中文摘要

SMT3 (Suppressor of MIF Two 3 protein) 首先是由研究酵母菌(Saccharomyces cerevisiae) 的中心節蛋白 (MIF2) 突變的抑制作用時所分離出的蛋白。因其基因結構類似Ubiquitin故而被稱為Ubiquitin-like protein。除了酵母菌外，在其他高低等真核生物亦發現SMT3的存在。在較低等的真核生物如酵母菌、線蟲、果蠅等僅發現一種SMT3的存在，而在較高等的真核生物如哺乳類則存在三種不同的SMT3 (SMT3A、SMT3B及SMT3C) 。在演化上，這些SMT3蛋白的胺基酸序列具相當強烈的保守性，顯示SMT3在生物體中功能的重要性。目前已知SMT3C為一個修飾作用的蛋白，可與細胞內許多蛋白進行修飾作用如PML、IκBα、P53等，故而掌控細胞內許多的重要生理功能。然而對於SMT3A、SMT3B的功能至今仍不明確，且與SMT3C功能的相關性亦無確切的証實。
為了瞭解SMT3B的功能，我們曾在酵母菌雜交系統中偵測到SMT3B可與Daxx (Fas結合蛋白) C-terminus產生作用。進一步發現三種人類的SMT3 isoforms (3A、3B、3C) 可分別與Daxx N-terminus (Daxx 1)及C-terminus (Daxx 4) 產生結合作用。在Truncated SMT3研究中發現，SMT3與Daxx N-terminus (Daxx 1) 及C-terminus (Daxx 4) 的結合方式並不相同。其中SMT3A、3B較為相似，而異於SMT3C。且在定點突變分析中發現SMT3C與Daxx N-terminus (Daxx 1) 的結合方式是藉由SMT3C C-terminus的 glycine97與Daxx N-terminus (Daxx 1) lysine60產生共價結合，此機制與SMT3C作用在其他目標蛋白經由Ubc9所辨認的SUMO-1一般序列ΨKXE (SUMO-1 consensus sequence) 的共價結合方式相同。此外，在SMT3自我作用 (self-reacting) 的反應中發現，人類的SMT3三個isoforms中僅SMT3C具有自我聚合的現象。
綜合以上結果，我們認為人類SMT3 (SMT3A、3B、3C) 雖然皆可與Daxx產生作用，但結合方式僅SMT3A/B較為相似，而異於SMT3C，但由truncated SMT3實驗顯示SMT3C作用在Daxx N- terminus (Daxx 1) 的方式與SMT3A/B作用在Daxx C-terminus (Daxx 4) 的方式相似，故而推測SMT3A/B似乎亦具有特殊的胺基酸辨認的序列位於Daxx C-terminus部分上。

Abstract

SMT3 (Suppressor of MIF2 3 protein) was identified as a mutation
suppressor in yeast centromere protein MIF2. It is also known as an
ubiquitin-like protein due to the smilarities of their primary structures that
is very conserved during the eukaryotic evolution. Although only one
SMT3 was found in low eukaryotes such as in yeast, three members of
SMT3 (SMT3A, SMT3B and SMT3C) have been identified in high
eukaryotes. It has been known that SMT3C plays an important role in post-translational modification. However, the functions of SMT3A and SMT3B are not well studied yet and the relationship among the SMT3 families remains unclear.
In the present study, Daxx, a Fas binding protein, was demonstrated to bind to SMT3B using yeast two-hybrid assay. It was found that the
N-terminal domain of Daxx (Daxx 1) and the C-terminal domain of Daxx
(Daxx 4), respecifitively, bound to all members of human SMT3
families (including SMT3A, SMT3B and SMT3C). Neverthless,
mechanisms of interactions between the SMT3 families and Daxx
domains remined unclear. Studies on truncated human SMT3 families
have shown that two glycines on the C-terminal end of human SMT3
families were required in the interaction between SMT3 and Daxx
domains, for example, SMT3A and SMT3B required C-terminal two glycines on the Daxx 4 domain where as SMT3C required C-terminal two glycines on the Daxx 1 domain. Morever, truncated SMT3C and Daxx 1 domain point mutations have also indicated that the the linkage of
glycine97 of SMT3C and the lysine60 of Daxx 1, in which the SMT3C/
SUMO-1 consensus sequence ΨKXE was found. Further, SMT3C was
the only member of the SMT3 families capable of self-reacting.
Results also suggested that similar mechanism of interaction
between SMT3A/B and Daxx 1, which is not in accordance with the model proposed in this study regarding the interaction mechanism between SMT3C and Daxx 1. Although two glycines on the C-terminal end of SMT3A/B were necessary for the interactions with Daxx 4 domains, the SMT3C/SUMO-1-consensus sequence ΨKXE was not detected in the Daxx 4 domain. It is therefore, suggested that the mechanism of the interaction between SMT3A/B and Daxx 4 is similar to that of SMT3C and Daxx 1, that may required different binding sequences that is specific for SMT3A/B.

目錄

中文摘要 …………………………………………………… i
英文摘要 …………………………………………………… iii
壹、緒 論
一、SMT3的發現及在酵母菌中的功能 …………………………. 1
二、酵母菌中心節蛋白MIF2與人類中心節蛋白的相關性 ……. 1
三、SMT3在演化上的相關性 ……………………………………. 2
四、SMT3C與Ubiquitin的相關性 ……………………………….. 3
五、SMT3C目前已知的功能 …………………………………….. 5
六、Daxx的發現及功能 ………………………………………...… 8
貳、研究目的 …………………………………………….. 11
參、研究策略及方法
一、研究策略 ……………………………………………..…… 12
二、研究材料及方法
(一)質体的建構 (Plasmid constructions)
1.Primers的設計及合成 ……………………………………… 13
2.聚合酶鏈反應PCR (Polymerase chain reaction) ..………….. 18
3.DNA電泳分析 …………………………………………….…. 20
4.DNA的轉殖 (DNA cloning) …………………………………. 20
(二)大腸桿菌的形質轉換 (E. coli transformation )
1.勝任細胞的製備 (Preparation of competent cells) ………….. 21
2.DH5α形質轉換 (Transformation) ……………..….….…… 22
(三)重組DNA的篩選 (Recombinant DNA selection) ……….…. 22
(四) DNA定序 (DNA sequencing)
1.定序膠片的製作 (Preparation of autosequence gel) ……….. 23
2.DNA定序分析 (DNA sequencing analysis) ……..………… 24
(五)酵母菌二次雜交分析 (Yeast two-hybrid assay)
1.酵母菌勝任細胞的配製 (Preparation of yeast competent cells) 24
2.酵母菌的形質轉換 (Yeast transformation) ………..………. 25
3.β-galactosidease Assay
3.1.Colony-Lift Filter Assay ………………………………. 26
3.2.Liquid Culture Assay …………………….………… 27
肆、結 果 ……………………………………………….. 29
伍、討 論 ……………………………………………………….. 34
陸、參考文獻 ………………………………………….….. 42
柒、圖 表
(一) 圖目錄
圖一、不同生物間的SMT3蛋白的胺基酸序列比較 …….….. 49
圖二、Ubiquitin與SMT3/SUMO的胺基酸序列比較 ……... 50
圖三、Ubiquitin與SUMO-1/SMT3C作用方式的比較 …….. 51
圖四、SUMO-1 作用在Daxx的結合區域 …….…………… 52
圖五、SMT3與Daxx作用可能產生的功能 ……………….... 53
(二) 表目錄
表一、Ubc9辨別Substrates的胺基酸序列 (SUMO-1-CS) ….. 54
表二、目前在酵母菌雜交系統中發現的Daxx作用蛋白 …… 55
表三、酵母菌雜交分析SMT3與Daxx的交互作用 ………… 56
表四、酵母菌雜交分析SMT3A與Daxx的作用區域 …….… 57
表五、酵母菌雜交分析SMT3B與Daxx的作用區域 …….… 58
表六、酵母菌雜交分析SMT3C與Daxx的作用區域 …….… 59
表七、酵母菌雜交分析Truncated SMT3A與Daxx的作用 60
表八、酵母菌雜交分析Truncated SMT3B與Daxx的作用 61
表九、酵母菌雜交分析Truncated SMT3C與Daxx的作用 62
表十、雜交分析Truncated SMT3A與Daxx 1及Daxx 4作用 63
表十一、雜交分析Truncated SMT3B與Daxx 1及Daxx 4作用 64
表十二、雜交分析Truncated SMT3C與Daxx 1及Daxx 4作用 65
表十三、突變分析探討SMT3C與Daxx 1 (Lys60)的作用 66
表十四、酵母菌二次雜交分析SMT3 與Daxx作用的強弱 67
表十五、酵母菌雜交分析探討SMT3與Ubiquitination的相關性68

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