This proposal is directed at scanning the human proteome to identify the downstream targets of caspase-2, -3 and -8 using a novel technique called mRNA display. Specifically, human proteome domain libraries displayed on their own mRNAs are generated and immobilized on the solid surface via the biotin residue specifically introduced near the N-terminus of each protein. Upon incubation with a purified caspase of interest, protein sequences that are specifically cleaved by the caspase are released and enriched, with the intact mRNA still covalently attached to the C terminus of each cleaved protein fragment. The selected protein sequences are then regenerated for iterative round of selection, by PCR amplification followed by in vitro transcription/translation, until the pool is dominated by sequences whose protein portions can be cleaved by the caspase. The identity of each protein is readily determined from its mRNA, by sequencing or cDNA microarray. To analyze the proteolysis of selected proteins by the caspase, free protein fragments and full-length proteins are transcribed/translated in vitro and incubated with the purified caspase of interest in the presence or absence of a specific inhibitor; and/or with cell-free extracts prepared from nonapoptotic and apoptotic cells. Such confirmed potential caspase downstream targets will be characterized to determine their cleavage sites using different approaches, including a focused mRNA displayed protein domain library generated by randomly priming the selected cDNA. Potential novel caspase substrates that have been demonstrated in vitro will be further analyzed in vivo, by western analysis of the potential caspase substrates in lysates from apoptotic cells. The specificity of their proteolysis will be determined by in vivo inhibitor studies. The biological significance of each confirmed novel caspase substrate will be studied by correlating the extent of its specific proteolysis with the degree of cell death and with the proteolysis of other important caspase substrates. The effect of over expression of the proteins on apoptosis will also be addressed. The proposed research expected to allow a systematic examination and identification of the downstream targets of caspase-2, -3 and -8 on a proteome-wide scale. It will have significant implications in understanding the molecular mechanisms that govern the caspase-induced cell death, whose malfunction or dysregulation may result in cancer, neurodegenerative diseases, or other pathological conditions. The simplicity and high throughput of the methodology involved will make the approach broadly applicable to rapid scan the human proteome for downstream targets of any other caspases.
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