| Summary: | 博士 === 國立臺灣大學 === 分子醫學研究所 === 95 === 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.
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