New Human Inducible Nitric Oxide Synthase-binding Proteins in Testis: Possible R

描述（由申请人提供）：一氧化氮（NO）是一种细胞信号分子，参与许多生物学过程，包括血管舒张、神经传递、巨噬细胞介导的免疫和致癌作用。一氧化氮（NO）在几乎所有类型的人类细胞中通过两种组成型和一种诱导型的一氧化氮合酶（NOS）而不同地形成。NO在生育中的可能作用已被许多最近的出版物提出。对大鼠的研究表明，NO对生殖和炎症性不孕都有正常的生理作用。NO具有明显的能力，以提高精子的活力和活动力在生育和不育的个人。然而，NO被认为是导致阻塞性无精子症的原因，阻塞性无精子症是不孕症的常见原因之一。诱导型一氧化氮合酶（induciblenitricoxidesynthase，iNOS）与组成型NOS一样，也参与蛋白质间的相互作用。进一步支持所有NOS的蛋白质-蛋白质相互作用，最近的定点诱变研究已经鉴定了人iNOS二聚化及其活性所必需的关键N-末端氨基酸残基。值得注意的是，三种NOS的氨基酸序列在它们的N-末端部分是不同的。利用iNOSs的N-末端作为酵母双杂交系统中的诱饵，可能会发现新的推定相互作用蛋白。由于NO在人体生理学中具有双重作用，因此在疾病预防方面需要NOS-同种型特异性抑制剂，例如，高血压，肺癌，不孕不育等，这个建议的目的是回答以下问题：什么其他蛋白质与iNOS相互作用，这种相互作用如何影响睾丸中NO的产生？该提案的假设是，iNOS与蛋白质相互作用，尚未确定，调节其在睾丸生理学中的作用。以下具体目标将研究这一假设：1）分离和鉴定编码在酵母双杂交系统中结合iNOS的蛋白质的基因; 2）使用哺乳动物细胞系在体内确认iNOS和推定的相互作用蛋白质之间的结合特异性;和3）使用RNA干扰研究表征iNOS调节对影响睾丸生理的NO合成的影响。因此，这项研究将扩大目前的知识，iNOS介导的NO合成在男性生育力的作用，并可能揭示其他致病机制的生殖系统，可能包括出生缺陷，由于男性生殖系突变。 公共卫生相关性：这项研究将扩大目前的知识的作用，诱导型一氧化氮合酶介导的NO合成在男性生育力，并可能揭示其他致病机制的生殖系统，可能包括出生缺陷，由于男性生殖系突变。

DESCRIPTION (provided by applicant): Nitric oxide (NO) is a cell signaling molecule and involved in numerous biological processes, including vasodilatation, neurotransmission, macrophage mediated immunity and carcinogenesis. Nitric oxide (NO) is differentially formed in almost all types of human cells by two constitutive and one inducible form of nitric oxide synthases (NOS). The possible role of NO in fertility has been proposed by many recent publications. Studies with rats have reported that NO has both normal physiological effect on reproduction and inflammation-based infertility. NO has apparent capability to improve sperm viability and motility in both fertile and infertile individuals. However, NO has been implicated as a cause to obstructive azoospermia, one of the common causes of infertility. Like constitutive NOSs, inducible nitric oxide synthase (iNOS) also takes part in protein-protein interaction. Further bolstering the protein-protein interaction of all NOSs, recent research with site- directed mutagenesis has identified critical N-terminal amino acid residues essential for dimerization of human iNOS and its activity. It is noteworthy that the amino acid sequence of three NOSs is different in their N-terminal portions. The use of iNOSs' N-terminus as bait in the yeast two-hybrid system could possibly identify new putative interacting proteins. Since NO has a dual role in human physiology, NOS-isoform specific inhibitors are needed in terms of disease prevention, e.g., hypertension, lung cancer, infertility, etc. This proposal is designed to answer the following question: What other proteins are interacting with iNOS and how does this interaction influence NO production in testis? The proposal's hypothesis is that iNOS interacts with proteins, not yet identified, that modulate its role in testis physiology. The following specific aims will investigate this hypothesis: 1) Isolate and identify genes that encode proteins that bind iNOS in the yeast two-hybrid system; 2) Confirm binding specificity between iNOS and putative interacting proteins in vivo using mammalian cell lines; and 3) Characterize the effects of iNOS modulation on NO synthesis affecting testis physiology employing RNA interference study. Consequently, this study will expand current knowledge of the role of iNOS mediated NO synthesis in male fertility, and possibly reveal other pathogenic mechanisms in the reproductive system that may include birth defects due to male germline mutations. PUBLIC HEALTH RELEVANCE: This study will expand current knowledge of the role of iNOS mediated NO synthesis in male fertility, and possibly reveal other pathogenic mechanisms in the reproductive system that may include birth defects due to male germline mutations.