Protein Amyloidogenesis in the Epididymis: Mechanisms and Biological Significance

描述(申请人提供)：我们研究的长期目标是以半胱氨酸氨基转移酶为分子模型，确定淀粉样蛋白在附睾腔内聚集的生物学意义及其调控机制。聚集蛋白的异常积累，也被称为淀粉样蛋白，在包括阿尔茨海默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，可以自我聚集并形成淀粉样蛋白，在聚集过程中采用共同的细胞毒性结构。由于蛋白质的活跃分泌和上皮对液体的大量排出，大分子可能在附睾管腔内发生大分子拥挤，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，必须建立监测/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聚。我们已经证实，胱抑素CRES和胱抑素C以高分子质量低聚物的形式存在于头腔中。我们还发现CRES与附睾腔中明确的结构有关。此外，在体外，CRES和胱抑素C形成可能具有细胞毒性的可溶性淀粉样前体，以及淀粉样纤维。我们还确定，表达突变的L68Q胱抑素C的雄性小鼠是不育的，可能是由于管腔中过量的胱抑素C寡聚复合体所致。这些新的发现强调了控制附睾中蛋白质聚集的关键性质。附睾可能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在无毒构象中。在支持方面，我们已经证明了管腔中的TG活性，CRES是TG的底物，并且TG将在头状液中形成CRES低聚物。基于这些研究，我们认为淀粉样蛋白聚集发生在附睾腔内，质量控制机制，如TG交联，防止有毒蛋白聚集，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可能会对精子成熟和功能产生负面影响。我们将通过：1)表征附睾腔中的淀粉型聚集；2)检查过度的淀粉样聚集的病理后果；以及3)检查附睾胞外质量控制的机制来解决这一假说。公共卫生相关性：本研究的目的是以半胱氨酸类药物为分子模型，确定附睾腔内淀粉样蛋白聚集的生物学意义及其调控机制。我们目标的完成将为我们了解淀粉样蛋白不仅在生殖道中的形成及其在不孕症中的潜在作用提供有价值的信息，并因此可能导致与细胞外聚集蛋白相关的疾病的新疗法和/或标记物，如阿尔茨海默病。

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.