The role of R2D2/AKAP interactions in fibrous sheath function.

R2D2/AKAP 相互作用在纤维鞘功能中的作用。

• 批准号: 8883664
• 负责人: DANIEL W CARR
• 金额: $ 6.14万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8883664
• 关键词: A kinase anchoring protein; Address; Animal Model; Appearance; Binding; Bronchi; Cells; Cilia; Clinic; Cyclic AMP-Dependent Protein Kinases; Data; Defect; Development; Dimerization; Disease; Docking; Enzymes; Event; Exhibits; Fertility; Fiber; Flagella; Frequencies; Genetic; Glean; Glycolysis; Goals; Human; Infertility; Knock-out; Knowledge; Laboratories; Lead; Link; Location; Male Contraceptions; Male Contraceptive Agents; Male Infertility; Mechanics; Medical; Modeling; Molecular; Movement; Mucociliary Clearance; Mus; Patients; Play; Positioning Attribute; Protein Binding; Protein Kinase; Protein Kinase Interaction; Proteins; Publishing; Regulation; Research; Role; Sampling; Signal Transduction; Sperm Tail; Spermatogenesis; Spermiogenesis; Structural defect; Structure; Tail; Testing; Translations; Tyrosine Phosphorylation; Ursidae Family; Work; asthenospermia; cell motility; fibrous protein; flexibility; human male; insight; male; men; mouse model; novel; prevent; protein kinase A kinase; protein protein interaction; public health relevance; scaffold; sperm cell; sperm function; treatment strategy

DESCRIPTION (provided by applicant): Flagella and cilia of all cells are regulated by the cyclic AMP (cAMP)-dependent protein kinase (PKA) though the mechanisms of this regulation are unclear. PKA is targeted by interactions with A-kinase anchoring proteins (AKAPs) via a dimerization/docking domain on the regulatory (R) subunit of PKA. We have identified four novel mammalian proteins that share the RII dimerization/docking (R2D2) domain of PKA and therefore also bind to AKAPs. Two of these proteins (ASP and ROPN1) interact with AKAPs in a manner similar to PKA; however, outside the docking domain, R2D2 proteins bear little or no similarity to PKA suggesting they have distinct functions. AKAPs 3 and 4, PKA and ROPN1 are all located in the fibrous sheath (FS) of spermatozoa. The FS is a flagellar cytoskeletal structure unique to sperm that surrounds the outer dense fibers and axoneme in the principal piece of the flagella. Originally thought to function primarily as a support structure for the flagella, we now know that the FS plays a critical role in motility regulation by acting as a scaffold for glycolyti and signaling enzymes such as PKA. Disruptions in FS-associated proteins are reliably associated with asthenozoospermia and impaired fertility both in the laboratory and in the clinic. Though few genetic factors have been directly linked to human male infertility, importantly for this work, defects in PKA, AKAPs and ROPN1 expression have all been directly linked to male-factor infertility in humans. We have recently published two studies using genetically modified mouse lines that lack ASP and/or ROPN1. Using these models, we have demonstrated that lack of ROPN1 results in a significant reduction in the percentage of progressively motile sperm and impaired fertility in male mice. In the absence of both ASP and ROPN1, males are infertile due to structural defects in the principal piece of the flagellum that appear to be contained to the fibrous sheath (FS), resulting in complete immotility. We hypothesize that ROPN1 participates in fibrous sheath formation by anchoring AKAPs during spermatogenesis. To test this hypothesis, we will characterize the expression and localization of these proteins during spermatogenesis, identify specific structural defects present in sperm lacking ASP/ROPN1, and determine how AKAP binding to PKA, ASP and ROPN1 is regulated. The goal of this proposal is to determine the role of ROPN1/ASP in the development and function of the fibrous sheath. Increased knowledge of essential protein interactions in FS will provide insight into asthenozoospermia and infertility in men. We have recently demonstrated that mice lacking ASP exhibit reduced basal ciliary beat frequency in the bronchus; thus our results may also have significance in ciliary function and disease. The long-term goal of our laboratory is to elucidate the functions and ASP and ROPN1 in the regulation of ciliary and flagellar motility with the ultimate objective of developing male contraceptives and treatments for diseases involving impaired mucociliary clearance and male factor infertility.

描述（由申请人提供）：所有细胞的鞭毛和纤毛受环环（CAMP）依赖性蛋白激酶（PKA）调节，尽管该调节的机制尚不清楚。 PKA是通过与PKA的调节（R）亚基上的二聚化/对接结构域与A-激酶锚定蛋白（AKAP）相互作用的目标。我们已经确定了四种新型的哺乳动物蛋白质，它们共享PKA的RII二聚/对接（R2D2）结构域，因此也与AKAP结合。这些蛋白质中的两个（ASP和ROPN1）以类似于PKA的方式与AKAP相互作用。但是，在扩展区之外，R2D2蛋白与PKA几乎没有或没有相似性，表明它们具有不同的功能。 Akaps 3和4，PKA和ROPN1都位于精子的纤维鞘（FS）中。 FS是鞭毛细胞骨架结构 围绕鞭毛的主要纤维和轴突的精子独有。最初认为主要是鞭毛的支撑结构，我们现在知道FS通过充当糖糖和信号酶（例如PKA）而在运动调控中起关键作用。 FS相关蛋白的干扰与嗜硫代植物经皮有可靠的相关性，并且在实验室和诊所中的生育能力受损。尽管很少有与人类男性不育症直接相关的遗传因素，但对于这项工作，PKA，AKAP和ROPN1表达的缺陷都与人类的男性因素不育直接相关。我们最近使用缺乏ASP和/或ROPN1的基因修饰的小鼠系发表了两项研究。使用这些模型，我们已经证明缺乏ROPN1导致逐渐运动精子的百分比显着降低，并且雄性小鼠的生育能力受损。在没有ASP和ROPN1的情况下，由于鞭毛的主要部分中的结构缺陷，男性是不育的，似乎包含在纤维鞘中（FS），从而导致完全免疫。我们假设ROPN1通过在精子发生过程中锚定AKAP来参与纤维鞘形成。为了检验该假设，我们将表征这些蛋白质在精子发生过程中的表达和定位，确定缺乏ASP/ROPN1精子中存在的特定结构缺陷，并确定如何调节AKAP与PKA，ASP和ROPN1的结合。该提案的目的是确定ROPN1/ASP在纤维鞘的开发和功能中的作用。 FS中对基本蛋白质相互作用的知识的增加将为您的痛苦症和男性不孕症提供洞察力。我们最近证明，缺乏ASP的小鼠在支气管中表现出降低的基底睫状跳频率。因此，我们的结果在睫状功能和疾病中也可能具有重要意义。我们实验室的长期目标是阐明在调节睫状和鞭毛运动中的功能和ASP和ROPN1，其最终目的是开发涉及粘膜清除率和男性因子不育的疾病的男性避孕药和治疗方法。