Identifying Calpain-10 Substrates from Human Proteome

描述（申请人提供）：最近，糖尿病易感基因的全基因组筛选和定位克隆表明，编码广泛表达的半胱氨酸蛋白酶钙蛋白酶-10的CAPN 10基因的遗传变异与易患2型糖尿病的墨西哥裔美国人群体以及芬兰的孤立群体中2型糖尿病风险增加相关。 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程度在不同人群中差异很大。虽然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，但其他研究表明其他种族人群中没有相关性，包括大型芬兰队列，日本人和斯堪的纳维亚高加索人。显然，如果没有详细的生物化学研究，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，钙蛋白酶-10的下游底物的鉴定是了解其作用机制及其与2型糖尿病关系的关键。该建议旨在使用称为mRNA展示的新型蛋白质组学技术平台扫描人类蛋白质组以识别钙蛋白酶-10的下游靶标。 首先，人钙蛋白酶-10将使用杆状病毒表达系统过表达并表征。 然后，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。在与纯化的钙蛋白酶-10孵育后，被钙蛋白酶-10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钙蛋白酶-10切割的序列占优势。通过测序或cDNA微阵列，可以容易地从其mRNA确定每种蛋白质的身份。 在存在或不存在钙蛋白酶抑制剂的情况下，将使用体外转录/翻译的游离蛋白（或片段）研究钙蛋白酶-10对选定蛋白的切割。 将绘制其裂解位点并研究其动力学。 该项目预计将允许在蛋白质组范围内系统地鉴定钙蛋白酶-10的潜在生理底物。这些结果将对解决在不同种族人群中观察到的calpain-10的争议具有重要意义。该技术的简单性和高通量将使该方法广泛用于扫描人类蛋白质组中其他钙蛋白酶家族成员的下游靶点，这些靶点与其他人类病理学状况有关，包括阿尔茨海默病，神经系统疾病和胃癌。

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.