KLHL 10-mediated uniquitination during spermiogenesis

描述（由申请方提供）：精子发生是圆形精子细胞分化为细长精子细胞并最终形成精子的过程。在精子发生晚期，转录停止，晚期精子细胞发育所需的蛋白质使用精子细胞伸长开始前产生和储存的mRNA进行翻译。因此，晚期精子细胞中的蛋白质水平主要在翻译和翻译后水平上受到控制。鉴于参与这一过程的蛋白质多达数百种（如果不是数千种的话），因此蛋白质周转必须受到严格的监管。泛素途径可能参与其中，因为它是调节蛋白质周转并从而控制蛋白质活性的有效且快速的机制。然而，很少有人知道这个途径的分子组成和特定的底物，它在精子发生过程中的目标。KLHL 10是BTB-Kelch蛋白大家族的新成员，我们的敲除研究表明KLHL 10对精子发生至关重要。我们最近的研究表明，KLHL 10是基于CULS的泛素E3连接酶复合物的一个组成部分。基于我们的初步数据和最近的报告，我们假设，KLHL 10调节一组蛋白质的营业额在精子形成过程中作为一个底物特异性的接头在CULS的泛素E3连接酶复合物。为了验证我们的假设，我们提出（1）检测KLHL 10-CUL 3-ROC 1复合物的泛素E3连接酶活性，并确定在我们的酵母双杂交测定中鉴定的8种Kelch结构域结合蛋白是底物还是衔接子，（2）检测野生型和KLHL 10敲除小鼠中KLHL 10-CUL 3 E3泛素连接酶靶向的蛋白的周转率，以及（3）在体外和体内确定BACK结构域在基于KLHL 10-CUL 3的泛素E3连接酶复合物中的作用。我们将使用各种各样的生物化学，遗传和分子生物学技术来实现这些目标。该研究将揭示KLHL 10调控精子发生的分子机制。由于KLHL 10在精子发生中起重要作用，因此KLHL 10介导的泛素化途径的失调可能与男性不育有关。另一方面，KLHL 10及其底物蛋白可能是未来非激素男性避孕药的良好靶点。

DESCRIPTION (provided by applicant): Spermiogenesis is the process by which round spermatids differentiate into elongated spermatids and eventually spermatozoa. During late spermiogenesis, transcription ceases and proteins required for late spermatid development are translated using mRNAs produced and stored before spermatid elongation starts. Therefore, protein levels in late spermatids are mainly controlled at translational and post- translational levels. Given that hundreds, if not thousands, of proteins participate in this process, protein turnover must be strictly regulated. The ubiquitin pathway is likely to be involved since it is an efficient and rapid mechanism for regulating protein turnover and thereby controlling protein activity. However, very little is known about the molecular composition of this pathway and the specific substrates that it targets during spermiogenesis. KLHL10, a new member of a large BTB-Kelch protein family, was first cloned by my lab and our knockout study demonstrates that KLHL10 is essential for spermiogenesis. Our recent study reveals that KLHL10 is a component of the CULS-based ubiquitin E3 ligase complexes. Based on our preliminary data and recent reports by others, we hypothesize that KLHL10 regulates the turnover of a set of proteins during spermiogenesis by acting as a substrate-specific adaptor in CULS-based ubiquitin E3 ligase complexes. To test our hypothesis, we propose (1) to examine the ubiquitin E3 ligase activity of the KLHL10-CUL3-ROC1 complex and to determine whether the 8 Kelch domain-binding proteins identified in our yeast two-hybrid assays are substrates, or adaptor, (2) to examine turnover rates of proteins targeted by the KLHL10-CUL3 E3 ubiquitin ligase in wild-type and Klhl10 knockout mice, and (3) to determine the role of the BACK domain in the KLHL10-CUL3-based ubiquitin E3 ligase complexes in vitro and in vivo. We will use a wide variety of biochemical, genetic, and molecular biology techniques to achieve these goals. The proposed study will reveal the molecular mechanism by which KLHL10 regulates spermiogenesis. Since KLHL10 plays an essential role in spermiogenesis, deregulation of the KLHL10-mediated ubiquitination pathway may be involved in male infertility. On the other hand, KLHL10 and its substrate proteins may be good targets for future non-hormonal male contraceptives.