Research On RNF167 Regulating Protein Ubiquitination And Related Inhibitor Of ERAD, SVIP, Regulating Autophagy

ERAD (endoplasmic reticulum- associated degradation) and autophagy were belived had hardly any relationship between the two major protein degradation pathways. More and more evidence has been shown that ERAD and autophagy have something to do with each other. At least, uiquitin or poly-ubiquitin chain is validated as protein degradation signal in both pathways. ERAD, in which K48 ubiquitin chains work as degradation signal, degrades soluble proteins in the cell. The ubiquination approach is catalysed by three enzymes sequencially: ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3, thus, poly-ubiquitinated proteins are subjected to proteasome for degradation. In contrast, protein aggragates or insoluble proteins and organelles are degraded by autophagy. But ubiquitin chains which linked to the sustrates are still degradation signal. Atg proteins and p62 and/or NBR1 take part in autophagy and send the substrates to lysosome for degradation. Although ubquitin chains are used as degradation signal in these two pathways, the type of ubquitin chains are different between ERAD and autophagy. K48 poly-ubiquitin chains are the signal of ERAD, whereas K63 type is specific for autophagy. Obviously, different type of poly-ubiquitin chains has different function, but now no technology can be used to observe specific ubiquitin chain's type in live cell. This thesis contains three sections: 1) ubiquitin ligase RNF167 takes part in ERAD, 2) the nature inhibitor of gp78, another ubiquitin ligase interacting with RNF167, named SVIP regulates autophagy, and 3) K48 ubiquitin chain imaging with UCS in live cell.Misfolded proteins are related to some diseases closely. The clearance of misfolded soluble proteins is relied on ERAD. Ubiquitin ligase is one kind of most important enzymes in ERAD pathway, which links ubiquitin or ubiquin chain to the substrate proteins. RNF167 is a ubiquitin ligase not well-known. Previous result showed that RNF167 may regulate the degradation of CFTRΔF508. In this research, RNF167 was found localized to lysosome mainly, and a small part of which localized to ER. In liver and spleen of mice, RNF167 expressed stongly, and a little lower level was observed in central nervous system. After post-translational modification, 24-peptide signal sequence was cleaved for RNF167 precursor, and N-linked glycosilation was achivied. RNF167 mutants were constructed by site-direct mutagenesis. RNF167rm was a RING domain mutant, which had non-active ubiquitin ligase activity sites, lost the E3 activity. Sorting signal mutant, RNF1673A, localized to cytoplasm rather than lysosome. The substrates'range was analysed in the research. RNF167 colocalized well with CFTRΔF508 in lysosome, furthermore, RNF167 regulated the degradation of CFTRΔF508, but had no effect on other two typical ERAD substrates NHK and CD3Δ. RNF167 and CFTRΔF508's interaction was confirmed with immuno-precipitation method. Extra evidence showed RNF167 may form a protein complex together with other ERAD componants, such as gp78/Hrd1, VCP and derlin.The small p97/VCP-interacting protein (SVIP) functions as an inhibitor of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. Here we show that overexpression of SVIP in HeLa cells leads to localization of p97/VCP at the plasma membrane, intracellular foci and juxtanuclear vacuoles. The p97/VCP-positive vacuolar structures colocalized or associated with LC3 and lamp1, suggesting that SVIP may regulate autophagy. In support of this possibility, knockdown of SVIP diminished, whereas overexpression of SVIP enhanced LC3 lipidation. Surprisingly, knockdown of SVIP reduced the levels of p62 protein at least partially through downregulation of its mRNA, which was accompanied by a decrease in starvation-induced formation of p62 bodies. Overexpression of SVIP, on the other hand, increased the levels of p62 protein and enhanced starvation-activated autophagy as well as promoted sequestration of polyubiquitinated proteins polyubiquitin and p62 in autophagosomes. These results suggest that SVIP plays regulatory roles of p97 subcellular localization and is able to regulate autophagy.Eight topologically distinct polyubiquitin chains signal for a remarkably diverse functions in cells, yet no technology is currently available for analysis of any of these chains in living cells. Here we developed a fluorescent ubiquitin chain sensor (UCS) based on ubiquitin-interacting motif-mediated bimolecular fluorescence complementation (BiFC). Application of the UCS enables imaging and quantification of the dynamic increases of K48-specific polyubiquitin chains in living cells under various stress conditions and during Parkin-dependent mitophagy.