Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-04-17
2003-02-04
Caputa, Anthony C. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069520, C435S091500, C435S325000
Reexamination Certificate
active
06515118
ABSTRACT:
FIELD OF THE INVENTION
The field of this invention is proteins involved in chromatin destabilization.
BACKGROUND
Apoptosis is executed through a suicide program that is built into all animal cells (reviewed by White,1996; Wyllie,1995). Cells undergoing apoptosis show distinctive morphological changes, including membrane blebbing, cytoplasmic and nuclear condensation, chromation aggregation and formation of apoptotic bodies (Wyllie, 1980). The biochemical hallmark of apoptosis is the cleavage of chromatin into nucleosomal fragments (Wyllie et al., 1980). Multiple lines of evidence indicate that apoptosis can be triggered by the activation of a family of cysteine proteases with specificity for aspartic acid residues, including CED-3 of C. elegans, CPP32/Yama/Apopain of humans, and DCP-1 of Drosophila (Yuan et al., 1993; Xue et al., 1996; Fernandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995; Song, et al., 1997). Recently, these proteins have been designated as caspases (Alnemri et al., 1996).
The most intensively studied apoptotic caspase is caspase-3, previously called CPP32/Yama/Apopain (Fernandes-Alnemri, et al., 1994; Tewari, et al., 1995; Nicholson, et al., 1995). Caspase-3 normally exists in the cytosolic fraction of cells as an inactive precursor that is activated proteolytically when cells are signaled to undergo apoptosis (Schlegel et al., 1996; Wang et al., 1996). Multiple apoptotic signals, including serum withdrawal, activation of Fas, treatment with granzyme B, ionizing radiation, and a variety of pharmacological agents, activate caspase-3 (Chinnaiyan et al., 1996; Darmon, et al., 1996; Datta et al., 1996, 1997; Erhardt and Cooper, 1996; Hasegawa et al., 1996; Jacobson et al., 1996; Martin et al., 1996; Schlegel et al, 1996). A caspase-3-specific tetrapeptide inhibitor, Ac-DEVD SEQ ID NO:3-CHO, can abolish the ability of cytosol from apoptotic cells to induce apoptosis in normal nuclei and block the initiation of the cellular apoptotic program in response to apoptotic stimuli (Nicholson et al., 1995; Dubrez, et al., 1996; Jacobson et al., 1996). Deletion of caspase-3 from the mouse genome through homologous recombination results in excessive accumulation of neuronal cells, owing to a lack of apoptosis in the brain (Kuida et al., 1996). Addition of active caspase-3 to normal cytosol activates the apoptotic program (Enari et al., 1996). These data indicate that caspase-3 is both necessary and sufficient to trigger apoptosis.
The identified substrates of caspase-3 include poly(ADP-ribose) polymerase (PARP) (Tewari et al. 1995; Nicholson et al., 1995), sterol-regulatory element binding proteins (SREBPs) (Wang et al., 1995; 1996), the U1 associated 70 kDa protein (Caciola-Rosen et al. 1996), D4-GDI (Na et al., 1996), huntingtin (Goldberg et al., 1996), and the DNA-dependent protein kinase (Casciola-Rosen et al., 1996; Song et al., 1996). It is not known whether the cleavage of any of these substrates plays a causal role in apoptosis.
Our laboratory recently established an experimental system in which DNA fragmentation characteristic of apoptosis can be triggered in vitro by incubation of normal nuclei with activated cytosolic extracts (Liu et al., 1996b). The activation occurred in two stages: first, cytosolic caspase-3 was cleaved and activated in a reaction that was triggered by cytochrome c released from mitochondria; and second, activated caspase-3 interacted with other cytosolic components to generate DNA fragmentation when added to isolated nuclei (Liu et al., 1996b; Yang et al., 1997). The present invention describes the purification, characterization, and cDNA cloning of a downstream factor that is activated by caspase-3 and in turn induces nuclear DNA fragmentation. We call this factor DNA Fragmentation Factor (DFF).
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to isolated DNA Fragmentation Factor (DFF) polypeptides, related nucleic acids, and polypeptide domains thereof having DFF-specific activity. DFF polypeptides can regulate chromatin stability and hence provide important regulators of cell viability. The polypeptides may be produced recombinantly from transformed host cells from the subject DFF encoding nucleic acids or purified from mammalian cells. The invention provides isolated DFF hybridization probes and primers capable of specifically hybridizing with the disclosed DFF gene, DFF-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for DFF transcripts), therapy (e.g. gene therapy to modulate DFF 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 DNA Fragmentation Factor (DFF) polypeptides of the invention include DFF-45, DFF-40, and polypeptides comprising domains thereof. Such DFF domains have at least 10, preferably at least about 12, more preferably at least about 14 consecutive residues of DFF-45 or DFF-40 and provide DFF domain specific activity or function, such as being a caspase-3 substrate, mediating DNA fragmentation, activating a nuclease and inhibiting a DFF polypeptide.
The nucleotide sequence of a natural cDNA encoding a human DFF-45 polypeptide is shown as SEQ ID NO:1, and the full conceptual translate is shown as SEQ ID NO:2. The DFF polypeptides 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 DFF-specific amino acid sequence and binding specificity or function.
DFF-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 DFF polypeptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as another DFF polypeptide, a DFF regulating protein, a DFF-activated nuclease, or other regulator that directly modulates DFF activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an DFF specific agent such as those identified in screening assays such as described below. DFF-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 polypeptide to function as negative mutants in DFF-expressing cells, to elicit DFF specific antibody in a heterologous host (e.g a rodent or rabbit), etc. In any event, the DFF binding specificity of the subject DFF polypeptides necessarily distinguishes AIF.
The claimed DFF polypeptides are isolated or pure: an “isolated” polypeptide 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 polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. The DFF polypeptides and polypeptide 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, NY) or that are otherwise known in the art.
The invention provides natural and non-natural DFF-specific binding agents, methods of identifying and
Liu Xuesong
Wang Xiaodong
Board of Regents , The University of Texas System
Caputa Anthony C.
Osman Richard Aron
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