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）：运动能力使精子能够到达卵以进行受精，而正常的精子运动在很大程度上取决于鞭毛的形成和功能。已经提出了细长的精子特异性人物，它在精子鞭毛形成中起着核心作用。减数分裂表达蛋白1（MEIG1）是一种关键蛋白质，可调节黑用于稳定性和精子鞭毛形成。该蛋白被鉴定为精子抗原16L（Spag16L）的结合伴侣，一种精子尾轴突蛋白。它还结合了帕克蛋白共同调节基因的蛋白质产物（PACRG）。 PACRG缺陷小鼠的生殖表型反映了Meig1突变小鼠的生殖表型。 PACRG在自动赛后表达，并定位于Manchette。 Meig1和Spag16L存在于精子细胞的细胞质中。然而，两种蛋白质都迁移到延长精子中的人类。 Meig1失去了其在PACRG缺乏伸长的精子中的固定位置。在Meig1缺陷型小鼠中，Spag16L失去了其固定量。这些观察结果表明MEIG1在蛋白质伴游/靶向中的作用。 Meig1仅由88种氨基酸组成，并且没有生物信息学工具鉴定出功能域。它在体内被磷酸化，磷酸化可能会调节MEIG1功能，尽管尚未确定。我们使用核磁共振（NMR）的初步研究表明，MEIG1形成了独特的结构，为与其他蛋白质相互作用提供了较大的表面积，以及几种氨基酸，包括在芳族和带电区域中的几个潜在磷酸化位点，可能会形成蛋白质蛋白质相互作用表面。这项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。我们提出三个目标：1）在体内和体外剖析Spag16L/Meig1/Pacrg复合物； 2）识别介导与PACRG和SPAG16L相互作用的Meig1域； 3）。确定翻译后修饰在MEIG1功能中的作用。我们假设Meig1是与多种蛋白相关联的伴侣，维持了Manchette的完整性，并在精子鞭毛的组装中起作用。 PACRG通过与Meig1的特定结构结合将Meig1招募到Meig1，Meig1通过不同的域结合其他蛋白质（例如Spag16L）。域的突变/缺失将减少或废除与这些蛋白质的相互作用。 Meig1与具有差异结合亲密关系的PACRG和SPAG16L结合，以促进与Maig1与Maig1与Manchette相关的PACRG和其货物卸载（例如Spag16L）。我们预计MEIG1在体内具有几种磷酸化的氨基酸，并且磷酸化控制其与货物蛋白的亲和力。关于导致这些分子适当靶向和组装精子鞭毛的机制知之甚少。本应用中提出的研究将首次揭示分子 蛋白质伴随到鞭毛组装部位的基础，以及对正常精子发生必不可少的独特伴侣的结构/功能关系。