Dissection of the structural basis of MEIG1 in assembling sperm flagella

剖析MEIG1组装精子鞭毛的结构基础

• 批准号: 9293129
• 负责人: Zhibing Zhang
• 金额: $ 17.64万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-15 至 2018-02-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9293129
• 关键词: Affinity; Amino Acids; Antibodies; Antigens; Area; Bacteria; Binding; Binding Proteins; Bioinformatics; Cells; Charge; Complex; Cytoplasm; Defect; Deletion Mutation; Dissection; Docking; Failure; Fertilization; Flagella; Genes; Globular Region; Hybrids; In Vitro; Individual; Knowledge; Lead; Maintenance; Mammalian Cell; Mediating; Meiosis; Microtubules; Modeling; Modification; Molecular; Molecular Chaperones; Mus; Mutant Strains Mice; Nuclear Magnetic Resonance; Organelles; Phenotype; Phosphorylation; Phosphorylation Site; Plasmids; Play; Post-Translational Protein Processing; Process; Proteins; Recruitment Activity; Reproductive Biology; Research; Role; Shapes; Site; Sperm Motility; Sperm Tail; Spermatids; Spermatocytes; Spermatogenesis; Spermiogenesis; Structure; Structure-Activity Relationship; Surface; Testing; Testis; Time; Translating; Yeasts; cell motility; egg; in vivo; liquid chromatography mass spectrometry; male; parkin gene/protein; protein protein interaction; public health relevance; reproductive; screening; sperm cell; sperm protein; tool; trafficking; yeast two hybrid system

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.

描述（由申请人提供）：运动性使精子能够到达卵子进行受精，正常的精子运动性在很大程度上取决于鞭毛的形成和功能。一个伸长的精子细胞特有的，manchette，已被提出在精子鞭毛的形成中发挥核心作用。减数分裂表达蛋白1（MEIG 1）是调节精子鞭毛形成和顶丝稳定性的关键蛋白。该蛋白被鉴定为精子相关抗原16 L（SPAG 16 L）（精子尾部轴丝蛋白）的结合伴侣;它还结合帕金共调节基因的蛋白产物（PACRG）。Pacrg缺陷小鼠的生殖表型反映了Meig 1突变小鼠的生殖表型。PACRG在减数分裂后表达，并定位于肩突。MEIG 1和SPAG 16 L存在于精母细胞的细胞质中。然而，这两种蛋白质迁移到长形精子细胞的柄。MEIG 1在Pacrg缺陷的伸长精子细胞中失去了其柄状定位。在Meig 1缺陷型小鼠中，SPAG 16 L失去了它的肩袖定位。这些观察结果表明MEIG 1在蛋白质护送/靶向中的作用。MEIG 1仅由88个氨基酸组成，并且没有用生物信息学工具鉴定出功能结构域。它在体内被磷酸化，磷酸化可能调节MEIG 1的功能，尽管这还没有建立。我们使用核磁共振（NMR）的初步研究表明，MEIG 1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大的表面积，并且几个氨基酸，包括芳香族和带电区域中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MEIG 1复合物在哺乳动物精子鞭毛发生中的作用。我们提出三个目标：1）在体内和体外解剖SPAG 16 L/MEIG 1/PACRG复合物; 2）鉴定MEIG 1介导与PACRG和SPAG 16 L相互作用的结构域; 3）。确定翻译后修饰在MEIG 1功能中的作用。我们推测MEIG 1作为一种伴侣蛋白，与多种蛋白质结合，保持了柄的完整性，并在精子鞭毛的组装中发挥作用。PACRG通过与MEIG 1的特定结构域结合将MEIG 1募集到肩托，并且MEIG 1通过不同的结构域与其他蛋白质如SPAG 16 L结合。结构域的突变/缺失将减少或消除MEIG 1与这些蛋白质的相互作用。MEIG 1以不同的结合亲和力结合PACRG和SPAG 16 L，以促进MEIG 1对接到与肩托相关的PACRG并卸载其货物（例如，SPAG16L）。我们预期MEIG 1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。很少有人知道的机制，导致适当的目标和组装这些分子到精子鞭毛。这项申请中提出的研究将首次揭示 蛋白质护送到鞭毛组装位点的基础，以及正常精子发生所必需的独特伴侣的结构/功能关系。