HSD-34(TRIM69)蛋白的功能研究
摘要
精子发生(spermatogenesis)是一个高度复杂而有序的过程,在这一分化过程中,细胞特异性和发育阶段特异性基因的转录和表达受到严格的时空调控。大量细胞特异性和发育阶段特异性蛋白质合成并被准确降解,以保证精子发生的顺利进行。研究表明,泛素系统在整个精子发生过程中都发挥着非常重要的作用,包括有丝分裂、减数分裂、染色质重塑、DNA损伤修复和细胞凋亡等过程。
最近研究发现,泛素化除了蛋白酶体降解之外,还具有一些新的功能,如调节蛋白质活性和亚细胞定位等。研究表明,泛素系统通过非蛋白质降解的功能,参与细胞内蛋白质功能的调控,进而影响细胞发育、凋亡、衰老等生命过程。
本室梁刚博士利用激光捕获显微切割(Laser Capture Microdissection,LCM)技术结合抑制性消减杂交(Suppressive Subtractive Hybridization,SSH)方法构建了人圆形精子细胞消减初级精母细胞cDNA文库。根据人初级精母细胞特异EST,设计引物,以人睾丸cDNA文库为模板进行PCR扩增,得到人睾丸组织中表达的新基因,其中一个命名为HSD-34,于2004年9月将序列提交GenBank,登录号为AY305385。
HSD-34 cDNA序列全长1544bp,具有1503bp的开放阅读框,编码500个氨基酸,定位于人类15号染色体(Location:15q21.1),GeneID为140691。HSD-34与其小鼠同源物氨基酸序列的同源性高达91%,HSD-34小鼠同源物在小鼠睾丸、心脏、肝脏和脑组织中均有较高的表达。结构域分析提示,HSD-34蛋白N-末端41-82位氨基酸残基构成RING finger结构域,110-144位氨基酸残基构成B-box结构域,175-195位和226-253位氨基酸残基构成coiled-coil结构域,C-末端305-500位氨基酸残基构成B30.2结构域,即HSD-34具有RING finger-B-box-coiled-coil(RBCC)三个连续的结构域,因此,该蛋白质属于TRIM/RBCC蛋白质家族成员。2006年底,GenBank将HSD-34及其同源基因(RNF36,Trif)统一命名为TRIM69。
RING finger结构域在介导泛素分子转移到特异底物和RING蛋白质自身的过程中发挥关键的作用。因此,RING结构域是很多泛素连接酶E3的特征性标志。而泛素连接酶E3具有自身泛素化的特性。我们将HSD-34的RING finger结构域中半胱氨酸残基(Cys)或组氨酸残基(His)进行突变,构建了一系列点突变体,进行体内(in vivo)泛素化实验。结果证明,HSD-34具有自身泛素化作用,是一个新的泛素连接酶E3;同时表明,RING finger结构域是其发挥催化功能的活性结构域;C61和C64是RING结构域的关键位点。
构建了去除RING结构域(ARING),去除RING-B-box结构域(ARING-B),保留N-端RBCC结构域(RBCC)和C-端B30.2结构域(B30.2)的缺失体。进行体内泛素化实验,进一步证实RING结构域是HSD-34泛素连接酶E3的活性结构域。
通过酵母双杂交实验,筛选HSD-34的相互作用蛋白质。为了防止HSD-34发生自激活,以N-端去除RING finger结构域(即1-246bp)的序列作为诱饵基因,进行酵母双杂交筛选,得到4个阳性克隆,分别是:细胞周期检验点Hus1蛋白(GenBank登录号为NM-004507);参与细胞骨架actin组装的RhoE蛋白(GenBank登录号为NM-005168);Bcl-2家族A1蛋白(Bcl-2A1)(GenBank登录号为NM-004049);以及白细胞介素2受体β蛋白(IL-2RB)(GenBank登录号为NM-000878)。
文献报道,HSD-34小鼠同源基因可以诱导细胞发生凋亡。对四个阳性克隆的生物信息学分析表明,细胞周期检验点Hus1蛋白,在细胞内与另外两个细胞周期检验点蛋白Rad9和Rad1形成Rad9-Hus1-Rad1(9-1-1)复合物,作为细胞周期G2/M期检验点,参与细胞DNA损伤修复和细胞凋亡的调控,并且Hus1蛋白在人睾丸组织中高表达,因此,我们选择Hus1蛋白进行深入研究。
通过免疫共沉淀方法证明了HSD-34蛋白与Hus1蛋白在细胞内存在相互作用;细胞免疫荧光结合激光共聚焦扫描结果显示:HSD-34蛋白与Hus1蛋白在HeLa细胞内共定位。进一步证实了二者的相互作用关系。
HSD-34蛋白在细胞浆和细胞核中均有分布,细胞浆中主要定位于细胞核周围,呈环状聚集;在细胞核中呈散点状分布;ARING缺失体聚集于整个细胞膜周围;ARING-B缺失体在整个细胞膜上聚集的趋势更加显著,并在细胞膜上伸出丝状细小伪足;B30.2缺失体在细胞膜周围分布;RBCC缺失体部分恢复HSD-34在细胞浆中的聚集现象,但边界不清晰;C61A/C64A突变体弥漫分布于整个细胞中,完全失去了HSD-34蛋白的定位特点。以上结果提示,RING finger结构域对于HSD-34蛋白在细胞内的定位起重要作用。
HSD-34对外源性和内源性Hus1蛋白的降解实验,表明HSD-34不参与Hus1蛋白的蛋白酶体降解。HSD-34转染HeLa细胞,引起内源性Hus1蛋白和磷酸化Hus1蛋白表达显著上调;同时激活caspase-3,导致细胞凋亡。提示,HSD-34对Hus1蛋白的功能具有特异的调节作用。
应用Hoechst33258进行细胞核染色,观察到转染HSD-34后,细胞发生核浓缩,出现明显的细胞凋亡现象。荧光显微镜观察到GFP-HSD-34融合蛋白在胞浆呈花环状分布或在核周两极聚集成括号状;然后转入胞核内,引起细胞核浓缩、碎裂。推测:HSD-34蛋白在细胞质中合成,然后穿梭进入细胞核,最终在细胞核内发挥促进细胞凋亡的作用。
转染HSD-34后,检测不同时间点细胞在分子水平上的改变。发现1)转染后12h,检测到HSD-34蛋白的表达,并呈现表达增加的趋势;同时,检测到活化的caspase-9和caspase-3的表达,且二者在该时间点表达量最高,随后逐渐降低;2)实验组12h检测到磷酸化的Hus1蛋白的表达,随着时间的延长,表达量显著增加,且与HSD-34表达趋势一致;3)实验组和对照组中,p53蛋白的表达没有发生显著变化。
应用PathDetect(?)顺式报告系统,检测HSD-34对NF-κB,NFAT,CRE,AP1,STAT1细胞信号转导通路的影响。结果显示:HSD-34可以激活CRE细胞信号转导通路。
综上结果,我们推测,HSD-34蛋白通过线粒体介导的细胞凋亡通路,活化caspase-9,caspase-3;同时在细胞核内,激活CRE信号通路,使Hus1蛋白表达上调,由于HSD-34与Hus1蛋白存在相互作用,催化Hus1蛋白发生磷酸化修饰,磷酸化的Hus1蛋白进一步活化细胞凋亡通路,最终导致凋亡的发生,而这一过程不依赖于p53蛋白的活化。
Spermatogenesis is a complex process,in which undifferentiated spermatogonia divide and differentiate into mature spermatozoa.During the process,many genes are transcribed and translated under precise temporal and spatial regulation.A large number of cell-specific and developmental stage-specific proteins were accurately degraded.Therefore,the ubiquitin-proteasome pathway plays a key role in spermatogenesis.
Research had found that the ubiquitin-proteasome system had some new functions,such as the regulation of protein activity and their subcellular localization.Recent studies have found that depend on its non-degradation function,the ubiquitin system was also involved in the regulation of cell growth,apoptosis,and aging.
The method of laser capture microdissection(LCM) combined with suppressive subtractive hybridization(SSH) was used to isolate the differentially expressed genes during germ cell differentiation.A new gene designated as HSD34 was obtained and characterized.
HSD-34 cDNA is 1544bp.Its open reading frame consists of 1506bp,which is encoding a 500 amino acids protein.The GenBank Accession No.of HSD-34 is AY305385 and localizes in human chromosome 15(Location:15q21.1).HSD34,containing a RING finger domain,a B-box,and a coiled-coil motif in the N-terminal region,belongs to the TRIM (Tripartite motif) or RBCC(RING,B-Box,coiled-coil) family.The identity of HSD-34 and its mouse homolog is about 91%.The mouse homolog of HSD-34 is observed with higher expression in mouse testis,heart,liver and brain tissue.
RING finger domain plays a key role in transferring the ubiquitin to specific substrate and RING protein itself.Therefore,the RING domain is a character of ubiquitin ligase E3. Ubiquitin ligase E3 can ubiquitinate itself.We construct a series of point mutants within the RING finger domain and some truncates.With the in vivo ubiquitination assay,we found that HSD-34 could ubiquitinate itself,which showed that HSD-34 was a new ubiquitin ligase E3. Meanwhile,RING finger domain is the active domain of HSD-34;C61 and C64 sites are the key sites of this domain.
Using a yeast two-hybrid assay,we had screened the interaction proteins of HSD-34.We use a truncate,which had been removed of the N-terminal RING finger domain(246bp),as the bait gene.Four positive clones were found,namely,the cell cycle checkpoint protein Hus1 (GenBank Accession No.NM-004507);RhoE(GenBank Accession No.NM-00516 8); Bcl-2A1(GenBank Accession No.NM-00404 9);and IL-2RB(GenBank Accession No. NM-0008 78).
We had chosen the checkpoint protein Hus1 for further study because checkpoint protein Hus1 interacts with two other checkpoint protein Rad9 and Rad1,which could form a G2/M cell cycle checkpoint complex,Rad9-Hus1-Rad1(9-1-1) complex.Rad9-Hus1 -Rad1 complex is confirmed to be involved in DNA repair and apoptosis.
Using the co-immunoprecipitation method,we had proved HSD-34 could interact with Hus1.Immunofluorescence and confocal laser scanning showed that HSD-34 co-localized with Hus1 in HeLa cells,which also confirmed the interaction between these two proteins.
HSD-34 protein was distributed in cytoplasm and nucleus.In cytoplasm,HSD-34 was mainly surrounding the nucleus as a ring;while in nucleus,it was in a speckled pattern.The deletions of different domain could result in different localization of this protein,for example, RING deletions mutant was surrounded the membrane;RING-B deletions mutant was gathered the membrane more apparently,and stretched out small feet from the membrane; B30.2 deletions was also surrounded the membrane;RBCC deletions was aggregated within the cytoplasm just as HSD-34,but the border was not clear;C61A/C64A mutant was diffused throughout the cell,completely lost the distribution pattern of HSD-34 protein.
The degradation experiment showed that HSD-34 did not participate in the degradation of Hus1 through the proteasome pathway.The transfection of HSD-34 plasmid into HeLa cells could cause the increased expression of Hus1 protein,which the level of phosphorylated Hus1 protein was also significantly increased.At the same time,HSD-34 could also active caspase-3 pathway,which could lead to apoptosis.These results suggested that HSD-34 can regulate the function of Hus1 protein in apoptosis.
Morphology analysis found that HSD-34 can promote cell apoptosis.Fluorescence microscopy found that GFP-HSD-34 fusion protein was first distributed as a ring in the cytoplasm;then was transferred to the nucleus,where it could cause the condensation and fragmentation of the nucleus.Then we had speculated that,HSD-34 protein was synthesized in the cytoplasm,and shuttled into the nucleus,from where it exerted its important role in apoptosis.
After the transfection of HSD-34 into HeLa cells,we detected different protein levels at different time points.We found that the expression of active caspase-9,active caspase-3,Hus1 and phosphorylation Hus1 were elevated upon the induction of HSD-34 within those test cells. However,the expression of p53 did not change between the test group and the control group.
The pathDetect(?) cis-reporting system was used to detect if HSD-34 could affect the cellular signal transduction pathways,such as NF-κB,NFAT,CRE,AP1,STAT1 pathway.The results showed that HSD-34 can activate CRE cellular signal transduction pathway.
These results suggested that HSD-34 may induce apoptosis through caspase-9,caspase-3 pathway;at the same time,HSD-34 could activate the CRE signaling pathway within the nucleus.After that,the expression of Hus1 could be up-regulated and phosphorylated.The phosphorylated Hus1 could activate the apoptosis pathway and lead the cells to apoptosis. This process is a p53-independent pathway.
最近研究发现,泛素化除了蛋白酶体降解之外,还具有一些新的功能,如调节蛋白质活性和亚细胞定位等。研究表明,泛素系统通过非蛋白质降解的功能,参与细胞内蛋白质功能的调控,进而影响细胞发育、凋亡、衰老等生命过程。
本室梁刚博士利用激光捕获显微切割(Laser Capture Microdissection,LCM)技术结合抑制性消减杂交(Suppressive Subtractive Hybridization,SSH)方法构建了人圆形精子细胞消减初级精母细胞cDNA文库。根据人初级精母细胞特异EST,设计引物,以人睾丸cDNA文库为模板进行PCR扩增,得到人睾丸组织中表达的新基因,其中一个命名为HSD-34,于2004年9月将序列提交GenBank,登录号为AY305385。
HSD-34 cDNA序列全长1544bp,具有1503bp的开放阅读框,编码500个氨基酸,定位于人类15号染色体(Location:15q21.1),GeneID为140691。HSD-34与其小鼠同源物氨基酸序列的同源性高达91%,HSD-34小鼠同源物在小鼠睾丸、心脏、肝脏和脑组织中均有较高的表达。结构域分析提示,HSD-34蛋白N-末端41-82位氨基酸残基构成RING finger结构域,110-144位氨基酸残基构成B-box结构域,175-195位和226-253位氨基酸残基构成coiled-coil结构域,C-末端305-500位氨基酸残基构成B30.2结构域,即HSD-34具有RING finger-B-box-coiled-coil(RBCC)三个连续的结构域,因此,该蛋白质属于TRIM/RBCC蛋白质家族成员。2006年底,GenBank将HSD-34及其同源基因(RNF36,Trif)统一命名为TRIM69。
RING finger结构域在介导泛素分子转移到特异底物和RING蛋白质自身的过程中发挥关键的作用。因此,RING结构域是很多泛素连接酶E3的特征性标志。而泛素连接酶E3具有自身泛素化的特性。我们将HSD-34的RING finger结构域中半胱氨酸残基(Cys)或组氨酸残基(His)进行突变,构建了一系列点突变体,进行体内(in vivo)泛素化实验。结果证明,HSD-34具有自身泛素化作用,是一个新的泛素连接酶E3;同时表明,RING finger结构域是其发挥催化功能的活性结构域;C61和C64是RING结构域的关键位点。
构建了去除RING结构域(ARING),去除RING-B-box结构域(ARING-B),保留N-端RBCC结构域(RBCC)和C-端B30.2结构域(B30.2)的缺失体。进行体内泛素化实验,进一步证实RING结构域是HSD-34泛素连接酶E3的活性结构域。
通过酵母双杂交实验,筛选HSD-34的相互作用蛋白质。为了防止HSD-34发生自激活,以N-端去除RING finger结构域(即1-246bp)的序列作为诱饵基因,进行酵母双杂交筛选,得到4个阳性克隆,分别是:细胞周期检验点Hus1蛋白(GenBank登录号为NM-004507);参与细胞骨架actin组装的RhoE蛋白(GenBank登录号为NM-005168);Bcl-2家族A1蛋白(Bcl-2A1)(GenBank登录号为NM-004049);以及白细胞介素2受体β蛋白(IL-2RB)(GenBank登录号为NM-000878)。
文献报道,HSD-34小鼠同源基因可以诱导细胞发生凋亡。对四个阳性克隆的生物信息学分析表明,细胞周期检验点Hus1蛋白,在细胞内与另外两个细胞周期检验点蛋白Rad9和Rad1形成Rad9-Hus1-Rad1(9-1-1)复合物,作为细胞周期G2/M期检验点,参与细胞DNA损伤修复和细胞凋亡的调控,并且Hus1蛋白在人睾丸组织中高表达,因此,我们选择Hus1蛋白进行深入研究。
通过免疫共沉淀方法证明了HSD-34蛋白与Hus1蛋白在细胞内存在相互作用;细胞免疫荧光结合激光共聚焦扫描结果显示:HSD-34蛋白与Hus1蛋白在HeLa细胞内共定位。进一步证实了二者的相互作用关系。
HSD-34蛋白在细胞浆和细胞核中均有分布,细胞浆中主要定位于细胞核周围,呈环状聚集;在细胞核中呈散点状分布;ARING缺失体聚集于整个细胞膜周围;ARING-B缺失体在整个细胞膜上聚集的趋势更加显著,并在细胞膜上伸出丝状细小伪足;B30.2缺失体在细胞膜周围分布;RBCC缺失体部分恢复HSD-34在细胞浆中的聚集现象,但边界不清晰;C61A/C64A突变体弥漫分布于整个细胞中,完全失去了HSD-34蛋白的定位特点。以上结果提示,RING finger结构域对于HSD-34蛋白在细胞内的定位起重要作用。
HSD-34对外源性和内源性Hus1蛋白的降解实验,表明HSD-34不参与Hus1蛋白的蛋白酶体降解。HSD-34转染HeLa细胞,引起内源性Hus1蛋白和磷酸化Hus1蛋白表达显著上调;同时激活caspase-3,导致细胞凋亡。提示,HSD-34对Hus1蛋白的功能具有特异的调节作用。
应用Hoechst33258进行细胞核染色,观察到转染HSD-34后,细胞发生核浓缩,出现明显的细胞凋亡现象。荧光显微镜观察到GFP-HSD-34融合蛋白在胞浆呈花环状分布或在核周两极聚集成括号状;然后转入胞核内,引起细胞核浓缩、碎裂。推测:HSD-34蛋白在细胞质中合成,然后穿梭进入细胞核,最终在细胞核内发挥促进细胞凋亡的作用。
转染HSD-34后,检测不同时间点细胞在分子水平上的改变。发现1)转染后12h,检测到HSD-34蛋白的表达,并呈现表达增加的趋势;同时,检测到活化的caspase-9和caspase-3的表达,且二者在该时间点表达量最高,随后逐渐降低;2)实验组12h检测到磷酸化的Hus1蛋白的表达,随着时间的延长,表达量显著增加,且与HSD-34表达趋势一致;3)实验组和对照组中,p53蛋白的表达没有发生显著变化。
应用PathDetect(?)顺式报告系统,检测HSD-34对NF-κB,NFAT,CRE,AP1,STAT1细胞信号转导通路的影响。结果显示:HSD-34可以激活CRE细胞信号转导通路。
综上结果,我们推测,HSD-34蛋白通过线粒体介导的细胞凋亡通路,活化caspase-9,caspase-3;同时在细胞核内,激活CRE信号通路,使Hus1蛋白表达上调,由于HSD-34与Hus1蛋白存在相互作用,催化Hus1蛋白发生磷酸化修饰,磷酸化的Hus1蛋白进一步活化细胞凋亡通路,最终导致凋亡的发生,而这一过程不依赖于p53蛋白的活化。
Spermatogenesis is a complex process,in which undifferentiated spermatogonia divide and differentiate into mature spermatozoa.During the process,many genes are transcribed and translated under precise temporal and spatial regulation.A large number of cell-specific and developmental stage-specific proteins were accurately degraded.Therefore,the ubiquitin-proteasome pathway plays a key role in spermatogenesis.
Research had found that the ubiquitin-proteasome system had some new functions,such as the regulation of protein activity and their subcellular localization.Recent studies have found that depend on its non-degradation function,the ubiquitin system was also involved in the regulation of cell growth,apoptosis,and aging.
The method of laser capture microdissection(LCM) combined with suppressive subtractive hybridization(SSH) was used to isolate the differentially expressed genes during germ cell differentiation.A new gene designated as HSD34 was obtained and characterized.
HSD-34 cDNA is 1544bp.Its open reading frame consists of 1506bp,which is encoding a 500 amino acids protein.The GenBank Accession No.of HSD-34 is AY305385 and localizes in human chromosome 15(Location:15q21.1).HSD34,containing a RING finger domain,a B-box,and a coiled-coil motif in the N-terminal region,belongs to the TRIM (Tripartite motif) or RBCC(RING,B-Box,coiled-coil) family.The identity of HSD-34 and its mouse homolog is about 91%.The mouse homolog of HSD-34 is observed with higher expression in mouse testis,heart,liver and brain tissue.
RING finger domain plays a key role in transferring the ubiquitin to specific substrate and RING protein itself.Therefore,the RING domain is a character of ubiquitin ligase E3. Ubiquitin ligase E3 can ubiquitinate itself.We construct a series of point mutants within the RING finger domain and some truncates.With the in vivo ubiquitination assay,we found that HSD-34 could ubiquitinate itself,which showed that HSD-34 was a new ubiquitin ligase E3. Meanwhile,RING finger domain is the active domain of HSD-34;C61 and C64 sites are the key sites of this domain.
Using a yeast two-hybrid assay,we had screened the interaction proteins of HSD-34.We use a truncate,which had been removed of the N-terminal RING finger domain(246bp),as the bait gene.Four positive clones were found,namely,the cell cycle checkpoint protein Hus1 (GenBank Accession No.NM-004507);RhoE(GenBank Accession No.NM-00516 8); Bcl-2A1(GenBank Accession No.NM-00404 9);and IL-2RB(GenBank Accession No. NM-0008 78).
We had chosen the checkpoint protein Hus1 for further study because checkpoint protein Hus1 interacts with two other checkpoint protein Rad9 and Rad1,which could form a G2/M cell cycle checkpoint complex,Rad9-Hus1-Rad1(9-1-1) complex.Rad9-Hus1 -Rad1 complex is confirmed to be involved in DNA repair and apoptosis.
Using the co-immunoprecipitation method,we had proved HSD-34 could interact with Hus1.Immunofluorescence and confocal laser scanning showed that HSD-34 co-localized with Hus1 in HeLa cells,which also confirmed the interaction between these two proteins.
HSD-34 protein was distributed in cytoplasm and nucleus.In cytoplasm,HSD-34 was mainly surrounding the nucleus as a ring;while in nucleus,it was in a speckled pattern.The deletions of different domain could result in different localization of this protein,for example, RING deletions mutant was surrounded the membrane;RING-B deletions mutant was gathered the membrane more apparently,and stretched out small feet from the membrane; B30.2 deletions was also surrounded the membrane;RBCC deletions was aggregated within the cytoplasm just as HSD-34,but the border was not clear;C61A/C64A mutant was diffused throughout the cell,completely lost the distribution pattern of HSD-34 protein.
The degradation experiment showed that HSD-34 did not participate in the degradation of Hus1 through the proteasome pathway.The transfection of HSD-34 plasmid into HeLa cells could cause the increased expression of Hus1 protein,which the level of phosphorylated Hus1 protein was also significantly increased.At the same time,HSD-34 could also active caspase-3 pathway,which could lead to apoptosis.These results suggested that HSD-34 can regulate the function of Hus1 protein in apoptosis.
Morphology analysis found that HSD-34 can promote cell apoptosis.Fluorescence microscopy found that GFP-HSD-34 fusion protein was first distributed as a ring in the cytoplasm;then was transferred to the nucleus,where it could cause the condensation and fragmentation of the nucleus.Then we had speculated that,HSD-34 protein was synthesized in the cytoplasm,and shuttled into the nucleus,from where it exerted its important role in apoptosis.
After the transfection of HSD-34 into HeLa cells,we detected different protein levels at different time points.We found that the expression of active caspase-9,active caspase-3,Hus1 and phosphorylation Hus1 were elevated upon the induction of HSD-34 within those test cells. However,the expression of p53 did not change between the test group and the control group.
The pathDetect(?) cis-reporting system was used to detect if HSD-34 could affect the cellular signal transduction pathways,such as NF-κB,NFAT,CRE,AP1,STAT1 pathway.The results showed that HSD-34 can activate CRE cellular signal transduction pathway.
These results suggested that HSD-34 may induce apoptosis through caspase-9,caspase-3 pathway;at the same time,HSD-34 could activate the CRE signaling pathway within the nucleus.After that,the expression of Hus1 could be up-regulated and phosphorylated.The phosphorylated Hus1 could activate the apoptosis pathway and lead the cells to apoptosis. This process is a p53-independent pathway.
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