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圆形精子细胞特异基因rsb66编码的蛋白质及睾丸新基因hsd14的功能研究
【作者】 陈叙；
【作者基本信息】 中国协和医科大学 ， 生物化学， 2007， 博士
【摘要】 为了分离、鉴定精子发生过程的新基因,本实验室在国际上首次用激光捕获显微切割（Laser Capture Microdissection）技术分离获得了大量的大鼠初级精母细胞和圆形精子细胞,提取了这两种细胞的总RNA后,通过抑制性消减杂交技术建立了初级精母细胞消减圆形精子细胞的消减cDNA文库（RSB）。本论文的研究对象rsb66基因就是从RSB文库分离得到的一条新基因。rsb66的cDNA全长为564bp,GenBank注册号为AY121839。开放阅读框编码含168个氨基酸残基的多肽链。Northern blot显示该基因特异性表达于大鼠睾丸中。rsb66和它的人同源基因在核苷酸水平上的同源性为88.2%,在氨基酸水平上的同源性高达92.3%,提示该基因在进化上高度保守。在先前的研究中,我们应用酵母双杂交系统,以全长的RSB66为诱饵,筛选人睾丸cDNA文库,共获得2个与RSB66相互作用的分子:HSD45（AY604176）和HSD46（AY604177）。hsd45的cDNA全长为985bp,开放读码框编码221个氨基酸残基的多肽链。BLAST分析发现hsd45与一新基因完全相同。该基因的编码产物能通过与cyclinA1相互作用抑制Cdk的活性,故命名为INCA1（Inhibitor of Cdk interacting with Cyclin A1）。本研究中,我们运用GST-pull down和免疫共沉淀首先分别在体外和体内两种体系中确证了RSB66和HSD45的相互作用。前期工作中对蛋白质晶体结构的解析结果提示:RSB66的Tyr-117和His-119残基可能形成一个活性位点。在进一步的功能研究中,我们探索了这两个位点对RSB66功能的影响。我们构建了点突变体mRSB66（117Ala119Ala）及两个截短体106RSB66和128RSB66的重组表达质粒。免疫共沉淀的结果显示:外源性的RSB66和mRSB66虽都能与HeLa细胞中内源性的HSD45相互作用,但RSB66全长与HSD45的相互作用明显较mRSB66强;然而实验中未检测到截短体106RSB66和128RSB66与HSD45的相互作用。结果提示:Tyr-117和His-119残基在RSB66与HSD45的相互作用中起到了一定的作用,但仅这两个位点的突变并不能完全抑制RSB66和HSD45之间的相互作用;位于RSB66结构C端的α-螺旋在二者的相互作用中也有重要作用。此外,免疫荧光的结果提示:HSD45在HeLa细胞的核和胞浆中都有表达,RSB66主要表达于HeLa细胞的胞浆中,二者在HeLa细胞的胞浆中共定位;突变体mRSB66、106RSB66和128RSB66也都主要定位于HeLa细胞的胞浆中;无论是点突变体还是截短体的定位与RSB66全长相比,没发生明显的改变。提示Tyr-117和His-119残基以及C端的α-螺旋对于RSB66的定位没有明显的影响。据报道:HSD45（INCA1）能与cyclin A1发生相互作用并抑制cyclin A1/Cdk2复合物的激酶活性,提示其可以通过对Cdk活性的调控影响细胞周期的进程。因此,我们推测RSB66也可能通过与HSD45的相互作用影响细胞周期。流式细胞分析的结果显示:过量表达RSB66能够引起HeLa细胞的细胞周期出现明显的G2/M期延迟的现象;mRSB66的过量表达也能引起了类似的现象,但G2/M期的延迟程度比全长RSB66轻。通过体外激酶活性实验我们发现:纯化的RSB66能抑制以histone H1为底物的cyclin A1/Cdk2复合物的激酶活性,Western Blotting显示cyclin A1/Cdk2激酶活性的下调与cyclin A1、Cdk2和HSD45（INCA1）表达水平的变化无关。纯化的mRSB66、106RSB66和128RSB66不同程度地抑制cyclin A1/Cdk2激酶活性。我们推测这可能是RSB66引起HeLa细胞的细胞周期G2/M期延迟的原因。hsd14基因是本组借助电子克隆的方法,以睾丸特异性的EST为基础克隆得到的一个睾丸特异性表达的新基因,其cDNA全长为1107bp,GenBank注册号为AY251164,读码框编码288个氨基酸,包括3个dsRNA结合结构域。BLASTP分析的结果表明HSD14的氨基酸序列除了缺少了N端的25个氨基酸外与PACT（PKR activating protein）蛋白完全一致,这提示HSD14和PACT有可能是同一基因在不同组织中的不同剪接体。PACT能通过激活PKR导致细胞蛋白质的合成受阻,从而诱导细胞凋亡,还可能参与RNAsilencing途径的调节。本研究旨在结合PACT已知的功能,探索HSD14在生精过程中的作用。在以纯化的His6-HSD14免疫BALB/c小鼠和以HSD14的多克隆抗体显微注射BALB/c小鼠睾丸曲细精管时,我们观察到小鼠睾丸曲细精管组织结构紊乱,生精细胞退化、凋亡的现象。此外,在应用酵母双杂交系统,以HSD14为诱饵筛选其相互作用蛋白质时,观察到HSD14的表达使酵母菌出现了明显的生长抑制（anti-growthphenotype）。利用精原细胞株GC-1spg,我们成功地构建了hsd14小鼠同源基因的内源性稳定RNAi细胞株。本实验组经过多年的研究和探索成功建立了小鼠体内睾丸曲细精管显微注射和以精原细胞系移植为基础的体内RNAi的技术平台。以上的初步结果和成功构建的RNAi稳定细胞株,为我们充分利用该技术平台,对HSD14在生精过程的功能进行深入的研究奠定了基础。
【Abstract】 In order to identify novel genes in spermatogenesis,tens of thousands of rat spermatocytes and round spermatids had been isolated by Laser Capture Microdisseetion (LCM) in our laboratory,and total RNA of these two cells had been extracted.Then the Suppression Subtracted Hybridization(SSH) library of round spermatid-specific cDNAs against those of primary spermatocyte(RSB) had been constructed,rsb66 which is investigated in present study was one of those genes obtained from RSB subtracted library.Its full-length cDNA comprises 564bp and was assigned the GenBank accession number AY121839.The open reading frame encodes a polypeptide consisting of 168 amino acid residues.Northern blot showed that rsb66 was specifically expressed in rat testis.The coding sequences of rsb66 and its human homolog showed 88.2%homology on the nucleotide and 92.3%identity on the amino acid level.Such highly homolog between human and rodents suggests that this gene is conserved in the evolution.In previous studies,a Yeast Two-Hybrid system had been used to screen the interaction partners of full-length RSB66 in human testis eDNA library.As a result,two positive clones were identified and designated as hsd45(AY604176) and hsd46(AY604177).The full-length cDNA of hsd45 comprises 985bp and the open reading frame encodes a polypeptide consisting of 221 amino acid residues.The result of BLASTP analysis showed that HSD45 was identical to INCA1(Inhibitor of CDK interacting with Cyelin A1),a novel cyclin A1/CDK2 interaction partner in a Yeast-Triple Hybrid approach.In our study the interaction between RSB66 and HSD45 were firstly eomfirmed both in vitro and in vivo by GST-pull down and co-immunoprecitetation,respectively.According to the previous results in crystal structure of RSB66,Tyr-117 and His-119 residues may form an active site which is essential for the function of RSB66.To investigate the influence of these two sites on the function of RSB66,one dot mutant mRSB66(117Ala119Ala) and tow deletions(106RSB66 and 128RSB66) were constructed.Results of co-immunoprecitetation showed that mRSB66 had weaker interaction with HSD45 compareing with wild-type RSB66, while the interaction between two deletions of RSB66 and HSD45 could not be detectable.It indicated Tyr-117 and His-ll9 residues had effected on the interaction between RSB66 and HSD45,and theα-helix in C-terminal was also crucial for the interaction.By immunoflouresence we found HSD45 located in both nuclear and cytoplasm of HeLa,it co-localized with RSB66 in cytoplasm.All the mutants showed similar localization to wild-type RSB66 which indicated that the mutants had no apparent effect on the localization of the protein.It was reported that HSD45(INCA1) can interacted with eyclin A1 and therefore inhibit the kinase activity of Cdk.This suggested its participation in regulation of the cell cycle.We presumed that RSB66 could also regulate the cell cycle through its interaction with HSD45. Results of flow cytometry showed that obvious G2/M delay was induced by overexpression of RSB66 in HeLa,which was also found in overexpression of mRSB66.However,the degree of G2/M delay was alleviated in the latter one.In vitro kinase avtivity assay using histone H1 as substrates demonstrated that cyclin A1/Cdk2 kinase activity was inhibited by purified RSB66.And this down-regulation of kinase activity did not due to the variation of the expression levels of cyclin A1,Cdk2 and HSD45. Moreover,the kinase activity was influenced by putified mRSB66,106RSB66 and 128RSB66 in different levels.Our results indicated that this inhibition in kinase activity might be cause of G2/M delay in cell cycle of HeLa.hsd14 was a novel gene obtained from a human testis-specific EST by silicon cloning.Its full-length eDNA comprises 1107bp and was assigned the Genbank accession number AY251164.The open reading frame encodes a polypeptide consisting of 288 amino acid residues and contains three dsRNA binding domain.The result of BLASTP analysis showed that amino acid sequence of HSD14 was identical to PACT(PKR activating protein) except the absence of 25 amino acids in the N-terminal.It indicated that HSD14 and PACT might be products of the same gene due to selected splicing.It was reported that PACT can activate PKR,inhibit protein synthesis,and therefore induce apoptosis of cells.It may also be involved in RNA silencing pathway.In this study,we try to explore the function of HSD14 in spermatogenesis on the base of the function of PACT.We immuned BALB/c mice with purified His6-HSD14 and injected mice testis with anti-HSD14 antibodies.To our surprise,the testes of mice were in severe disorder. Degeneration and apoptosis were observed in many germ cells.Moreover,anti-growth phenotype was observed in yeast when we screen for interaction partner of HSD14 in a Yeast-Two Hybrid system using HSD14 as bait.We constructed stable RNAi cell line of endogenous prkra(hsd14 mouse homologue) successfully using GC-1spg.Our group has established a novel technique of microinjection into seminiferous tubules of mouse and spermatogonial cell line(GC-1spg) transplantation.Our work of serum microinjection and constructin of stable RNAi cell line will lay a foundation for our dedication to function of HSD14 in spermatogenesis through the novel technique.