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

基本信息

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
项目类别：
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

项目摘要

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（申请人提供）：精子的活力使精子能够到达卵子进行受精，正常的精子活力很大程度上取决于鞭毛的形成和功能。人们提出，一种细长的精子细胞特有的 manchette 在精子鞭毛的形成中发挥着核心作用。减数分裂表达蛋白 1 (MEIG1) 是调节 manchette 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) 的结合伴侣，SPAG16L 是一种精子尾轴丝蛋白；它还结合 Parkin 共调控基因的蛋白质产物 (PACRG)。 Pacrg 缺陷小鼠的生殖表型与 Meig1 突变小鼠的生殖表型相似。 PACRG 在减数分裂后表达并定位于 manchette。 MEIG1 和 SPAG16L 存在于精母细胞的细胞质中。然而，这两种蛋白质在伸长的精子细胞中都会迁移到 manchette。 MEIG1 在 Pacrg 缺陷的伸长精子细胞中失去了其 manchette 定位。在 Meig1 缺陷小鼠中，SPAG16L 失去了 manchette 定位。这些观察结果表明 MEIG1 在蛋白质护送/靶向中发挥作用。 MEIG1仅由88个氨基酸组成，并且利用生物信息学工具未鉴定出任何功能域。它在体内被磷酸化，磷酸化可能会调节 MEIG1 功能，尽管这一点尚未确定。我们使用核磁共振 (NMR) 进行的初步研究表明，MEIG1 形成了一种独特的结构，为与其他蛋白质相互作用提供了大的表面积，并且一些氨基酸，包括芳香区和带电区域中的几个潜在磷酸化位点，可以形成蛋白质-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。我们提出三个目标：1）在体内和体外解剖SPAG16L/MEIG1/PACRG复合物； 2) 鉴定介导与 PACRG 和 SPAG16L 相互作用的 MEIG1 结构域； 3）。确定翻译后修饰在 MEIG1 功能中的作用。我们假设 MEIG1 作为伴侣，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 通过与 MEIG1 的特定结构域结合将 MEIG1 招募到 manchette，而 MEIG1 通过不同的结构域结合其他蛋白，例如 SPAG16L。结构域的突变/删除将减少或消除 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 在体内具有多个磷酸化氨基酸，磷酸化控制其与货物蛋白的亲和力。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。本申请中提出的研究将首次揭示分子蛋白质护送至鞭毛组装位点的基础，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。