Dissection of the structural basis of MEIG1 in assembling sperm flagella

描述(申请人提供)：活动性使精子能够到达卵子受精，而正常的精子活动性在很大程度上取决于鞭毛的形成和功能。一种细长的精子细胞特异性的套筒被认为在精子鞭毛的形成中起着中心作用。减数分裂表达蛋白1(MEIG1)是调节套囊稳定性和精子鞭毛形成的关键蛋白。该蛋白被鉴定为精子相关抗原16L(SPAG16L)的结合伙伴，SPAG16L是一种精子尾轴丝蛋白，它也与Parkin共同调节基因的蛋白产物(PACRG)结合。Pacrg缺陷小鼠的生殖表型与Meig1突变小鼠相似。PACRG在减数分裂后表达，并定位于地幔。MEIG1和SPAG16L存在于精母细胞的细胞质中。然而，在伸长的精子细胞中，这两种蛋白质都迁移到套筒中。MEIG1在Pacrg缺陷的伸长精子细胞中失去了其套筒定位。在Meig1基因缺陷小鼠中，SPAG16L失去了其外套架定位。这些观察表明，MEIG1在蛋白质护送/靶向中发挥了作用。MEIG1由88个氨基酸组成，生物信息学工具未发现MEIG1的功能结构域。它在体内被磷酸化，而磷酸化可能调节MEIG1的功能，尽管这一点尚未确定。我们的核磁共振初步研究表明，MEIG1形成了一个独特的结构，为与其他蛋白质的相互作用提供了较大的表面积，几个氨基酸，包括芳香区和带电区的几个潜在的磷酸化位点，可能形成蛋白质-蛋白质相互作用面。本研究的长期目标是研究MEIG1复合体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)在体内和体外剖析SPAG16L/MEIG1/PACRG复合体；2)确定MEIG1与PACRG和SPAG16L相互作用的结构域；3)。确定翻译后修饰在MEIG1功能中的作用。我们推测，MEIG1是一种伴侣蛋白，与多种蛋白质结合，维持外套膜的完整性，并在精子鞭毛的组装中发挥作用。PACRG通过与MEIG1的特定结构域结合将MEIG1招募到壁炉架上，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/缺失将减少或取消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与与套筒相关的PACRG的对接和卸货(例如，SPAG16L)。我们推测MEIG1在体内有几个磷酸化的氨基酸，磷酸化控制着它与货物蛋白的亲和力。人们对导致这些分子正确靶向并组装到精子鞭毛中的机制知之甚少。在这项申请中提出的研究将首次揭示分子 蛋白质护送到鞭毛组装部位的基础，以及对正常精子发生至关重要的独特伴侣的结构/功能关系。

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.