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

• 批准号: 7992363
• 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
• 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/7992363
• 关键词: Address; Adopted; Affect; Agglutination; Alzheimer' 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; Health; 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

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.

描述（由申请人提供）：我们研究的长期目标是使用胱抑素作为分子模型来确定淀粉样蛋白聚集的生物学意义及其在附睾腔中的控制机制。聚集蛋白（也称为淀粉样蛋白）的异常积累在包括阿尔茨海默氏病在内的退行性疾病中很常见。睾丸和附睾中的淀粉样蛋白也与人类不育有关。蛋白质，包括半胱氨酸蛋白酶抑制剂，可以自我聚集并形成淀粉样蛋白，在聚集过程中采用常见的细胞毒性结构。由于上皮细胞对蛋白质的活跃分泌和对液体的深度清除，附睾管腔内很可能发生大分子拥挤，导致淀粉样蛋白聚集。然而，由于其在精子成熟中的关键作用，必须建立监视/清除机制来控制这一过程并防止细胞毒性聚集物的病理性积累。我们已经确定半胱氨酸蛋白酶抑制剂 CRES 和半胱氨酸蛋白酶抑制剂 C 作为高分子质量寡聚复合物存在于头腔中。我们还表明，CRES 与附睾管腔中的特定结构相关。此外，在体外，CRES 和胱抑素 C 形成可溶性淀粉样蛋白前体（可能具有细胞毒性）以及淀粉样原纤维。我们还确定，表达突变体 L68Q 半胱氨酸蛋白酶抑制剂 C（一种不稳定且高度淀粉样蛋白形成形式）的雄性小鼠不育，可能是由于管腔中过量的半胱氨酸蛋白酶抑制剂 C 寡聚复合物所致。这些新发现强调了控制附睾中蛋白质聚集的关键性质。附睾控制聚集的一种机制是通过转谷氨酰胺酶（TG）交联，导致蛋白质聚集成无毒构象。作为支持，我们显示了管腔中的 TG 活性，CRES 是 TG 的底物，并且 TG 将在头液中形成 CRES 低聚物。基于这些研究，我们提出淀粉样蛋白聚集发生在附睾腔内，并且TG交联等质量控制机制可防止有毒蛋白聚集体的积累，从而维持正常的附睾功能。我们还提出，损害这些保护机制的条件会对精子的成熟和功能产生负面影响。我们将通过以下方式解决这一假设：1）表征附睾腔中淀粉样蛋白类型的聚集； 2）检查淀粉样蛋白过度聚集的病理后果； 3）检查附睾细胞外质量控制机制。公共健康相关性：我们研究的目的是使用半胱氨酸蛋白酶抑制剂作为分子模型来确定淀粉样蛋白聚集的生物学意义及其在附睾腔中的控制机制。我们目标的完成将为我们了解淀粉样蛋白的形成提供有价值的信息，不仅在生殖道中及其在不孕中的潜在作用，而且在一般情况下，可能会导致与细胞外聚集蛋白相关的疾病（如阿尔茨海默病）的新疗法和/或标记物。