Identification of Phospho-proteins Regulating Sperm Function
调节精子功能的磷酸蛋白的鉴定
基本信息
- 批准号:9333123
- 负责人:
- 金额:$ 18.73万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:Adenylate CyclaseAllelesAntibodiesBicarbonatesBindingBiochemicalCatalytic DomainChemicalsCyclic AMPCyclic AMP-Dependent Protein KinasesCyclic NucleotidesDetectionEnzymesEpididymisEventFertilityFertilizationGenesGenetic EngineeringGerm CellsGoalsHandImpairmentIntracellular Second MessengerKnock-inKnock-in MouseKnockout MiceKnowledgeLeadMale InfertilityMass Spectrum AnalysisModificationMusMutant Strains MicePhosphoproteinsPhosphorylationPhosphotransferasesProcessProtein SubunitsProteinsRoleSecond Messenger SystemsSperm CapacitationSperm MaturationSperm MotilityTestisValidationadenylate kinaseanalogcell motilitychemical geneticsgenetic approachinorganic phosphatemalemutantnovelnovel strategiesreproductive tractsperm cellsperm functionsperm proteinzygote
项目摘要
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.
抽象的
众所周知,cAMP激活的蛋白激酶A(PKA)对于精子运动至关重要
和生育。 PKA的精子特异性催化亚基的靶向破坏(Cα2)或可溶性腺苷酸
循环酶(SAC)导致男性不育症。这两种敲打老鼠的精子都有运动障碍和缺乏
受精所需的能力和过度激活的能力。而PKA在精子中的作用
关于PKA的蛋白质底物知之甚少。确定身份
这些蛋白质中将使我们能够理解精子运动和生育能力的基础机制。
我们将使用一种新型的化学遗传方法来识别精子PKA底物。这种方法使用
PKA催化亚基的ATP结合域的一般设计的修改,使得
改良的酶识别其他激酶不接受的特定ATP类似物。磷酸化
在ATP模拟存在下底物的底物可以相对轻松地识别。我们有
手,带有针对PKA和小鼠催化亚基的修饰基因的敲入小鼠
精子囊的破坏。我们正在生成双重突变小鼠,表达模拟敏感的PKA
囊无背景。
我们提出了两个具体目标。在模拟敏感的敲入小鼠的第一个目标精子蛋白中
并且双突变小鼠将用ATP类似物磷酸化。磷酸化蛋白将是
通过模拟特异性抗体检测到使用串联MS进行鉴定。在第二个目标中
将确认蛋白质是有效的PKA底物,并确定其磷酸化的变化如何变化
发生在附睾的精子成熟期间和受精所需的精子激活事件中。
这些目标的成功执行应导致对CAMP和PKA的机械理解
调节精子功能。
项目成果
期刊论文数量(7)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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:
- 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
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:
- 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:
- 影响因子:0
- 作者:Dey S;Eisa A;Kline D;Wagner FF;Abeysirigunawardena S;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:
- 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
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SRINIVASAN VIJAYARAGHAVAN其他文献
SRINIVASAN VIJAYARAGHAVAN的其他文献
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{{ truncateString('SRINIVASAN VIJAYARAGHAVAN', 18)}}的其他基金
A knock-in mouse model for male fertility: basis for the mammal-specific protein phosphatase isoform PP1y2 in sperm
雄性生育能力的敲入小鼠模型:精子中哺乳动物特异性蛋白磷酸酶亚型 PP1y2 的基础
- 批准号:
10527437 - 财政年份:2022
- 资助金额:
$ 18.73万 - 项目类别:
A knock-in mouse model for male fertility: basis for the mammal-specific protein phosphatase isoform PP1y2 in sperm
雄性生育能力的敲入小鼠模型:精子中哺乳动物特异性蛋白磷酸酶亚型 PP1y2 的基础
- 批准号:
10675027 - 财政年份:2022
- 资助金额:
$ 18.73万 - 项目类别:
Protein Phosphatase Action in Mammalian Spermatogenesis and Sperm Function
蛋白磷酸酶在哺乳动物精子发生和精子功能中的作用
- 批准号:
8289861 - 财政年份:2012
- 资助金额:
$ 18.73万 - 项目类别:
Regulation of Sperm Function by Protein Phosphorylation
蛋白质磷酸化调节精子功能
- 批准号:
8051037 - 财政年份:2010
- 资助金额:
$ 18.73万 - 项目类别:
Regulation of Sperm Function by Protein Phosphorylation
蛋白质磷酸化调节精子功能
- 批准号:
7846469 - 财政年份:2009
- 资助金额:
$ 18.73万 - 项目类别:
The Role of 14-3-3 Proteins in Oogenesis and Early Development
14-3-3 蛋白在卵子发生和早期发育中的作用
- 批准号:
9170889 - 财政年份:2009
- 资助金额:
$ 18.73万 - 项目类别:
REGULATION OF SPERM FUNCTION BY PROTEIN PHOSPHORYLATION
蛋白质磷酸化对精子功能的调节
- 批准号:
6637061 - 财政年份:2001
- 资助金额:
$ 18.73万 - 项目类别:
REGULATION OF SPERM FUNCTION BY PROTEIN PHOSPHORYLATION
蛋白质磷酸化对精子功能的调节
- 批准号:
6521294 - 财政年份:2001
- 资助金额:
$ 18.73万 - 项目类别:
Regulation of Sperm Function by Protein Phosphorylation
蛋白质磷酸化调节精子功能
- 批准号:
7534804 - 财政年份:2001
- 资助金额:
$ 18.73万 - 项目类别:
Regulation of Sperm Function by Protein Phosphorylation
蛋白质磷酸化调节精子功能
- 批准号:
7659309 - 财政年份:2001
- 资助金额:
$ 18.73万 - 项目类别:
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