Identifying Calpain-10 Substrates from Human Proteome

描述（由申请人提供）：最近，对糖尿病易感基因的全基因组筛选和定位克隆表明，编码普遍表达的半胱氨酸蛋白酶calpain-10的CAPN10基因的遗传变异与易患2型糖尿病的墨西哥裔美国人和芬兰孤立人群中2型糖尿病风险增加有关。然而，后续研究表明，这种假定的糖尿病易感基因与2型糖尿病之间的关联水平在不同人群中差异很大。尽管一些研究证实了非裔美国人、英国人、波兰人和南印度人的相关性，但其他调查显示在其他种族人群中没有相关性，包括大量的芬兰人群、日本人和斯堪的纳维亚高加索人。显然，如果没有详细的生化研究，最初的假设既不能被最终证实，也不能被推翻。人们普遍认为，确定calpain-10的下游底物是了解其作用机制及其与2型糖尿病关系的关键。该建议旨在扫描人类蛋白质组，利用一种称为mRNA显示的新型蛋白质组学技术平台来识别calpain-10的下游靶标。首先，人类calpain-10将使用杆状病毒表达系统进行过表达并进行表征。然后，通过在每个蛋白质的n端附近特异性引入生物素残基，生成显示在其自身mrna上的人类蛋白质组结构域文库，并将其固定在固体表面。与纯化的calpain-10孵育后，被calpain-10特异性切割的蛋白序列被释放并富集，完整的mRNA仍共价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，直到池中占主导地位的序列的蛋白质部分可以被calpain-10切割。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身份。在存在或不存在calpain抑制剂的情况下，将使用体外转录/翻译的游离蛋白（或片段）研究calpain-10对选定蛋白的切割。它们的裂解位点将被绘制，动力学将被研究。

DESCRIPTION (provided by applicant): Recently, genome-wide screen and positional cloning for diabetes-susceptibility genes implicate that genetic variation in the CAPN10 gene, which encodes ubiquitously expressed cysteine protease calpain-10, is associated with increased risk of type 2 diabetes in a population of Mexican Americans who are susceptible to the disease, and also in an isolated population from Finland. Follow-up studies show, however, that levels of association between this putative diabetes-susceptibility gene and type 2 diabetes vary greatly in different populations. Whereas some studies have confirmed an association in African American, British, Polish, and South Indian populations, other investigations showed no association in other ethnic populations, including a large Finnish cohort, Japanese, and Scandinavian Caucasians. Clearly, the original hypothesis can be neither conclusively validated nor overturned without detailed biochemical studied. It is widely believed that identification of downstream substrates of calpain-10 holds the key to understand its action mechanisms and its relationship to type 2 diabetes. This proposal is directed at scanning the human proteome to identify the downstream targets of calpain-10 using a novel proteomic technology platform called mRNA display. First, human calpain-10 will be over-expressed using a baculovirus expression system and characterized. Then, 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 purified calpain-10, protein sequences that are specifically cleaved by calpain-10 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 calpain-10. The identity of each protein can be readily determined from its mRNA, by sequencing or cDNA microarray. The cleavage of selected proteins by calpain-10 will be studied using in vitro transcribed/ translated free proteins (or fragments) in the presence or absence of calpain inhibitors. Their cleavage sites will be mapped and kinetics will be studied. The project is expected to allow a systematic identification of potential physiological substrates for calpain-10 on a proteome-wide scale. The results will have significant implications in addressing the controversy of calpain-10 observed in different ethnic populations. The simplicity and high throughput of the technology will make the approach broadly used to scan the human proteome for downstream targets of other family members of calpains, which have been linked to other human pathological conditions, including Alzheimer's disease, neurological disorders, and gastric cancer.