Role of Klotho Ectodomain Release in Suppression of Aging

描述(申请人提供)：美国人的预期寿命一直在不断提高。因此，社会面临着与年龄相关的疾病的挑战。另一方面，仍有人因过早衰老而过早死亡。为了解决这些问题，需要更好地了解衰老和与年龄相关的疾病的潜在机制。携带失活Klotho基因等位基因的小鼠表现出人类衰老的特征，这表明Klotho蛋白是延长寿命和抑制与年龄相关的疾病所必需的。Klotho蛋白是以跨膜蛋白的形式生物合成的，其胞外结构域在细胞表面释放。Klotho胞外结构域释放的机制和生物学意义目前尚不清楚。这项研究的长期目标是确定负责产生可溶性Klotho的蛋白酶，并确定Klotho胞外区域释放在抑制衰老和与年龄相关的疾病中的作用。初步研究表明，Klotho胞外结构域在培养细胞中由肿瘤坏死因子-a转换酶(TACE)释放。这项工作将进一步确定TACE是否在生理上对Klotho胞外结构域的释放负责。这个问题将通过确定siRNA介导的TACE表达下调对培养细胞中Klotho释放的影响，并通过使用已建立的Klotho放射免疫分析方法比较TACE阴性和野生型小鼠的血浆Klotho水平来解决。研究人员还将通过质谱学确定跨膜Klotho中的TACE裂解位点。他们将通过构建包含裂解位点突变的Klotho变体来确认裂解位点，并确定这些突变是否会影响细胞内和合成肽底物中的Klotho裂解。最后，这些研究人员将通过确定体内Klotho释放的活性是否与年龄相关来研究TACE介导的Klotho释放在衰老中的作用。这项工作将揭示Klotho抗衰老活性背后的机制。它可能将Klotho释放确定为操纵Klotho活性的目标，Klotho活性可用于延长寿命，并用于预防和治疗与年龄有关的疾病。

DESCRIPTION (provided by applicant): The life expectancy for Americans has been rising continuously. As a result, society faces challenges of age-related diseases. On the other hand, there are still people who suffer untimely death from premature aging. A better understanding of mechanisms underlying aging and age-related diseases is needed to solve these issues. Mice carrying inactivated klotho gene alleles exhibit hallmarks of human aging, suggesting that the Klotho protein is required for the extension of life and the suppression of age-related diseases. The Klotho protein is biosynthesized as a transmembrane protein whose ectodomain is released at the cell surface. The mechanisms for and the biological significance of Klotho ectodomain release are currently unknown. The long-term goal of this study is to identify the protease responsible for generating soluble Klotho and to determine the role of Klotho ectodomain release in suppression of aging and age-related diseases. Preliminary studies have shown that the Klotho ectodomain is released in cultured cells by the tumor necrosis factor-a converting enzyme (TACE). This work will further determine whether TACE is physiologically responsible for Klotho ectodomain release. This question will be addressed by determining the effect of siRNA-mediated TACE expression knockdown on Klotho release in cultured cells, and by comparing the plasma Klotho levels in TACE-null and wild-type mice using an established Klotho radioimmunoassay. The investigators will also identify the TACE cleavage site in transmembrane Klotho by mass spectrometry. They will confirm the cleavage site by constructing a Klotho variant containing mutations at the cleavage site, and determine if the mutations affect Klotho cleavage in cells and with synthetic peptide substrates. Finally, these researchers will investigate the role of TACE-mediated Klotho release in aging by determining whether the activity of Klotho release in vivo correlates with age. This work will reveal insights into the mechanisms that underlie the anti-aging activity of Klotho. It may identify Klotho release as a target for manipulating Klotho activity, which can be used for lifespan extension, and for the prevention and treatment of age-related diseases.