Polynucleotides encoding activators of caspases

Details Polynucleotides encoding activators of caspases Polynucleotides encoding activators of caspases

2000-07-28

2003-03-18

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100, C536S023200, C536S023500

Reexamination Certificate

active

06534267

ABSTRACT:

FIELD OF THE INVENTION
The field of the invention is regulators of enzymes involved in cellular apoptosis.
BACKGROUND
One of the key regulatory steps for apoptosis is the activation of caspases, leading to the characteristic morphological changes associated with apoptotic cells including chromatin condensation, DNA fragmentation into nucleosomal fragments, nuclear membrane break down, externalization of phosphotidylserine and formation of apoptotic bodies that are readily phagocytosed (Liu et al., 1997 Cell 89, 175-184; Enari et al., 1998 Nature 391, 43-50; Sahara, et al., 1999 Nature 401, 168-173; Lazebnik, et al., 1995 Proc. Natl. Acad. Sci. USA 92, 9042-9046; Martin et al., 1996 J. Biol. Chem. 271, 28753-28756; Zhang et al., 1999 J. Cell Biol. 145, 99-108).
One major apoptotic caspase activation cascade is triggered by cytochrome c, a protein that normally functions in the electron transfer chain in mitochondria (Liu et al., 1996 Cell 86, 147-157). In living cells, holocytochrome c exists exclusively in the intermembrane space of mitochondria, and is therefore sequestered away from its deadly cytosolic partner, Apaf-1 (Zou et al., 1997 Cell 90, 405-413). Upon receiving apoptotic stimuli, such as serum deprivation, activation of cell surface death receptors, and excessive damage of DNA, the outer membrane of mitochondria becomes permeable to cytochrome c (Reviewed by Reed, 1997 Cell 91, 559-562). Once released to cytosol, cytochrome c binds to Apaf-1 with 2:1 stoichiometry and forms an oligomeric Apaf-1/cytochrome c complex in the presence of dATP or ATP (Purring et al., 1999 J. American Chem. Soc. 121, 7435-7436; Zou et al., 1999 J. Biol. Chem. 274, 11549-11556). This oligomerized Apaf-1/cytochrome c complex then recruits and activates the apical caspase of this pathway, procaspase-9 (Li et al., 1997 Cell 91, 479-489; Zou et al., 1999). Caspase-9 in turn activates downstream caspases such as caspase-3, -6 and -7 that constitute the major caspase activity in an apoptotic cell (Li et al., 1997; Srinivasa et al., 1998 Mol. Cell 1, 949-957; Faleiro et al., 1997 EMBO J; 16, 2271-2281).
Here we disclose the identification, purification, molecular cloning, and characterization of a novel factor that promotes cytochrome c/Apaf-1-dependent caspase activation. Like cytochrome c, this protein is normally located in mitochondria and released into cytosol when cells undergo apoptosis. We named this protein Smac, for the second mitochondria-derived activator of caspase, after cytochrome c. Addition of Smac to cytosolic extracts causes robust caspase activation in these extracts even without the addition of dATP. Smac also allows caspase activation in the presence of physiological levels of potassium salt.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to polypeptide regulators (activators and inhibitors) of enzymes involved in cellular apoptosis, particularly caspases In a particular aspect, the invention provides polypeptide and polynucleotide sequences diagnostic of caspase activators. These sequences and polypeptides and polynucleotides embodying these sequences find a wide variety of diagnostic and therapeutic applications involving detecting and/or modulating expression and/or function of activators or caspases or genes or transcripts encoding such activators. In more particular aspects, the invention provides genetic and immuno probes specific to activators of caspases.
Since undesirable activation or inactivation of apoptosis has been associated with many human diseases such as cancer, autoimmune disease and neurodegenerative diseases, the disclosed caspase regulatory polypeptides and polynucleotides provide both drug targets and regulators to promote or inhibit apoptosis. Also, since disclosed native Smac proteins naturally translocate from mitochondria to cytosol during apoptosis, Smac proteins can be used as diagnostic markers for apoptosis during normal or disease stages, e.g. using labeled Smac proteins such as fusion proteins or using detectable Smac-specific binding agents.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones described herein.
The subject polypeptide sequences find a wide variety of applications. In one embodiment, the subject sequences are used to synthesize polypeptides which in turn provide a number of applications, including use in proteomic microarrays (e.g. Silzel J W, et al. Clin Chem 1998 September;44(9):2036-43), models for rationale drug design, immunogens for antibody elicitation, etc. The polypeptide sequences are also used to specifically detect sequences comprising SEQ ID NO:2, or fragments thereof, particularly at least one of SEQ ID NO:2, residues 1-78 or SEQ ID NO:2, residues 176-239, or fragments thereof, or polypeptides comprising such sequences. Any convenient sequence detection method may be used, including computational methods for direct sequence detection (e.g. BLAST-type algorithms, alignments, etc.) and physical methods for inferential sequence detection of polymers (e.g. mass spectroscopy, etc.).
In addition to direct synthesis, the subject polypeptides can also be expressed in cell and cell-free systems (e.g. Jermutus L, et al., Curr Opin Biotechnol. 1998 October;9(5):534-48) from encoding polynucleotides, such as the corresponding parent polynucleotides or naturally-encoding polynucleotides isolated with degenerate oligonucleotide primers and probes generated from the subject polypeptide sequences (“GCG” software, Genetics Computer Group, Inc, Madison Wis.) or polynucleotides optimized for selected expression systems made by back-translating the subject polypeptides according to computer algorithms (e.g. Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166).
The subject polypeptides include fragments of the recited sequences which have Smac-specific amino acid sequence, binding specificity or function. Preferred fragments comprise at least 8, preferably at least 10, preferably at least 15, more preferably at least 25, more preferably at least 35, most preferably at least 50 consecutive residues of SEQ ID NO:2, particularly of at least one of SEQ ID NO:2, residues 1-78 or SEQ ID NO:2, residues 176-239, and have corresponding-polypeptide-specific antibody binding, elicitation or binding or elicitation inhibitory activity.
Specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, etc. Binding assays encompass any assay where the molecular interaction of a subject polypeptide with a binding target is evaluated. The binding target may be a natural binding target such as a regulating protein or a non-natural binding target such as a specific immune protein such as an antibody, or a specific agent such as those identified in screening assays such as described below. 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 caspase activation or apoptosis assays, by the ability of the subject polypeptide to function as negative mutants in expressing cells, to elicit specific antibody in a heterologous host (e.g a rodent or rabbit), etc. In a particular embodiment, the subject polypeptide fragments provide specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides 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 specific antibodies is assayed

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