Ubiquitin C-Terminal Hydrolase L1 regulates autophagy by inhibiting autophagosome formation through its deubiquitinating enzyme activity

Ubiquitination modification has been shown to play a key role in autophagy. Increasing studies reported the involvement of de-ubiquitinating enzymes (DUBs) in autophagy pathway. To systematically search how DUBs manipulate autophagy, we utilized a double fluorescence tagged LC3 stable HeLa cell line, and did a genome wide screen of 55 human DUBs which is about 60% coverage of the DUB family. We found a bunch of DUBs have impact on autophagy by either changing the LC3 puncta formation or the autophagy flux. One of them, Ubiquitin C-Terminal Hydrolase L1 (UCHL1) correlated to Parkinson's disease, strongly affects autophagy by inhibiting autophagosome formation. We found UCHL1 overexpression inhibits LC3 puncta formation and is dependent on its DUB activity. Knockdown of UCHL1 significantly promotes LC3 puncta formation. Further study revealed that UCHL1 may affect autophagy by interacting with LC3 but not other autophagy related proteins. Interestingly, a Parkinson's disease related mutant UCHL1 I93 M defects its DUB activity and can no longer inhibit autophagosome formation. We further screened 22 commercially available DUB inhibitors and found two potent UCHL1 inhibitors LDN-57444 (LDN) and NSC632839 (NSC), when treating cells, both strongly induce LC3 puncta formation. Taken together, our results indicated a new insight into the manner in which DUB regulates autophagy and provided potential drugs for the Parkinson's disease.