LMBRD1調節細胞核質運輸蛋白質CAS及Importin-alpha之細胞內分布

Abstract

先前的研究指出NESI（nuclear export signal interacting protein）為一參與D 型肝炎病毒大型delta抗原（HDAg-L）核輸出的蛋白質。NESI 蛋白質屬於lmbrd1基因的其中一個異體蛋白質（protein isoform），NESI蛋白質的胺基酸序列與lmbrd1基因中的最大異體蛋白質LMBD1的第74至540的胺基酸序列完全相同。在利用程式運算的結果中指出，兩者皆可能為細胞膜蛋白質，其中NESI具有七個可能的穿膜區域（transmembrane domains），而LMBD1有九個可能的穿膜區域。在本研究中，首先在人類肝癌細胞株Huh7中分別表現NESI及LMBD1蛋白質，並觀察其細胞分布情形。結果發現NESI主要分布於接近細胞核膜區域（perinuclear region），而LMBD1主要分布於細胞胞器（organelles）。綜合先前有關D型肝炎病毒蛋白質核輸出的研究，NESI蛋白質可能參與細胞核與細胞質之間的傳輸。 在實驗室過去的研究中，利用免疫沈澱（immunoprecipitation）及液態層析串聯式質譜儀（LC-MS/MS）分析可能與LMBRD1蛋白質（NESI與LMBD1的總稱）有交互作用的候選蛋白質，鑑定出CAS（cellular apoptosis susceptibility protein）與LMBRD1具有形成蛋白質複合體的能力。已知CAS為攜帶importin-alpha從細胞核運輸出至細胞質的穿梭蛋白質（nuclear shuttling protein）。為了研究LMBRD1與CAS之間的關係，將帶有lmbrd1 shRNA的質體轉染至Huh7細胞株中進行觀察，結果發現在lmbrd1基因降低表現的實驗組中，CAS和importin-alpha具有在細胞核中累積的現象，然而在lmbrd1基因表現量降低但經轉染對shRNA具有抗性的NESI表現質體的rescue實驗中，可觀察到其主要分布位置又回到細胞質。另外，在過去的文獻中指出當細胞受到過氧化氫處理時importin-alpha有集中到細胞核的現象，而這樣的現象在轉染高度表現NESI的細胞中發現有部分返回細胞質的情形。NF-kB具有NLS（nuclear localization signal），其進入細胞核的機制是由importin-alpha所協助執行的。在一般狀態下，NF-kB在Huh7細胞中是均勻分布於細胞質及細胞核之中，在將lmbrd1基因表現降低的Huh7細胞中發現NF-kB主要分布在細胞質，並且在rescue的實驗中發現NF-kB的分布於細胞核中。過去的研究指出，去磷酸化的CAS傾向分布在細胞核。本研究中，利用anti-phosphoserine/theronine的抗體進行免疫沈澱，進一步利用Western blot偵測磷酸化的CAS，結果顯示磷酸化的CAS在lmbrd1基因降低表現的實驗組中有下降的情形。綜合以上結果，在Huh7細胞株中，NESI在調節細胞核質運輸中扮演重要的角色可能是透過影響CAS及importin-alpha運送回細胞質的機制。

Previous studies have revealed that nuclear export signal-interacting protein (NESI) participatesin the nuclear export of the large hepatitis delta antigen with a proline-rich nuclear export signal (NES). NESI is an isoform of lmbrd1 geneproducts,Its amino acid sequence is identical to the amino acid residues74 to 540 of the largest isoform of lmbrd1 geneproductLMBD1. Functions of the NESI and LMBD1 proteins in human cells are largely unknown, but both were predicted as membrane proteins. LMBD1 contains 9 putative transmembrane domains,whereas NESI contains 7 putative transmembrane domains. In this study, different subcellular localizations of the LMBD1 and NESI proteins were detected in Huh7 cells. NESI was mainly localized at the perinuclear region, whereas LMBD1 was localized in organelles. These indicate that NESI may participate in the transport between nucleus and cytoplasm. By performing immunoprecipitation followed by LC-MS/MS, our laboratory has previously identifiedcellular apoptosis susceptibility protein (CAS), also named as exportin 2, as one of the LMBRD1-associated proteins. CAS is known to be a nuclear shuttling protein involving in the nuclear export of importin-alpha. To investigate the function of NESI in the regulation of nuclear shuttling, shRNA specific for lmbrd1gene was introduced into Huh7 cells. The results showed that CAS and its cargoimportin-alpha were accumulated in the nuclei of lmbrd1 knockdown cells. Nevertheless, redistribution to the cytoplasm was observed when the lmbrd1 knockdown cells weretrasfected with shRNA-resistanctplasmid expressing NESI protein.On the other hand, overexpression of NESI rescued the nuclear accumulation of importin-alpha induced by hydrogen peroxide. In addition, NF-kBpossesses nuclear localization signal (NLS), its nuclear import is mediated by importin-alpha. The subcellular localization of NF-kB wasdetected both in the nucleus and the cytoplasm of Huh7 cells. In lmbrd1 knockdown cells, NF-kB was distributed mainly in the cytoplasm, buttranslocated to the nucleus in cells rescued by overexpession of the NESI protein. In addition, it was reported that the dephosphorylated CAS prefer to locate to the nucleus. In this study,immunoprecipitation and Western blot analysis with anti-phosophoserine/threonine and CAS antibodiesdemonstrated a down regulation of the phosphorylated CAS inlmbrd1 knockdown cells. Taken together, these results indicate that NESI plays a critical role in the regulation of nucleocytoplasmic transport through the CAS-mediated importin-alpha recycling pathway in Huh7 cells.