蛋白质酰基转移酶 ZDHHC3 在卵母细胞成熟过程中的功能研究

发布时间：2018-01-01 14:38

本文关键词：蛋白质酰基转移酶 ZDHHC3 在卵母细胞成熟过程中的功能研究　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：有性生殖生物通过配子来传递遗传信息到子代。其配子的产生需要通过减数分裂从而在受精后维持体细胞染色体数目稳定。绝大多数脊椎动物的卵母细胞在生长完成后长期停滞于减数分裂的前期(也称为G2期)。G2期停滞是卵母细胞的关键检验点之一。在激素的刺激下,卵母细胞重启减数分裂进程的过程被称为G2/M期转换。减数分裂G2/M期转换的调控对于卵子质量和受精至关重要。G2/M期转换是如何调控的是发育生物学的重要科学问题之一。G蛋白偶联(GPCR-Gas-Adenylyl cyclase-cAMP)信号通路在卵母细胞减数分裂停滞中具有重要作用,该通路在维持人、小鼠和非洲爪蛙卵母细胞减数分裂停滞中具有高度保守性。蛋白质修饰是很多生物学过程的调控方式,蛋白质棕榈酰化也调控了GPCR信号通路,我们此前的研究表明长链脂酰辅酶A合成酶Acs11b是非洲爪蛙卵母细胞减数分裂停滞所必需的。Acs11b可能是为下游蛋白酰基转移酶提供棕榈酰辅酶A,从而棕榈酰化修饰减数分裂停滞所必需的关键蛋白。在本研究中,提供的实验证据表明ZDHHC3符合上述蛋白棕榈酰基转移酶的特征,在卵母细胞中敲降ZDHHC3会导致成熟加快和对孕酮反应更加敏感。在ZDHHC3敲降卵母细胞中Gas蛋白的棕榈酰化水平显著下降。进一步的研究发现,爪蛙卵母细胞中Gas蛋白发生棕榈酰化修饰的主要位点是第三位的半胱氨酸残基,而缺乏棕榈酰基转移酶活性的ZDHHC3突变体不能够棕榈酰化Gas。因此,ZDHHC3是Gas棕榈酰化所必需的因子,也是通过调控Gas蛋白的棕榈酰化参与卵母细胞减数分裂停滞的维持的关键蛋白质。为了验证蛋白质棕榈酰化修饰是脊椎动物卵母细胞成熟保守的调控机制,随后的实验用特异抑制剂直接抑制蛋白质的棕榈酰化,结果也导致了小鼠卵母细胞的减数分裂恢复,而同时添加酰苷酸环化酶激动剂可以部分阻断抑制剂处理所导致的减数分裂恢复。以上结果说明蛋白质的棕榈酰化也参与了小鼠卵母细胞G2期的维持,蛋白质棕榈酰化可能也是通过GPCR-Gas-Adenylyl cyclase-cAMP信号通路来调控小鼠卵母细胞的减数分裂进程。功能缺失实验结果表明,酰基转移酶ZDHHC3和ZDHHC7参与维持小鼠卵母细胞G2期停滞,并很可能棕榈酰化其中的关键调控蛋白。进一步的研究显示这一调控的主要目标蛋白质可能是Gas,而且酰基转移酶通过调控Gαs在卵母细胞中的定位来影响其功能的发挥。为了深入研究蛋白酰基转移酶ZDHHC3和ZDHHC7的生理功能,构建了ZDHHC3和ZDHHC7基因敲除小鼠。虽然单独敲除ZDHHC3或者ZDHHC7对雌鼠的生殖能力没有显著影响,但同时敲除ZDHHC3和ZDHHC7的小鼠却无法存活,提示ZDHHC3和ZDHHC7具有功能补偿效应,或者ZDHHC3和ZDHHC7在小鼠发育过程中有其它重要的生理功能。ZDHHC3和ZDHHC7在小鼠卵母细胞发育过程中的具体功能将需要建立ZDHHC3和ZDHHC7卵母细胞条件性敲除小鼠进一步研究获得答案。本研究揭示了蛋白酰基转移酶ZDHHC3在卵母细胞减数分裂停滞中的重要作用以及G蛋白信号通路调控的新机制。这一发现将有助于更好地理解卵母细胞的发育过程,并且对于阐明蛋白质棕榈酰化修饰调控机制具有重要的意义。
[Abstract]:Sexual reproduction by biological gametes to transfer genetic information to offspring. The gametes through meiosis to maintain somatic chromosome number stability after fertilization. In the vast majority of early vertebrate oocytes from meiotic arrest in growth after long-term (also known as G2).G2 arrest is one of the key inspection point oocytes. In hormone stimulated oocytes during the resumption of meiosis process called G2/M transition. Control of meiosis G2/M transition to the quality of eggs and fertilized vital.G2/M transition is how regulation is an important question in developmental biology of.G protein coupled signal (GPCR-Gas-Adenylyl cyclase-cAMP) pathway plays an important role in oocyte meiotic arrest in this pathway in the maintenance of human, mouse and Xenopus frog oocytes meiosis arrest It is highly conserved. Protein modification is the regulation of many biological processes, palmitoylation also regulates GPCR signaling pathway, our previous study indicates that long chain acyl coenzyme A synthetase Acs11b is the African clawed frog oocytes meiosis arrest required.Acs11b may provide a palmitoyl coenzyme A downstream protein acyltransferase, thereby palmitoylation modification of meiosis key protein required for stagnation. In this study, experimental evidence is provided to demonstrate the characteristics of ZDHHC3 with the above protein palmitoyltransferase, knockdown of ZDHHC3 will lead to faster and more sensitive to the mature progesterone response in oocytes. Palmitoylation level of knockdown of Gas protein in oocytes the ZDHHC3 decreased significantly. Further research found that the claw frog oocytes of Gas protein in the primary sites of palmitoylation modification of cysteine is third bits The amino acid residues, and the lack of ZDHHC3 mutant palmitoyl transferase activity can not palmitoylated Gas. therefore, factor ZDHHC3 is required for Gas palmitoylation, also through regulating Gas protein palmitoylation of key proteins in oocyte meiotic arrest maintenance. In order to verify the palmitoylation modification regulation mechanism the vertebrate oocyte maturation conservative, subsequent experiments with specific inhibitors directly inhibit protein palmitoylation, results also led to the meiosis of mouse oocytes split recovery, while adding adenylyl cyclase agonist could partially block meiotic inhibitor treatment caused by the split recovery. The above results showed that the protein of palm acylation is also involved in the maintenance of mouse G2 oocytes, palmitoylation may also through the cyclase-cAMP pathway to GPCR-Gas-Adenylyl The regulation of mouse oocyte meiosis process. The experimental results show that the loss of function, ZDHHC3 and ZDHHC7 acyltransferase involved in the maintenance of mouse oocyte G2 arrest, and is likely to be a key regulatory protein palmitoylation of them. Further research shows that the main purpose of this subject is a regulatory protein may be Gas, but also through the positioning control of G acyltransferase alpha s in the oocytes to affect its function. In order to study the physiological function of protein acyltransferase ZDHHC3 and ZDHHC7, constructed ZDHHC3 and ZDHHC7 gene knockout mice. Although separate knockdown of ZDHHC3 or ZDHHC7 on the reproductive capacity of female rats had no significant effect, but at the same time ZDHHC3 and ZDHHC7 knockout mice are can not survive, suggesting that ZDHHC3 and ZDHHC7 have the function of compensation effect, or ZDHHC3 and ZDHHC7 during mouse development has other important physiological functions of.ZDHHC3 and ZDHHC 7 specific functions in the process of mouse oocyte development will require the establishment of ZDHHC3 and ZDHHC7 oocytes conditional knockout mice for further study to get answers. This study reveals a new mechanism of protein acyltransferase ZDHHC3 in oocyte meiotic arrest and the important role of G protein signal pathway. This discovery will have the development process of helping to better understand the oocytes, and is of great significance to clarify the palmitoylation modification regulation mechanism.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q26

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