节点文献
睾丸蛋白酶体的鉴定及其功能研究
【作者】 杜伯雨；
【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士
【摘要】 真核生物26S蛋白酶体由两部分构成,负责催化底物降解的20S催化颗粒和连接在20S两个末端的19S调节颗粒,其中20S催化颗粒是由四个环状结构叠合在一起形成的对称的桶状结构(αββα),由于其内表面PSMB5、PSMB7和PSMB6三个亚基的存在,两个β环具有chymotrypsin-like、trypsin-like和caspase-like三种催化活性。在IFN-γ的诱导下,这三个亚基可被PSMB8、PSMB10和PSMB9替换,成为可增强抗原递呈作用的免疫蛋白酶体的一部分。本实验室前期研究结果显示家兔睾丸中除含有普通的26S蛋白酶体外,还可能存在两种不同类型的蛋白酶体。它们包含功能未知的蛋白酶体激活因子PSME4,其20S催化颗粒还存在着不同的亚基(比如,尚未见报道的PSMA7-like亚基和存在于免疫蛋白酶体的的亚基)。我们在纯化牛肌肉、睾丸和脾脏中的蛋白酶体时发现,从这三种组织来源的蛋白酶体在从阴离子交换柱上洗脱的时候所需的离子强度不同。利用短肽荧光底物对我们从牛肌肉、睾丸和脾脏中纯化的26S蛋白酶体进行酶活性分析的时候发现,我们初步发现,从牛睾丸中纯化出的蛋白酶体的chymotrypsin like活性要显著低于从牛肌肉和脾脏中纯化出的蛋白酶体的活性(P<0.05),睾丸蛋白酶体的trypsin like活性显著高于肌肉和脾脏中的蛋白酶体的活性(P<0.05),而肌肉蛋白酶体的caspase like活性要显著高于睾丸和脾脏蛋白酶体的活性(P<0.05)。但是应用两种蛋白质底物Histone H1和Casein来测定这三种组织来源的蛋白酶体的降解活性却未发现存在着何种差异(P>0.05)。在应用针对蛋白酶体三种活性位点的抑制剂(MG132、Velcade、Leupeptin和Ac-ApnLD-Al)对肌肉和睾丸组织来源的蛋白酶体进行活性抑制时也未发现对这两种蛋白酶体的抑制效应存在差异(P>0.05)。在应用体外培养的生精细胞系和体细胞系进行实时定量PCR实验和Western blot实验时发现,在所选的细胞系中都存在着表达水平类似的PSMB5基因,但是在培养的精原细胞株和精母细胞株(GC-1spg和GC-2spd)中存在着较高水平的PSMB8基因的表达,而在其他两种体细胞株(NIH 3T3和C2C12)中却并未发现这种基因的表达。应用PSME4的抗体在精母细胞系GC-2spd中进行免疫沉淀实验来确认同PSME4结合的20S蛋白酶体类型时我们发现,蛋白酶体的催化亚基中PSMB8、PSMB9、PSMB6和PSMB7都可以同PSME4共沉淀下来,而蛋白酶体的组成亚基PSMB5和在IFN-γ诱导下产生的亚基PSMB10都不能同PSME4共沉淀,提示由这两种催化亚基参与所构成的20S蛋白酶体可能不与PSME4发生结合。而应用PSME4的抗体在体细胞系NIH 3T3中进行免疫沉淀实验时却并未沉淀下来蛋白酶体的催化亚基,提示在体细胞中虽然也存在着PSME4的表达,但是可能并不参与组成蛋白酶体。应用原位杂交技术观察了小鼠睾丸组织切片中PSME4、PSMB8和PSMB5在生精细胞不同发育阶段中的表达情况。发现PSME4、PSMB8和PSMB5的基因表达水平随生精细胞处于不同的发育阶段而存在着差异,以生精细胞发生周期中的Ⅷ期为转折点,在此期之前,PSME4、PSMB8和PSMB5在圆形精子中的表达呈现阳性反应,而粗线期精母细胞(Pachytene spermatocyte)中的表达为阴性反应；Ⅷ期之后,这三种基因在粗线期精母细胞中的表达转为阳性反应,而在圆形精子和细线期精母细胞(Leptotene spermatocyte)中的表达为阴性,此外这三种基因在各期的Sertoli细胞和长形精子细胞中的表达呈现阴性反应。此外应用PSMB8和PSMB5的抗体进行免疫组织化学实验时我们初步发现,PSMB5在一些间质细胞内存在着高水平的表达,PSMB8在睾丸组织内的各种细胞中均有表达,在生精细胞的细胞核中均未发现有PSMB5的表达,除了长形精子细胞的细胞核,PSMB8在各生精细胞核中均发现存在着表达。这些结果提示在睾丸内精子细胞发生的不同时期及其不同的细胞器内,蛋白酶体的亚基组成模式可能存在着差别,这可能同精子细胞在发育的不同时期所经历的细胞事件有一定的关系。真核细胞内即将被26S蛋白酶体降解的蛋白质底物一般都携带有一个泛素标签,这个泛素标签是通过一系列的酶促反应——泛素化反应而实现的,参与这一系列反应的酶分别被命名为E1、E2和E3。E1是泛素激活酶(Ubiquitin-activating enzyme),可以利用ATP降解所产生的能量激活泛素以形成高能的硫脂键,接着激活的泛素分子可以被转移到E2泛素载体蛋白(Ubiquitin carrier protein),最后在E3泛素连接酶(Ubiquitin ligases)的协同作用下,将多聚泛素链转移到底物分子上,之后这种“标记”的蛋白质底物分子可以为26S的蛋白酶体所识别并继而被降解。研究表明一些参与细胞凋亡途径的蛋白质在果蝇精子细胞的发育阶段发挥着非常重要的作用。同时具有泛素载体蛋白(E2)和泛素连接酶(E3)活性的巨型凋亡抑制蛋白(Inhibitor of Apoptosis Protein) BRUCE/Apollon被证明可以保护精子细胞核免于过度的浓缩和退化,因此推测其在生精过程中可能具有非常重要的功能。最新研究表明,BRUCE还控制有丝分裂后子细胞的最终分离。由于BRUCE蛋白是泛素连接酶Nrdp1/RNF41的底物,我们观察了Nrdp1/RNF41蛋白及BRUCE蛋白的表达水平同细胞周期进行的关系,发现Nrdp1/RNF41蛋白的表达水平随细胞周期的变化而变化,其表达水平在G2期早期(同步化释放后3-6小时间)出现升高,在同步化释放后18-24小时间升至最高。而BRUCE蛋白的表达水平同Nrdp1蛋白的表达水平较为类似,但似乎落后于Nrdp1蛋白水平的变化,在G2期(同步化释放后6小时)开始出现升高,之后表达水平一直处于升高状态,在同步化释放的早期,细胞处于S期时表达水平最低。这些结果表明,Nrdp1可能通过控制BRUCE的泛素化和降解而参与细胞周期的调控。这两种蛋白质在精子发生中的作用尚待进一步研究。综上所述,我们在哺乳动物睾丸中发现了新型的、具有特异组成和活性的蛋白酶体。同时,我们发现泛素连接酶Nrdp1/RNF41很可能参与细胞周期的调控。
【Abstract】 The proteasome in the eukaryotic cells is composed of two parts,20S catalytic complex, which cleaves protein substrates, and the 19S regulatory complex, which binds one or both ends of 20S complex and regulates the activity of the 20S particle. The 20S catalytic complex is a symmetrical, cylinder-shaped particle composed of four rings (αββα). Three catalytic subunits PSMB6, PSMB7 and PSMB5 with chymotrypsin-like, trypsin-like and caspase-like activities lie within the two inside packedβrings. Under the induction of IFN-γ, these three catalytic subunits could be replaced by PSMB9, PSMB10 and PSMB8, which are a part of the immunoproteasome involved in antigen presentation.Previous results from our group have shown that two types of proteasomes different from the 26S proteasome can be purified from rabbit testes. These proteasomes contain PSME4 (a proteasomal activator with unknown functions), a new PSMA7-like subunit, and a few subunits usually present in the immunoproteasome. In the process of purification of the bovine muscle, testis and spleen proteasomes, we found that different concentrations of NaCl were needed to elute these proteasomes from an anion exchange column. By using the peptide fluorescence substrate, we determined the activities of the proteasome from these tissues and found that the chymotrypsin-like activity of testis proteasome was significantly lower than that from muscle and spleen (P＜0.05). The trypsin-like activity of the testis proteasome was significantly higher than that of the proteasome purified from muscle and spleen (P＜0.05). But the caspase like activity of muscle proteasome was significantly higher than that of the proteaosmes purified form testis and spleen (P＜0.05). No significant differences were observed when the two protein substrates histone H1 and casein were used to determine the degradation efficiency of these three kinds of proteasome (P>0.05). When several proteasome inhibitors (MG132, Velcade, Leupeptin and Ac-ApnLD-Al) were used to incubate with the proteasomes, no significant differences in the inhibition of the activities were observed among the proteasomes from testis, muscle and spleen (P>0.05).By using the real-time PCR and western blot experiments, we found similar expression levels of PSMB5 in the spermatogonia cell line(GC-1 spg), spermatocyte cell line(GC-2 spd) and somatic cell lines (NIH 3T3 and C2C12). But PSMB8 was found highly expressed in the spermatogonia cell line and the spermatocyte cell line and not detectable in the two somatic cell lines. We further found that the catalytic subunits PSMB8, PSMB9, PSMB6 and PSMB7, but not PSMB5 and PSMB10, could co-precipitate with PSME4. However, when the similar immunoprecipitation experiment was performed in NIH 3T3 cell lysates, no catalytic subunits were co-precipitated with PSME4 antibody, raising the possibility that PSME4 mostly does not associate with the 20S proteasome in somatic cells.Using the in-situ hybridization teniques, we also observed the expression pattern of PSME4, PSMB8 and PSMB5 in mouse testis. The expression patterns of the three selected genes were quite different in different stages of spermatogenesis. Prior to theⅧstages of mouse spermatogenesis, PSME4, PSMB8 and PSMB5 were observed in the round spermatids, but not in pachytene spermatocytes. After theⅧstages, the expression of PSME4, PSMB8 and PSMB5 was observed in pachytene spermatocytes, but not in spermatids, leptotene spermatocytes and sertoli cells. Moreover, according to the initial results of immunohistochemistry with anti-PSMB8 and anti-PSMB5, we observed high expression level of PSMB5 in the leydig cells, while PSMB8 was extensively expressed in the testis tissues. The above results suggested that in the germ cells of different developmental stages, the different compostions of proteasome might exist.In eukaryotic cells, the protein substrate of the 26S proteasome usually carries an ubiquitin tag, which is added through ubiquitination. The enzymes involved in the ubiquitination include ubiquitin-activating enzyme (E1), ubiquitin carrier protein (E2) and ubiquitin-protein ligase (E3). E1 activates the ubiquitin by promoting the formation of high-energy sulfur-ester bond in the presence of ATP. Then the activated ubiquitin molecule is transferred to an E2. At last the ubiquitin is transferred to the substrate by E3. After that, the substrate tagged with polyubiquitin chain is targeted for degradation by the 26S proteasome. Certain apoptotic proteins played important roles during sperm development. For example, the giant Inhibitor of Apoptosis Protein (IAP) BRUCE/Apollon, which acts as both E2 and E3, can protect the sperm nucleus from high dense condensation and degeneration in Droshophila, indicating that this protein might play a very important role during spermatogenesis. Recent studies showed that BRUCE controls the midbody ring formation at the final stages of mitotic cell cycle. As BRUCE was the substrate of the E3 ubiquitin ligase, Nrdp1/RNF41, we examined the protein levels of Nrdp1/RNF41 and BRUCE throughout the cell cycle progression. The protein levels of Nrdp1/RNF41 increased in the early G2/M phase (3 hours after the release of synchronization), reached its peak at 18-24 hours after the release. The change of BRUCE protein levels appeared a little behind that of Nrdpl. Its levels increased at 6 hours after the release and kept increasing afterwards. During the early stages of the release of synchronization (S stage), the lowest BRUCE protein levels were observed. Our results demonstrate that Nrdp1 might be involved in the regulation of the cell cycle by promoting ubiquitination and degradation of BRUCE. However, more experiments were still needed to address the roles of these proteins in the progression of male meiotic cell cycle during spermatogenesis.Taken together, our results suggest that there are specialized forms of proteasome in mammalian testis with distinct compositions and activity. In addition, we found that the ubiquitin ligase Nrdpl/RNF41 was probably involved in cell cycle regulation.