探討SIK2與p97/VCP的交互作用對於調節內質網蛋白質降解的影響以及開發奈米鑽石在蛋白質體研究上的應用

Functional interaction between Salt-inducible kinase 2 and p97/VCP regulates ER-associated degradation and application of nanodiamond in proteomic research

陳韋豪(Wei-Hao Chen)

指導教授 ： 呂勝春

共同指導教授 ： 韓肇中

國立臺灣大學/醫學院/分子醫學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02130

摘要

本文的第一部份主要在探討SIK2在內質網蛋白質降解路徑中扮演的功能以及其如何被調控的機制. 在這裡我們觀察到一個與許多細胞生理活動相關,名為p97/VCP的ATPase, 會與SIK2交互反應. 我們發現p97/VCP在in vitro實驗中能被SIK2磷酸化, 而此磷酸化會造成p97/VCP本身的ATPase活性上升. 另外, SIK2以及p97/VCP在本研究中被發現會共同存在於內質網上, 代表它們之間的交互作用與內質網上的生理活動有關. 如果我們表現基因轉殖的SIK2蛋白質在細胞中, 發現會減少一個由內質網蛋白質降解路徑降解的蛋白質CD3d在293T細胞中存在的量. 但是如果表現無激脢活性的SIK2, 將不會觀察到此現象. 為了了解SIK2如何對細胞中CD3d量造成影響, 我們利用藥物抑制細胞中蛋白質的新合成, 發現SIK2會促進CD3d降解的速度. 此外,我們的研究顯示SIK2會降低CD3d殘餘在內質網中的數量, 證明了SIK2的活性與即將被降解的蛋白質被抽離內質網進入細胞質有關. 這些證據支持了SIK2會經由活化p97/VCP來調節內質網蛋白質降解路徑的論點. 另外, 利用質譜分析, 我們的研究更發現了SIK2上面有4個轉譯後修飾的位置, 分別是K53與K144位置的醋酸基化以及S358與S587位置的磷酸化. 在這些修飾中, K53的醋酸基化發現會降低SIK2的活性, 進而影響到內質網蛋白質降解路徑, 而這個位置的醋酸基的修飾與否則發現是經由p300以及HDAC6的活性進行反應而造成.
本文的第二部份則是提出了一個由奈米鑽石進行蛋白質固相萃取的方法. 與傳統方法相比較, 此法對於濃縮以及純化蛋白質, 進而進行質譜分析或是蛋白質電泳分析, 具有快速以及不需分離吸附載體的優點. 此外, 吸附在奈米鑽石上的蛋白, 可直接以化學物還原以及修飾蛋白質上的雙硫鍵, 接著進行酵素的切割. 本研究進一步利用此法進行尿液蛋白質體分析, 並展示了未來應用於尋找體液中與疾病相關之生物標記的可能性.

關鍵字

蛋白質降解 ； 醋酸基化 ； 磷酸化

並列摘要

The first part of this study is to investigate function and regulation of salt-inducible kinase-2 (SIK2) in ER-associated degradation (ERAD). Here we identified p97/VCP, an ATPase associated with a variety of cellular activities, as an SIK2 interacting protein. We found that p97/VCP is an in vitro substrate of SIK2, and the ATPase activity of p97/VCP was enhanced when phosphorylated by SIK2. Furthermore, SIK2 and p97/VCP were colocalized to the ER membrane, suggesting their functional interaction may be associated with events in the ER. Expression of wild-type, but not kinase-dead, recombinant SIK2 decreased the level of recombinant CD3d, a known substrate for ER-associated degradation (ERAD) in 293T cells. The degradation rate of CD3d was increased by SIK2 when protein synthesis was inhibited. Decreased levels of recombinant CD3d retained in the microsomal fraction in the presence of SIK2 suggest that the kinase activity of SIK2 is required for retrotranslocation of ERAD substrate. These findings suggest that SIK2 participates in the regulation of ERAD through activation of p97/VCP. Furthermore, we characterized post-translational modifications at K53, K144, S358, and S587 of SIK2. Among the modifications, K53 acetylation was demonstrated in this study to down-regulate the activity of SIK2. Acetylation statues of this residue were shown to be involved in regulation of ERAD, and were controlled by the enzymatic activity of p300/CBP and HDAC6.
The second part of our research presents a new solid-phase extraction and elution platform based on surface-functionalized diamond nanocrystallites. Compared with conventional methods, the platform facilitates purification and concentration of intact proteins and their enzymatic digests for ensuing sodium dodecyl sulfate-polyacrylamide gel electrophoresis or matrix-assisted laser desorption/ionization mass spectrometry analysis without prior removal of the adsorbent. One-pot work flow involving reduction of disulfide bonds, protection of free cysteine residues, washing off residual chemicals, and proteolytic digestion of adsorbed proteins can be performed directly on the particles. The utility and versatility of this protein workup platform were demonstrated with liquid chromatography-electrospray ionization tandem mass spectrometry in proteome analysis of human urine. The proteome analysis of each urine sample can be completed in 8 h.

並列關鍵字

ERAD ； SIK2 ； p97 ； p300 ； CBP ； HAT ； HADC

參考文獻

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