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: 8392182
• 负责人: Gail A Cornwall
• 金额: $ 28.37万
• 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 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8392182
• 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; 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

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.

我们研究的长期目标是确定淀粉样蛋白的生物学意义 聚集和机制，其控制在附睾腔使用半胱氨酸蛋白酶抑制剂作为分子模型。 聚集蛋白（也称为淀粉样蛋白）的异常积累在退行性变中很常见。 包括阿尔茨海默病在内的疾病。睾丸和附睾中的淀粉样蛋白也与 人类不育蛋白质，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， 在聚集过程中常见的细胞毒性结构。由于蛋白质的积极分泌和深刻的 上皮细胞清除液体，大分子拥挤可能发生在肾小管腔中。 附睾引起淀粉样蛋白聚集。然而，由于其在精子中的关键作用 成熟，监督/清除机制必须到位，以控制这一过程，并防止 细胞毒性聚集体的病理性积聚。我们已经确定，半胱氨酸蛋白酶抑制剂克雷斯和半胱氨酸蛋白酶抑制剂 C作为高分子量低聚复合物存在于头腔中。我们还表明， 克雷斯与附睾腔中的明确结构相关。此外，体外克雷斯和半胱氨酸蛋白酶抑制剂 C形成可溶性淀粉样蛋白前体，其可能是细胞毒性的，以及淀粉样蛋白原纤维。我们还 确定表达突变型L 68 Q胱抑素C的雄性小鼠，一种不稳定的和高度淀粉样蛋白生成的形式， 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。这些新发现 强调控制附睾中蛋白质聚集的关键性质。的一种机制 附睾可以通过转氨酶（TG）交联控制聚集，从而产生蛋白质 以无毒构象聚集。作为支持，我们已经显示了管腔中的TG活性，即克雷斯是一种 TG底物，且TG将在头流体中形成克雷斯低聚物。基于这些研究，我们建议 淀粉样蛋白聚集发生在附睾腔， 如TG交联，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可能会产生负面影响。 影响精子成熟和功能。我们将通过以下方式解决这一假设：1）表征淀粉样蛋白型 附睾腔内的聚集; 2）检查过量淀粉样蛋白的病理后果 聚集;和3）检查附睾细胞外质量控制的机制。叙事 我们研究的目的是确定淀粉样蛋白聚集的生物学意义， 以半胱氨酸蛋白酶抑制剂为分子模型，探讨其在附睾腔的调控机制。的完成 我们的目标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 生殖道及其在不孕症中的潜在作用，但一般而言，因此可能导致新的治疗方法和/或 与细胞外聚集蛋白相关的疾病如阿尔茨海默病的标志物。

项目成果

Nonpathological extracellular amyloid is present during normal epididymal sperm maturation.

• DOI: 10.1371/journal.pone.0036394
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Whelly S;Johnson S;Powell J;Borchardt C;Hastert MC;Cornwall GA
• 通讯作者: Cornwall GA

Amyloid properties of the mouse egg zona pellucida.

• DOI: 10.1371/journal.pone.0129907
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Egge N;Muthusubramanian A;Cornwall GA
• 通讯作者: Cornwall GA

Fertility defects in mice expressing the L68Q variant of human cystatin C: a role for amyloid in male infertility.

表达人胱抑素 C L68Q 变体的小鼠的生育缺陷：淀粉样蛋白在男性不育中的作用。

• DOI: 10.1074/jbc.m113.515759
• 发表时间: 2014
• 期刊: The Journal of biological chemistry
• 作者: Whelly,Sandra;Serobian,Gaiane;Borchardt,Clinton;Powell,Jonathan;Johnson,Seethal;Hakansson,Katarina;Lindstrom,Veronica;Abrahamson,Magnus;Grubb,Anders;Cornwall,GailA
• 通讯作者: Cornwall,GailA