Identification of Phospho-proteins Regulating Sperm Function

调节精子功能的磷酸蛋白的鉴定

• 批准号: 9333123
• 负责人: SRINIVASAN VIJAYARAGHAVAN
• 金额: $ 18.73万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-17 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333123
• 关键词: Adenylate Cyclase; Alleles; Antibodies; Bicarbonates; Binding; Biochemical; Catalytic Domain; Chemicals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotides; Detection; Enzymes; Epididymis; Event; Fertility; Fertilization; Genes; Genetic Engineering; Germ Cells; Goals; Hand; Impairment; Intracellular Second Messenger; Knock-in; Knock-in Mouse; Knockout Mice; Knowledge; Lead; Male Infertility; Mass Spectrum Analysis; Modification; Mus; Mutant Strains Mice; Phosphoproteins; Phosphorylation; Phosphotransferases; Process; Protein Subunits; Proteins; Role; Second Messenger Systems; Sperm Capacitation; Sperm Maturation; Sperm Motility; Testis; Validation; adenylate kinase; analog; cell motility; chemical genetics; genetic approach; inorganic phosphate; male; mutant; novel; novel strategies; reproductive tract; sperm cell; sperm function; sperm protein; zygote

Abstract It has been long known that protein kinase A (PKA) activated by cAMP is essential for sperm motility and fertility. Targeted disruption of the sperm specific catalytic subunit of PKA (Cα2) or the soluble adenylyl cyclase (sAC) results in male infertility. Sperm from both these knock out mice have impaired motility and lack the ability to undergo capacitation and hyperactivation required for fertilization. While the role of PKA in sperm function is well established little is known about the protein substrates of PKA. Determination of the identities of these proteins will enable us to understand mechanisms underlying sperm motility and fertility. We will use a novel chemical-genetic approach to identify sperm PKA substrates. This approach uses a genetically engineered modification of the ATP binding domain of the catalytic subunit of PKA such that the modified enzyme recognizes specific ATP analogues that are not accepted by other kinases. Phosphorylation of substrates in the presence of the ATP analog enables their identification with relative ease. We have, in hand, the knock-in mice harboring the modified gene for the catalytic subunit of PKA and mice with a targeted disruption of the sperm sAC. We are generating double mutant mice expressing the analog sensitive PKA on a sAC null background. We propose two specific aims. In the first aim sperm proteins from the analog sensitive knock-in mice and the double-mutant mice will be phosphorylated with an ATP analog. Phosphorylated proteins will be detected by analog-specific antibodies followed by their identification using tandem MS. In the second aim we will confirm that the proteins are valid PKA substrates and determine how changes in their phosphorylation occur during sperm maturation in the epididymis and during sperm activation events required for fertilization. Successful execution of these aims should lead to a mechanistic understanding of how cAMP and PKA regulate sperm function.

摘要 众所周知，cAMP激活的蛋白激酶A(PKA)对精子运动是必不可少的。 和生育能力。靶向破坏精子特异性蛋白激酶A催化亚单位(Cα2)或可溶性腺苷 环化酶(SAC)导致男性不育。这两种基因敲除小鼠的精子都削弱了活力，缺乏活力。 受精能力受精所需的获得能力和过度激活的能力而PKA在精子中的作用 功能已经确定，对PKA的蛋白底物知之甚少。身份的确定 这些蛋白质的研究将使我们能够了解精子运动和生育的潜在机制。 我们将使用一种新的化学遗传学方法来鉴定精子PKA底物。此方法使用 对PKA催化亚单位的ATP结合域进行基因工程修饰，使其 修饰的酶识别其他酶不能接受的特定的ATP类似物。磷酸化 在ATP类似物存在的情况下对底物进行识别，使其能够相对轻松地进行识别。我们有，在 另一方面，携带PKA催化亚单位修饰基因的敲入小鼠和具有靶向 破坏精子囊。我们正在产生双突变小鼠，表达模拟敏感的PKA SAC空背景。 我们提出了两个具体目标。第一个目标是来自模拟敏感敲入小鼠的精子蛋白 双突变小鼠将被一种三磷酸腺苷类似物磷酸化。磷酸化的蛋白质将是 用模拟特异性抗体进行检测，然后用串联质谱仪进行鉴定。 将确认这些蛋白质是有效的PKA底物，并确定它们的磷酸化如何变化 发生在附睾精子成熟期间和受精所需的精子激活事件期间。 成功执行这些目标应该导致对CAMP和PKA如何机械地理解 调节精子功能。

项目成果

Cyclic AMP and glycogen synthase kinase 3 form a regulatory loop in spermatozoa.

• DOI: 10.1002/jcp.26557
• 发表时间: 2018-09
• 期刊: Journal of cellular physiology
• 影响因子: 5.6
• 作者: Dey S;Goswami S;Eisa A;Bhattacharjee R;Brothag C;Kline D;Vijayaraghavan S
• 通讯作者: Vijayaraghavan S

The protein YWHAE (14-3-3 epsilon) in spermatozoa is essential for male fertility.

• DOI: 10.1111/andr.12865
• 发表时间: 2021-01
• 期刊: Andrology
• 影响因子: 4.5
• 作者: Eisa A;Dey S;Ignatious A;Nofal W;Hess RA;Kurokawa M;Kline D;Vijayaraghavan S
• 通讯作者: Vijayaraghavan S

Regulators of the protein phosphatase PP1γ2, PPP1R2, PPP1R7, and PPP1R11 are involved in epididymal sperm maturation.

• DOI: 10.1002/jcp.27130
• 发表时间: 2019-03
• 期刊: Journal of cellular physiology
• 影响因子: 5.6
• 作者: Goswami S;Korrodi-Gregório L;Sinha N;Bhutada S;Bhattacharjee R;Kline D;Vijayaraghavan S
• 通讯作者: Vijayaraghavan S

Roles of glycogen synthase kinase 3 alpha and calcineurin in regulating the ability of sperm to fertilize eggs.

• DOI: 10.1096/fj.201902163r
• 发表时间: 2020-01
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Dey S;Eisa A;Kline D;Wagner FF;Abeysirigunawardena S;Vijayaraghavan S
• 通讯作者: Vijayaraghavan S