Protein Amyloidogenesis in the Epididymis: Mechanisms and Biological Significance

附睾中蛋白质淀粉样蛋白生成：机制和生物学意义

基本信息

批准号：
8197861
负责人：
Gail A Cornwall
金额：
$ 29.89万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8197861
关键词：
Address Adopted Affect Agglutination Alzheimer&apos s Disease Amyloid Amyloid Fibrils Antibodies Apical Biochemical Biological Biological Assay Biological Process Calcium Cell Culture Techniques Cell physiology Cells Complex Crowding Cystatins Defect Degenerative Disorder Deposition Detection Disease Disease Marker Dyes Electron Microscopy Endocytosis Enzymes Epididymis Epithelium Excision Exhibits Exposure to Gel Goals Human Immunohistochemistry Impairment In Vitro Incubated Infertility L68Q cystatin C Lead Link Liquid substance Mechanics Messenger RNA Modeling Molecular Molecular Chaperones Molecular Conformation Molecular Models Molecular Sieve Chromatography Mus Mutant Strains Mice Nature Negative Staining Neurobiology Neurodegenerative Disorders Outcome Parkinson Disease Patients Process Protein Family Protein Precursors Protein Secretion Proteins Proteolysis Quality Control Recombinants Reproduction Role Sperm Agglutination Sperm Maturation Sperm Motility Structure Techniques Testing Testis Transglutaminases Trypsin Tubular formation Wild Type Mouse amyloid formation amyloidogenesis base biological systems cerebral artery crosslink cystatin As cytotoxic cytotoxicity extracellular in vivo male molecular mass molecular modeling monomer mutant novel post gamma-globulins prevent protein aggregate protein aggregation protein crosslink protein structure reproductive research study sperm analysis sperm cell sperm function

项目摘要

The long range objective of our studies is to determine the biological significance of amyloid-type protein aggregation and mechanisms for its control in the epididymal lumen using the cystatins as molecular models. The abnormal accumulation of aggregated protein, also known as amyloid, is common in degenerative diseases including Alzheimer's disease. Amyloid in the testis and epididymis has also been implicated in human infertility. Proteins, including the cystatins, which can self-aggregate and form amyloid adopt a common cytotoxic structure during their aggregation. Because of the active secretion of proteins and profound removal of fluid by the epithelium, macromolecular crowding is likely to occur in the tubular lumen of the epididymis causing amyloid-type protein aggregation. However, because of its critical role in sperm maturation, surveillance/clearance mechanisms must be in place to control this process and prevent a pathological accumulation of cytotoxic aggregates. We have established that the cystatins CRES and cystatin C are present in the caput lumen as high molecular mass oligomeric complexes. We have also shown that CRES is associated with defined structures in the epididymal lumen. Furthermore, in vitro CRES and cystatin C form soluble amyloid precursors, which may be cytotoxic, as well as amyloid fibrils. We have also determined that male mice expressing the mutant L68Q cystatin C, an unstable and highly amyloidogenic form, are infertile possibly due to excess cystatin C oligomeric complexes in the lumen. These novel findings emphasize the critical nature of controlling protein aggregation in the epididymis. One mechanism by which the epididymis may control aggregation is by transglutaminase (TG) crosslinking resulting in protein aggregates in a nontoxic conformation. In support we have shown TG activity in the lumen, that CRES is a substrate for TG, and that TG will form CRES oligomers in caput fluid. Based on these studies we propose that amyloid-type protein aggregation occurs in the epididymal lumen and that quality control mechanisms, such as TG crosslinking, prevent the accumulation of toxic protein aggregates thereby maintaining normal epididymal function. We also propose that conditions that impair these protective mechanisms can negatively impact sperm maturation and function. We will address this hypothesis by: 1) characterizing amyloid-type aggregation in the epididymal lumen; 2) examine the pathological consequences of excessive amyloid aggregation; and 3) examine mechanisms of extracellular quality control in the epididymis. Narrative The objective of our studies is to determine the biological significance of amyloid-type protein aggregation and mechanisms for its control in the epididymal lumen using the cystatins as molecular models. A completion of our aims will provide valuable information for our understanding of amyloid formation not only in the reproductive tract and its potential role in infertility but in general and as such may lead to new therapies and/or markers for diseases associated with extracellular aggregated proteins such as Alzheimer's disease.

我们研究的远程目标是确定淀粉样蛋白蛋白的生物学意义使用伴半胱氨酸蛋白酶作为分子模型，在附属管腔中对其控制的聚集和机制。聚集蛋白的异常积累，也称为淀粉样蛋白，在退行性中很常见包括阿尔茨海默氏病在内的疾病。睾丸和附睾中的淀粉样蛋白也与人类不孕症。蛋白质，包括囊蛋白，可以自我聚集并形成淀粉样蛋白采用在聚集过程中，常见的细胞毒性结构。由于蛋白质和深刻的积极分泌通过上皮去除液体，大分子拥挤可能发生在附睾引起淀粉样蛋白蛋白聚集。但是，由于其在精子中的关键作用必须建立成熟，监视/清除机制来控制这一过程并防止细胞毒性骨料的病理积累。我们已经确定Cystatins Cres和Cystatin C存在于高分子腔中，作为高分子质量寡聚复合物。我们还表明 CRE与附睾腔中定义的结构有关。此外，体外CRE和胱抑素 C形成可溶性淀粉样蛋白前体，可能是细胞毒性的，以及淀粉样蛋白原纤维。我们也有确定表达突变体L68Q胱抑素C的雄性小鼠，一种不稳定且高度淀粉部的形式，不育可能是由于管腔中过量的胱抑素C寡聚复合物所致。这些新颖的发现强调控制附睾中蛋白质聚集的临界性质。一种机制附睾可能控制聚集是通过转谷氨酰胺酶（TG）交联，导致蛋白质无毒构象的聚集体。在支持下，我们显示了管腔中的TG活性，Cres是一个 TG的底物，TG将在冠状流体中形成CRES低聚物。基于这些研究，我们提出淀粉样蛋白蛋白聚集发生在附睾腔内，质量控制机制，例如TG交联，防止有毒蛋白聚集体的积累，从而保持正常附子功能。我们还建议损害这些保护机制的条件可能会负面影响精子成熟和功能。我们将通过：1）表征淀粉样蛋白型的假设附睾腔中的聚集； 2）检查过度淀粉样蛋白的病理后果聚合; 3）检查附睾细胞外质量控制的机制。叙述我们研究的目的是确定淀粉样蛋白蛋白聚集的生物学意义和使用伴半胱氨酸蛋白酶作为分子模型在附睾管腔中进行控制的机制。完成我们的目标将为我们理解淀粉样蛋白形成提供宝贵的信息生殖道及其在不孕症中的潜在作用，但总的来说，可能会导致新的疗法和/或与细胞外骨外蛋白（如阿尔茨海默氏病）相关的疾病标记。