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. ? ? ?
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