Suppressors of death domains

Details Suppressors of death domains Suppressors of death domains

1998-03-05

2002-08-20

Caputa, Anthony C. (Department: 1642)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C435S069100, C435S325000

Reexamination Certificate

active

06437113

ABSTRACT:

FIELD OF THE INVENTION
The field of this invention is proteins involved in cell signal transduction.
BACKGROUND
Tumor necrosis factor (TNF) is an important cytokine involved in the signaling of a number of cellular responses including cytotoxicity, anti-viral activity, immun-regulatory activities and the transcriptional regulation of a number of genes. The TNF receptors (TNFR1 and TNFR2) are members of the larger TNF receptor superfamily which also includes the Fas antigen, CD27, CD30, CD40, and several other receptors (Smith et al., 1996, Cell 75, 959-962). Members of this family have been shown to participate in a variety of biological properties, including programmed cell death, antiviral activity and activation of the transcription factor NF-&kgr;B in a wide variety of cell types. In particular, death domain containing members of this family, such as TNFR1 and Fas, can induce programmed cell death through a shared ~80 amino acid death domain (Tartaglia et al., 1993, Cell 74, 845-853; Itoh et al., 1993, J. Biol. Chem. 258, 10932-10937).
Additional intracellular death domain containing proteins are identified through yeast two-hybrid interaction cloning by virtue of their interactions with the death domains of death domain containing members of the TNF receptor superfamily. For example, TRADD has been shown to interact specifically with TNFR1 (Hsu, et al., 1995, Cell 81, 495-504) and FADD (Boldin et al., 1995, J. Biol. Chem. 270, 7795-7798; Chinnaiyan et al., 1995, Cell 81, 505-512) and RIP (Stanger et al., 1995, Cell 81, 513-523) interact specifically with Fas. In fact, death domains define interaction domains that provide both homotypic and heterotypic associations and can function as adapters to couple members of the TNFR superfamily with other signaling proteins (see, e.g. Hsu et al. (1996) Cell 84, 299-308).
Accordingly, the ability to exogenously modulate the activity of death domain containing proteins would yield therapeutic application for numerous clinical indications. In addition, components of such pathways would provide valuable target reagents for automated, cost-effective, high throughput drug screening assays and hence would have immediate application in domestic and international pharmaceutical and biotechnology drug development programs. The present invention provides novel modulators of death domains, their use, e.g. in drug screens, modulating cellular function, etc.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to isolated Suppressor of Death Domain (SODD) polypeptides, related nucleic acids, polypeptide domains thereof having SODD-specific structure and activity and modulators of SODD function, particularly death domain binding activity. SODD polypeptides can regulate death domain containing proteins, including members of the TNFR superfamily and hence provide important regulators of cell function such as NF&kgr;B activation. The polypeptides may be produced recombinantly from transformed host cells from the subject SODD polypeptide encoding nucleic acids or purified from mammalian cells. The invention provides isolated SODD gene hybridization probes and primers capable of specifically hybridizing with the disclosed SODD-encoding genes, SODD-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. nucleic acid hybridization screens for SODD transcripts), modulating cellular physiology (e.g. modulating intracellular SODD activity to modulate TNF signal transduction) 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 sequence of a natural cDNA encoding a human SODD polypeptide is shown as SEQ ID NO:1, and the full conceptual translate is shown as SEQ ID NO:2. The SODD polypeptides of the invention include one or more functional domains of SEQ ID NO:2, which domains comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 contiguous residues of SEQ ID NO:2 including at least one, preferably at least two, more preferably at least 4 and most preferably all of said contiguous residues contained within at least one of residues 1-122, residues 180-237 and residues 450-457, and have human SODD-specific amino acid sequence and activity. SODD domain specific activities include TNFR superfamily death domain-binding and/or binding inhibitory activity and SODD-specific immunogenicity and/or antigenicity.
SODD-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 SODD polypeptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as an SODD binding target, a SODD regulating protein or other regulator that directly modulates SODD activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an SODD specific agent such as those identified in screening assays such as described below. SODD-binding specificity may be 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 NF&kgr;B reporter expression, by apoptosis assays, by the ability of the subject polypeptide to function as negative mutants in SODD-expressing cells, to elicit SODD specific antibody in a heterologous host (e.g. a rodent or rabbit), etc.
For example, deletion mutagenesis is used to defined functional SODD domains which bind and sequester TNFR superfamily death domains, inhibit apoptosis or inhibit NF&kgr;B expression in SODD-modulated NF&kgr;B activation assays. See, e.g. Table 1.
TABLE 1
Exemplary SODD deletion mutants defining
SODD functional domains.
Death
NF&kgr;B
Domain
Apoptosis
Inhi-
Mutant
Sequence
Binding
Inhibition
bition
&Dgr;N1
SEQ ID NO:2, residues 23-457
+
+
+
&Dgr;N2
SEQ ID NO:2, residues 68-457
+
+
+
&Dgr;N3
SEQ ID NO:2, residues 118-457
+
+
+
&Dgr;N4
SEQ ID NO:2, residues 185-457
+
+
+
&Dgr;N5
SEQ ID NO:2, residues 261-457
+
+
+
&Dgr;C1
SEQ ID NO:2, residues 14-56
+
+
+
&Dgr;C2
SEQ ID NO:2, residues 1-420
−
−
−
&Dgr;C3
SEQ ID NO:2, residues 1-373
−
−
−
&Dgr;C4
SEQ ID NO:2, residues 1-277
−
−
−
In a particular embodiment, the subject domains provide SODD-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides corresponding to SODD- and human SODD-specific domains are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of SODD-specific antibodies is assayed by solid phase immunosorbant assays using immobilized SODD polypeptides of SEQ ID NO:2, see, e.g. Table 2.
TABLE 2
Immunogenic SODD polypeptides eliciting SODD-specific rabbit
polyclonal antibody: SODD polypeptide-KLH conjugates
immunized per protocol described above.
SODD Polypeptide Sequence
Immunogenicity
SEQ ID NO:2, residues 1-10
+++
SEQ ID NO:2, residues 12-21
+++
SEQ ID NO:2, residues 25-37
+++
SEQ ID NO:2, residues 42-59
+++
SEQ ID NO:2, residues 62-71
+++
SEQ ID NO:2, residues 72-85
+++
SEQ ID NO:2, residues 88-89
+++
SEQ ID NO:2, residues 105-112
+++
SEQ ID NO:2, residues 116-122
+++
SEQ ID NO:2, residues 120-128
+++
SEQ ID NO:2, residues 175-182
+++
SEQ ID NO:2, residues 180-195
+++
SEQ ID NO:2, residues 201-2

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