Identification of Caspase Substrates from Human Proteome

描述（由申请人提供）：本提案旨在使用称为mRNA展示的新技术扫描人类蛋白质组以鉴定半胱天冬酶-2、-3和-8的下游靶标。具体地，产生在其自身mRNA上展示的人蛋白质组结构域文库，并通过在每种蛋白质的N-末端附近特异性引入的生物素残基将其固定在固体表面上。在与纯化的感兴趣的胱天蛋白酶一起孵育后，被胱天蛋白酶特异性切割的蛋白质序列被释放并富集，其中完整的mRNA仍然共价连接到每个切割的蛋白质片段的C末端。然后，通过PCR扩增，接着体外转录/翻译，再生所选择的蛋白质序列用于迭代轮的选择，直到库由其蛋白质部分可以被胱天蛋白酶切割的序列占主导地位。通过测序或cDNA微阵列，从其mRNA容易地确定每种蛋白质的身份。为了分析胱天蛋白酶对所选蛋白质的蛋白水解，将游离蛋白质片段和全长蛋白质在体外转录/翻译，并在存在或不存在特异性抑制剂的情况下与纯化的感兴趣的胱天蛋白酶一起孵育;和/或与从非凋亡和凋亡细胞制备的无细胞提取物一起孵育。将使用不同的方法表征这些确认的潜在胱天蛋白酶下游靶标以确定其切割位点，包括通过随机引发所选cDNA产生的聚焦mRNA展示蛋白质结构域文库。已经在体外证实的潜在的新型半胱天冬酶底物将通过对来自凋亡细胞的裂解物中的潜在半胱天冬酶底物的Western分析在体内进一步分析。其蛋白水解的特异性将通过体内抑制剂研究确定。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胱天蛋白酶底物的蛋白水解相关联来研究每个确认的新型胱天蛋白酶底物的生物学意义。过表达的蛋白质对细胞凋亡的影响也将得到解决。 拟议的研究有望在蛋白质组范围内系统地检查和鉴定caspase-2，-3和-8的下游靶点。这将对理解半胱天冬酶诱导的细胞死亡的分子机制具有重要意义，其功能障碍或失调可能导致癌症，神经退行性疾病或其他病理条件。所涉及的方法的简单性和高通量将使该方法广泛适用于快速扫描人类蛋白质组的下游目标的任何其他半胱天冬酶。

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.