細胞壓力反應下轉穀醯胺酶催化蛋白質修飾作用之研究

轉穀醯胺酶(TG)是一群催化蛋白質鏈合、胺類修飾以及去胺反應的酵素，而TG的蛋白質受質在各項研究中持續地被發現，TG的修飾作用會影響蛋白質受質的功能，並且有越來越多的證據顯示TG對蛋白質進行轉譯後蛋白質修飾的重要性。由於蛋白質體學的技術以及觀念的發展，鑑定TG的蛋白質受質身分也相對的便捷亦準確，TG重要的生化角色也逐漸的被發現在各種細胞生理的作用中。在此蛋白質體的研究中以酵素以及蛋白質受質之間的交互作用為理論基礎，以部份純化的方法分離出含有富含TG活性的蛋白質樣本，預期在這個樣本中能發現尚未被發現的TG的蛋白質受質。經過質譜鑑定後，目前篩選出二十九個推定的TG的蛋白質受質，研究過程中，其中的VCP、ERp72、GRP78、PDI、14-3-3、PRDX1、PSC2以及glyoxalase I (GlxI)經實驗首次證實結構上為TG的蛋白質受質。另外，GRP78為目前已知可能是TG的蛋白質受質，由質譜以及酵素催化的實驗結果驗證了之前的發現，並且在純化GRP78蛋白質的同時，發現具有活性的TG2也同時被純化，結果顯示GRP78與TG之間除了存在酵素與受質的關係，也可能存在有蛋白質間的交互作用。在整個研究過程中，除了尋找新的TG蛋白質受質之外，更重要的是找尋這些已知或者新鑑定的TG受質在細胞壓力反應之下是否會參與細胞的生理作用，因此同時也建立細胞壓力反應下造成TG活化的細胞模式。初次發現有TG受質的特性的14-3-3，除了證實會受到TG的修飾，部分結果發現14-3-3可能經由TG修飾所進行的蛋白質鏈合作用而形成二聚體並且改變在細胞中的位置。實驗中當細胞以A23187造成細胞內鈣離子上升藉以活化細胞內TG時，14-3-3由單體轉向成為二聚體之後，傾向從細胞萃取的上清液部分進入沉積物的部分。最後，在這些新定出的TG的蛋白質受質中，最清楚的發現在於GlxI的修飾與調節，GlxI除了具有TG的蛋白質受質的特性之外，GlxI並且會結合精素(spmermidine)以及精胺(spermine)的多胺類，而結合的強度可以抗sodium dodecyl sulfate (SDS)的變性作用。在TG催化之下，GlxI並不形成二聚體反而進行多胺類的轉胺作用或者去胺作用。同時，結合多胺類也會影響GlxI的活性以及穩定性，經由TG催化之後，多胺類的轉胺作用以及去胺作用更增加GlxI的酵素活性以及穩定性，其中多胺類的轉胺作用更使得GlxI在鹼性溶液中有更好的穩定性。更重要的是TG的作用可以在細胞內調控GlxI對於甲基乙二醛(MG)的去毒作用，以MG處理過的培養細胞，TG的活性會有明顯的增加並且會受到TG抑制劑的抑制，當加入TG的抑制劑到培養細胞後以MG處理，發現抑制TG之後細胞對於MG的抗性有明顯的下降。同時，以RNA干擾的方式降低細胞內TG2的表現量，發現也可以減低細胞對於MG的抗性。另外，細胞內多胺類的含量也會影響細胞對MG的抗性改變，加入二乙基正精胺(DENSPM)到培養細胞以降低細胞內多胺類的含量，再以MG處理細胞則造成了更嚴重的細胞死亡。這些結果顯示，TG的在細胞壓力反應中，可能具有藉由修飾作用來調節下游蛋白質受質的能力，其中，經由TG所催化的轉胺以及去胺作用來調節細胞內的GlxI的活性與穩定性，使細胞足以反應並去除過盛的MG所造成的細胞毒性。另外，這個結果可能也說明為何癌細胞對於抗癌藥物產生抗藥性的機制，癌細胞過度地表現TG以及GlxI而使癌細胞具有較好的能力可以移除藥物所引發細胞內上升的MG，因而使癌細胞對於抗癌藥物造成細胞內MG增加引起細胞凋亡產生抗藥性。

關鍵字

細胞壓力反應；轉榖醯胺酶蛋白質修飾

並列摘要

Transglutaminases (TGs) have been characterized as a group of enzymes designated for catalyzing protein cross-linking, polyamine incorporation, and deamidation. Meanwhile, an emerging group of proteins has been cataloged as TG protein substrates in numerous studies, and their functions are affected by the post-translational modifications catalyzed by TGs. This proteomics study was performed in a protein sample enriched in TG2 and protein substrates based on the theoretical interactions of enzymes and substrates. The fraction enriched in TG2 obtained by ion exchanger column chromatography was subjected to proteomics identification of potential TG2 protein substrates. Twenty nine putative TG2 protein substrates of interest were documented from the proteomics identification. VCP, ERp72, PDI, 14-3-3, PRDX1, PSC2, and glyoxalase I (GlxI) were verified as novel TG protein substrates by in vitro transamidation assay. Among the putative TG2 protein substrates, GRP78 has been identified as a TG2 protein substrate. Our results from mass spectrometry analysis and in vitro transamidation experiments confirmed previous descriptions. Moreover, TG2 activity was co-purified with GRP78 by affinity column chromatography indicating that the relationship between TG2 and GRP78 may go beyond that of enzyme and substrates. On the other hand, 14-3-3 was identified as a TG protein substrate for the first time. Dimerization of 14-3-3 was found in cells treated with calcium ionophore, possibly caused as TG2-catalyzed cross-linking and this rendered 14-3-3 insoluble. We focused on on glyoxalase I (GlxI) and studied in more detail the effect of TG2 on the substrates. Except for being a TG2 protein substrate, GlxI was found as a polyamine binding protein and the interaction between the enzyme and spermidine or spermine was SDS-resistant. Binding of polyamines alone increased the enzyme activity and protein stability in alkaline solution of GlxI. Moreover, polyamine incorporation and deamidation of GlxI were demonstrated by in vitro transamidation assay and competitive transamidation assay, respectively. TG2 catalyzed polyamine incorporation and deamidation of GlxI, instead of protein cross-linking. With TG2 catalysis, polyamine incorporation or deamidation of GlxI enhanced the enzyme activity. However, polyamine incorporation of GlxI not only increased the enzyme activity but also protein stability. More importantly, the modifications of GlxI by TG2 and up-regulation of Glx enzyme activity were found to increase cells’ resistence to methylglyoxal (MG)-induced cytotoxicity. The TG activity of cultured cells was elevated with MG and suppressed with TG inhibitors treatments. The inhibition of endogenous TG activity by TG inhibitors in cultured cells reduced the cell viability on cells challenged with MG. Furthermore, knockdown of TG2 by RNA interference also reduced the cell resistance to MG treatments. Besides, the endogenous polyamines also modulate the endogenous GlxI activity. Depletion of endogenous polyamine by DENSPM caused more cell death when cells were treated with MG. The results indicated that TG2 is able to regulate downstream protein substrates by posttranslational modifications. In this study, polyamine incorporation and deamidation of GlxI by TG2 catalysis are essential for cells to respond to the increasing MG and remove the toxicity of excessive MG. The results also imply that the drug resistance of cancer cells against MG-induced apoptosis may result from the overexpression of TG2 and GlxI.

並列關鍵字

cell stress response ； transglutaminase ； protein modification ； glyoxalase I

參考文獻

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3. Pisano, J. J., Finlayson, J. S., and Peyton, M. P. (1968) [Cross-link in fibrin polymerized by factor 13: epsilon-(gamma-glutamyl)lysine.], Science 160, 892-893.

4. Lorand, L. (2001) Factor XIII: structure, activation, and interactions with fibrinogen and fibrin, Ann N Y Acad Sci 936, 291-311.

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細胞壓力反應下轉穀醯胺酶催化蛋白質修飾作用之研究

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