Mechanism of viral hepatitis-mediated hepatocarcinogenes

细胞动态平衡受到多种细胞蛋白活性的严格调控。这些蛋白质的亚细胞定位和稳定性对它们的活性至关重要。例如，特定受体通过识别核定位信号(NLS)和/或核输出信号(NES)将蛋白质运入或运出细胞核。最近发现的一个NES是一个短的、疏水的、富含亮氨酸的基序，它是介导大载体蛋白和mRNAs核输出所必需的，也是足够的。据报道，许多细胞转录因子、癌蛋白、细胞周期调节因子和肿瘤抑制蛋白的活性受它们的NES调节。有趣的是，许多病毒蛋白也利用CRM1/RAN介导的途径，尽管其中一些病毒蛋白被认为足够小，可以被动地通过核孔复合体(NPC)扩散。这些致癌病毒蛋白已获得NES活性和修饰的核输出，这一事实暗示核输出可能是病毒介导的肿瘤发生的有效靶点。 乙肝病毒X蛋白(HBx)和丙型肝炎病毒核心蛋白(HC-core)是肝细胞癌的两个主要危险因素，它们可能通过改变CRM1/RAN介导的途径诱导肿瘤转化。最近，我们发现HBx和HC-core蛋白含有功能性Ness。与其他细胞内含有NES的蛋白质不同，HBX与细胞质中的CRM1结合并隔离，从而调节CRM1介导的其他细胞蛋白的核输出，包括NFkB/IkBA复合体。同样，HC-core介导的NFkB/IkBA复合体的激活也依赖于Ness的存在。这些发现表明，与HBx或HC-core相关的多种细胞功能可能部分归因于它们对CRM1/RAN介导的途径的影响。由于CRM1及其辅因子RAN GTP酶在有丝分裂的启动过程中也起着关键作用，HBX或HC-core使CRM1失活也可能导致基因组不稳定。 目前，我们正在验证HBX和HC-core可能通过与CRM1/RAN复合体的相互作用而诱导基因组不稳定的假设。最近，我们发现CRM1与中心体共定位。使用CRM1特异的抑制剂或通过致癌HBx蛋白将CRM1隔离在细胞质中会诱导中心粒意外分裂，并产生更多的有丝分裂纺锤体核中心。过早分裂会导致额外分离的中心粒，这些中心粒可能作为启动中心体复制的模板，导致这些细胞中额外的中心体。这种效应在有丝分裂细胞中优先观察到，与其p53或p21状态无关，并导致多极有丝分裂。相反，缺乏将CRM1隔离在细胞质中的能力的HBX突变体不会诱导额外的中心体。这些结果揭示了CRM1在维持中心体完整性方面的新作用，并暗示HBx破坏这一过程可能有助于乙肝病毒介导的致病机制。上述研究使我们产生了一个新的可检验的假说，即CRM1可能是病毒性肝炎介导的致癌作用的共同靶点，并为CRM1可能参与人类癌症的发生提供了基础。

Cellular homeostasis is regulated tightly by the activities of many cellular proteins. The subcellular localization and stability of these proteins are critical for their activities. Examples include proteins that are transported into or out of the nucleus by specific receptors through the recognition of nuclear-localization signals (NLS) and/or nuclear export signals (NES). One recently identified NES is a short, hydrophobic, leucine-rich motif that is necessary and sufficient to mediate nuclear export of large carrier proteins and mRNAs. The activity of many cellular transcription factors, oncoproteins, cell cycle regulators, and tumor suppressor proteins has been reported to be regulated by their NES. Interestingly, many viral proteins also utilize the Crm1/Ran-mediated pathway, even though some of these viral proteins are thought to be small enough to passively diffuse through the nuclear pore complex (NPC). The fact that these oncogenic viral proteins have acquired NES activity and modified nuclear export implies that nuclear export may be an efficient target for viral-mediated oncogenesis. We are examining the hypothesis that both the X protein (HBx) of hepatitis B virus (HBV) and core protein (HC-core) of hepatitis C virus (HCV), two major risk factors for hepatocellular carcinoma, may induce neoplastic transformation by altering the Crm1/Ran-mediated pathways. Recently, we discovered that the HBx and HC-core proteins contain functional NESs. Unlike other cellular NES-containing proteins, HBx binds to and sequesters Crm1 in the cytoplasm, thereby modulating Crm1-mediated nuclear export of other cellular proteins including the NFkB/IkBa complex. Similarly, HC-core-mediated activation of the NFkB/IkBa complex also depends on the presence of NESs. These findings suggest that multiple cellular functions associated with HBx or HC-core may be due, in part, to their influence on the Crm1/Ran-mediated pathway. Because Crm1 and its cofactor Ran GTPase also play a key role in mitosis initiation, the inactivation of Crm1 by HBx or HC-core also may induce genomic instability. Currently, we are testing the hypothesis that HBx and HC-core may induce genomic instability through their interactions with the Crm1/Ran complex. Recently, we found that Crm1 colocalizes with centrosomes. Use of a Crm1-specific inhibitor or sequestering of Crm1 in the cytoplasm by the oncogenic HBx protein induces unscheduled centriole splitting and the creation of an increased number of mitotic spindle nucleating centers. Premature splitting results in extra segregated centrioles that may serve as a template to initiate centrosome duplication, leading to supernumerary centrosomes in these cells. This effect is preferentially observed in mitotic cells, regardless of their p53 or p21 status and results in multipolar mitotic divisions. In contrast, an HBx mutant lacking the ability to sequester Crm1 in the cytoplasm does not induce supernumerary centrosomes. These results reveal a novel role for Crm1 in the maintenance of centrosome integrity, and imply that HBx disruption of this process may contribute to HBV-mediated pathogenesis. The above studies led us to generate a novel testable hypothesis that Crm1 may be a common target for viral hepatitis-mediated oncogenicity and provide a foundation for a possible involvement of Crm1 in human carcinogenesis.