhibits TNF- and Fas-induced cell death (Chinnaiyan et al., 1996a; Hsu et al., 1996a). The N-terminal domain of FADD, termed death effector domain (DED), induces apoptosis after overexpression, suggesting the DED of FADD may activate a downstream cell death signaling component (Chinnaiyan et al., 1996a; Hsu et al., 1996a).
A recently identified a cysteine protease, caspase-8, (previously called Mch5, MACH and FLICE, Fernandes-Alnemri et al., 1996; Boldin et al., 1996; Muzio et al., 1996) may represent the missing link between FADD and the basic cell death machinery. The N-terminal domain of caspase-8 contains two DED-like modules through which it interacts with FADD (Boldin et al., 1996; Muzio et al., 1996). The C-terminal domain of caspase-8 is homologous to members of the caspase family and has protease activity towards most known caspases and PARP (Fernandes-Alnemri et al., 1996; Srinivasula et al., 1996; Muzio et al., 1996). The present invention provides a novel family of proteins termed Casper (
csasep
ase-
e
ight-
r
elated protein), which are structurally related to caspase-8.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to natural isolated apoptosis regulating proteins called Casper proteins, related nucleic acids, and protein domains thereof having Casper-specific activity. The proteins may be produced recombinantly from transformed host cells from the subject Casper encoding nucleic acids or purified from mammalian cells. The invention provides isolated Casper hybridization probes and primers capable of specifically hybridizing with the disclosed Casper gene, Casper-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for Casper transcripts), therapy (e.g. gene therapy to modulate Casper gene expression) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating other transcriptional regulators, reagents for screening chemical libraries for lead pharmacological agents, etc.).
DETAILED DESCRIPTION OF THE INVENTION
The nucleotide sequences of a natural cDNA encoding a human Casper protein is shown as SEQ ID NO:1, and the full conceptual translate is shown as SEQ ID NO:2. The Casper proteins of the invention include incomplete translates of SEQ ID NO:1 and deletion mutants of SEQ ID NO:2, which translates and deletion mutants have Casper-specific amino acid sequence and assay-discernable Casper-specific binding specificity or function. Such active Casper deletion mutants, Casper peptides or protein domains comprise at least about 6, preferably at least about 8, more preferably at least about 10 consecutive residues of SEQ ID NO:2. For examples, Casper protein domains identified below are shown to provide protein-binding domains which are identified in and find use, inter alia, in solid-phase binding assays as described below.
Casper-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of an Casper protein with a binding target is evaluated. The binding target may be a natural intracellular binding target such as a FADD, TRAF1, TRAF2, Caspase-3 or Caspase-8 protein, or other regulator that directly modulates Casper activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an Casper specific agent such as those identified in screening assays such as described below. Casper-binding specificity may assayed by binding equilibrium constants (usually at least about 10
7
M
−1
, preferably at least about 10
8
M
−1
, more preferably at least about 10
9
M
−1
), by the ability of the subject protein to function as negative mutants in Casper-expressing cells, to elicit Casper specific antibody in a heterologous host (e.g a rodent or rabbit), etc. In any event, the Casper binding specificity of the subject Casper proteins necessarily distinguishes Caspase-8 and Mch4.
The claimed Casper proteins are isolated or pure: an “isolated” protein is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total protein in a given sample and a pure protein constitutes at least about 90%, and preferably at least about 99% by weight of the total protein in a given sample. The Casper proteins and protein domains may be synthesized, produced by recombinant technology, or purified from mammalian, preferably human cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, N.Y.) or that are otherwise known in the art.
The invention provides natural and non-natural Casper-specific binding agents, metho

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