Identification of Caspase Substrates from Human Proteome

描述(申请人提供)：这项建议旨在扫描人类蛋白质组，以识别caspase-2，-3和-8的下游靶标，使用一种名为mRNA显示的新技术。具体地说，展示在自己的mRNAs上的人类蛋白质组域文库是通过在每个蛋白质的N末端附近特别引入的生物素残基产生并固定在固体表面上的。当与纯化的目的半胱氨酸天冬氨酸酶孵育时，被半胱氨酸天冬氨酸酶特异性切割的蛋白质序列被释放和丰富，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扩增和体外转录/翻译，重新生成选定的蛋白质序列以进行迭代选择，直到池中主要是其蛋白质部分可被caspase切割的序列。通过测序或cDNA微阵列，可以很容易地从其mRNA中确定每种蛋白质的特性。为了分析caspase对所选蛋白质的蛋白分解作用，在体外转录/翻译游离蛋白质片段和全长蛋白质，并在存在或不存在特定抑制剂的情况下与纯化的目的caspase孵育，和/或与从非凋亡和凋亡细胞制备的无细胞提取物孵育。这些已确认的潜在caspase下游靶标将被表征，以使用不同的方法来确定它们的切割位置，包括通过随机启动所选的cdna而产生的聚焦的mRNA显示的蛋白结构域文库。已在体外证明的潜在的新型caspase底物将通过Western分析来自于凋亡细胞的裂解物中的潜在caspase底物，在体内进一步分析。它们蛋白分解的特异性将通过体内抑制剂研究来确定。每一种已证实的新caspase底物的生物学意义将通过将其特异性蛋白分解的程度与细胞死亡的程度以及其他重要caspase底物的蛋白分解程度相关联来研究。此外，还将讨论蛋白质过度表达对细胞凋亡的影响。 这项拟议的研究有望在蛋白质组范围内对caspase-2、-3和-8的下游靶标进行系统检查和鉴定。这对于理解caspase诱导的细胞死亡的分子机制具有重要意义，caspase诱导的细胞死亡的功能障碍或调节失调可能导致癌症、神经退行性疾病或其他病理情况。该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以寻找任何其他caspase的下游靶标。

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.