Identification of Caspase Substrates from Human Proteome

描述（由申请人提供）：该提案旨在扫描人类蛋白质组，以使用一种称为mRNA显示的新技术识别caspase-2， -3和-8的下游靶标。具体来说，人类蛋白质组结构域文库显示在自己的mrna上，通过在每个蛋白质的n端附近特异性引入的生物素残基，产生并固定在固体表面。用纯化的caspase孵育后，被caspase特异性切割的蛋白序列被释放并富集，完整的mRNA仍然共价地附着在每个被切割的蛋白片段的C端。然后，通过PCR扩增和体外转录/翻译，再生选定的蛋白质序列进行迭代选择，直到库中占主导地位的序列的蛋白质部分可以被caspase切割。通过测序或cDNA微阵列，可以很容易地从其mRNA确定每种蛋白质的身份。为了分析caspase对选定蛋白质的蛋白水解作用，在体外转录/翻译游离蛋白片段和全长蛋白，并在存在或不存在特定抑制剂的情况下与纯化的caspase孵育；和/或从非凋亡细胞和凋亡细胞制备的无细胞提取物。这些已确认的潜在caspase下游靶点将使用不同的方法进行表征，以确定其切割位点，包括通过随机引物所选cDNA生成的聚焦mRNA显示蛋白结构域文库。已经在体外证明的潜在的新型caspase底物将在体内进一步分析，通过western分析凋亡细胞裂解物中潜在的caspase底物。它们蛋白水解的特异性将通过体内抑制剂研究来确定。每一种已确认的新型caspase底物的生物学意义将通过将其特异性蛋白水解程度与细胞死亡程度以及与其他重要caspase底物的蛋白水解程度相关联来研究。过表达的蛋白质对细胞凋亡的影响也将被讨论。

DESCRIPTION (provided by applicant): 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.