Dissection of the structural basis of MEIG1 in assembling sperm flagella

描述（由申请人提供）：活力使精子能够到达卵子进行受精，正常的精子活力在很大程度上取决于鞭毛的形成和功能。一个延长的精子特异性，manchette，已经被提出在精子鞭毛形成中发挥核心作用。减数分裂表达蛋白1 （Meiosis expressed protein 1, MEIG1）是调控精子manchette稳定性和鞭毛形成的关键蛋白。该蛋白被鉴定为精子尾轴素蛋白精子相关抗原16L （SPAG16L）的结合伴侣；它还结合Parkin共调节基因的蛋白产物（PACRG）。pacrg缺失小鼠的生殖表型与Meig1突变小鼠相似。PACRG在减数分裂后表达，并定位于胸膜。MEIG1和SPAG16L存在于精母细胞的细胞质中。然而，这两种蛋白质在拉长的精子中迁移到manchette。MEIG1在缺乏pacg的细长精细胞中失去了它的manchet定位。在meig1缺陷小鼠中，SPAG16L失去了它的下颌定位。这些观察结果表明MEIG1在蛋白质护送/靶向中的作用。MEIG1仅由88个氨基酸组成，没有生物信息学工具鉴定出功能域。它在体内被磷酸化，磷酸化可能调节MEIG1的功能，尽管这还没有得到证实。我们使用核磁共振（NMR）的初步研究表明，MEIG1形成了一种独特的结构，为与其他蛋白质的相互作用提供了很大的表面积，并且几种氨基酸，包括芳香区和带电区的几个潜在磷酸化位点，可能形成蛋白质-蛋白质相互作用表面。本研究的长期目标是研究MEIG1复合物在哺乳动物精子鞭毛发生中的作用。我们提出了三个目标：1)在体内和体外解剖SPAG16L/MEIG1/PACRG复合物；2)鉴定MEIG1中介导PACRG和SPAG16L相互作用的结构域；3)。目的：确定翻译后修饰在MEIG1功能中的作用。我们假设MEIG1作为伴侣蛋白与多种蛋白质结合，维持manchette的完整性，并在精子鞭毛的组装中发挥作用。PACRG通过结合MEIG1的特定结构域将MEIG1招募到manchette上，而MEIG1通过不同的结构域与其他蛋白质如SPAG16L结合。结构域的突变/缺失将减少或消除MEIG1与这些蛋白质的相互作用。MEIG1以不同的结合亲和力与PACRG和SPAG16L结合，以促进MEIG1与与manchette相关的PACRG对接并卸载其货物（例如SPAG16L）。我们预计MEIG1在体内有几个磷酸化的氨基酸，磷酸化控制其对货物蛋白的亲和力。对于导致这些分子正确定位和组装到精子鞭毛中的机制知之甚少。本应用程序中提出的研究将首次揭示分子机制

DESCRIPTION (provided by applicant): Motility enables sperm to reach the egg for fertilization, and normal sperm motility is largely dependent on formation and function of the flagella. An elongating spermatid-specific, the manchette, has been proposed to play a central role in sperm flagella formation. Meiosis expressed protein 1 (MEIG1) is a key protein that regulates manchette stability and sperm flagella formation. The protein was identified as a binding partner of Sperm Associated-Antigen 16L (SPAG16L), a sperm tail axoneme protein; it also binds the protein product (PACRG) of the Parkin co-regulated gene. The reproductive phenotype of Pacrg-deficient mice mirrors that of the Meig1 mutant mice. PACRG is expressed post-meiotically and is localized to the manchette. MEIG1 and SPAG16L are present in the cytoplasm of spermatocytes. However, both proteins migrate to the manchette in elongating spermatids. MEIG1 loses its manchette localization in the Pacrg-deficient elongating spermatids. In Meig1-deficient mice, SPAG16L loses its manchette localization. These observations suggest a role for MEIG1 in protein escort/targeting. MEIG1 consists of only 88 amino acids, and no functional domains were identified with bioinformatic tools. It is phosphorylated in vivo, and phosphorylation might modulate MEIG1 function, although this has not been established. Our preliminary studies using nuclear magnetic resonance (NMR) revealed that MEIG1 forms an unique structure that provides a large surface area for interaction with other proteins, and several amino acids, including several potential sites for phosphorylation in the aromatic and charged regions, may form protein-protein interaction surfaces. The long-term objective of this research is to investigate the role of MEIG1 complexes in mammalian sperm flagellogenesis. We propose three aims: 1) To dissect the SPAG16L/MEIG1/PACRG complex in vivo and in vitro; 2) To identify domains of MEIG1 that mediate interactions with PACRG and SPAG16L; 3). To determine the role of post-translational modification in MEIG1 function. We hypothesize that MEIG1 functions as a chaperone that associates with multiple proteins, maintains the integrity of the manchette, and plays a role in assembly of the sperm flagella. PACRG recruits MEIG1 to the manchette through binding to a specific domain of MEIG1, and MEIG1 binds other proteins such as SPAG16L through a different domain. Mutations/deletions of the domains will reduce or abolish MEIG1 interaction with these proteins. MEIG1 binds to PACRG and SPAG16L with differential binding affinities to facilitate docking of MEIG1 to PACRG associated with the manchette and off-loading of its cargo (e.g., SPAG16L). We anticipate that MEIG1 has several phosphorylated amino acids in vivo, and phosphorylation controls its affinity to cargo proteins. Little is known about the mechanisms that lead to the proper targeting and assembly of these molecules into the sperm flagellum. The research proposed in this application will, for the first time, reveal the molecular basis of the escort of proteins to the site of flagellum assembly, and the structure/function relationships of a unique chaperone that is essential for normal spermiogenesis.