Thioredoxin進入神經母細胞瘤SH-SY5Y細胞之機制研究

Abstract

1.研究背景: 神經母細胞瘤SH-SY5Y細胞系 (D cell) 在缺乏血清培養或處理MPP+ (1-methyl-4-phenylpyridinium) 會細胞凋亡，若添加外源性Thioredoxin (Trx) 會抑制細胞凋亡 (Andoh, Chock and Chiueh, 2002)。另一株以多重逆境處理篩選出表現幹細胞標誌CD133的pre-conditioned SH-SY5Y cell (N cell) 比D cell表現較多Trx，在抗癌藥物etoposide作用下的細胞凋亡程度較D cell低 (Hu, 2013)；若以DNCB (2,4-Dinitrochlorobenzene) 抑制Trx redox cycling則N cell細胞凋亡的情形會大幅提升，證明Trx redox cycling與癌細胞產生抗藥性有關。推測分子量11.7 kDa的Trx在低濃度處理 (1 μM) 之下能夠進入並透過Trx redox cycling產生活性來保護細胞。已有文獻指出Trx會在細胞之間運移 (Kondo et al., 2004)，但是詳細機制並不清楚。所以本論文以探討研究人類Trx進入細胞的機制為主題。 2.研究目的: 希望了解氧化還原蛋白Trx進入SH-SY5Y細胞的可能機制，擬結合生物資訊分析的結果，找出與Trx進入細胞有關的胺基酸位置。一、Trx可能透過細胞膜特殊運輸系統運移。二、或與細胞膜表面膜蛋白交互作用後進入細胞行使功能。 3.研究方法: 為了區別SH-SY5Y細胞內源性蛋白Trx，本實驗利用基因工程技術構築質體，以大腸桿菌生產人類重組蛋白His-tag Trx與其突變型蛋白。透過生物資訊與點突變技術分析與Trx運移有關的胺基酸位置，推測Trx與特定膜蛋白交互作用之後能進入細胞行使功能。最後使用西方墨點法與細胞免疫染色技術偵測His-tag Trx進入細胞內之結果。 4.結果: 實驗結果顯示，(1) 外加1 μM Trx可以抑制etoposide (83 μM) 在SH-SY5Y細胞產生的細胞凋亡，為了區分細胞內源性Trx，以基因工程技術生產His-tag Trx蛋白。(2) 利用西方墨點法證明外源性的His-tag Trx不論是氧化態或者還原態都會進入SH-SY5Y細胞內，提升外源性His-tag Trx的濃度會增加細胞攝取的蛋白質量。(3) 利用細胞免疫染色與共軛焦顯微鏡觀察，His-tag Trx出現在細胞膜上也存在於細胞質與細胞核中。(4) 使用生物資訊工具分析Trx的序列，找到兩種可能與蛋白質運移相關的特徵。Trx帶有可辨認adaptor proteins的tyrosine-based sorting signal (Tyr49-X-X-Val52)，與clathrins主導之內吞作用 (endocytosis) 有關。序列Phe27-X-X-X-Trp31則是與peroxisome translocation有關。若將Tyr49與Val52突變成alanine則會抑制Trx進入細胞，但是Trp31突變並不會影響Trx運移。 5.結論: 本研究證明外加之外源性His-tag Trx可以進入細胞，在細胞膜、細胞質與細胞核中都可找到。結合生物資訊分析與點突變技術，初步結果找出氧化還原蛋白Trx胺基酸序列上與運輸相關的位置，包括：(1) 與endocytosis相關的tyrosine-based sorting signal (Tyr49-X-X-Val52)。(2) peroxisome membrane docking molecule結合的位置 (Phe27-X-X-X-Trp31)。實驗證明外源性的Trx可經由endocytosis運移到細胞內的細胞質與細胞核中，使細胞產生抗氧化及抗化療並且抑制細胞凋亡。

1.Backgrounds: The recent results of our research laboratory (Hu, 2013) has confirmed the early findings of an NIMH research team headed by Prof Chiueh (Andoh, Chock and Chiueh, 2002) that the exogenously administered human redox protein Trx (MW: 11.7 kDa; < 1 μM) can enter SH-SY5Y cells to produce its anti-apoptotic and cyto-protective effects against oxidative stress evoked by serum deprivation, neurotoxins and chemotherapeutics through a thiol-sensitive redox mechanism. The possible uptake and/or transport mechanisms of Trx are not fully understood at the present. 2.Objective: The present research aim is to investigate possible transport mechanisms for the human redox protein Trx to enter the SH-SY5Y cells for producing biological functions. The primary research goal is to create point mutations on His-tag Trx for searching by which mechanism of Trx to enter the cell membrane through a specific transport system. 3.Methods: We searched the functional sequence associated with the transportation of Trx by utilizing the bioinformatics analysis of Trx peptide sequence and synthesized His-tag Trx mutants for studying interactions between Trx and plasma membrane proteins. The end point was to detect the entry of exogenously administered His-tag Trx in SH-SY5Y cells by the western blotting, the immunocytochemistry and the confocal imaging. 4.Results: The present results show that (i) the extracellularly administered Trx (< 1μM) suppressed the etoposide (83 μM)-induced apoptosis in SH-SY5Y cells. We synthesized recombinant protein His-tag Trx to distinguish from the endogenous Trx in SH-SY5Y cells and thus verified that (ii) both the extracellularly administered Trx in oxidized and reduced form entered the cells in a dose-dependent manner and the entry of oxidized Trx were more than the reduced Trx. (iii) The extracellularly administered Trx localized in cell membrane, cytosol and cell nucleus. (iv) The mutations on the tyrosine-based sorting signal (Tyr49-X-X-Val52) blocked the entry of His-tag Trx. The modification of peroxisome membrane protein docking site (Phe27-X-X-X-Trp31) did not alter the entry of His-tag Trx from extracellular space to the cytosol. 5.Conclusions: The study utilized the bioinformatic analysis and point mutation technique to search the transport-related peptide on Trx protein, including (i) the tyrosine-based sorting signal (Tyr49-X-X-Val52) interacts with adaptor proteins in endocytosis and (ii) the peroxisome membrane docking site (Phe27-X-X-X-Trp31) for the translocation of cytosol proteins. The present results show that Trx entered the cells through endocytosis and subsequently entered nucleus to mediate the anti-oxidative cyto-protection. These results infer that the levels of Trx in cancer cells may also play a role in drug resistance.