The ubiquitin isopeptidase USP10 deubiquitinates LC3B to increase LC3B levels and autophagic activity

Abstract

Components of the autophagy machinery are subject to regulation by various posttranslational modifications. Previous studies showed that monoubiquitination of LC3B catalyzed by the ubiquitin-activating enzyme UBA6 and ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 targets LC3B for proteasomal degradation, thus reducing LC3B levels and autophagic activity under conditions of stress. However, mechanisms capable of counteracting this process are not known. Herein, we report that LC3B ubiquitination is reversed by the action of the deubiquitinating enzyme USP10. We identified USP10 in a CRISPR-Cas9 knockout screen for ubiquitination-related genes that regulate LC3B levels. Biochemical analyses showed that silencing of USP10 reduces the levels of both the LC3B-I and LC3B-II forms of LC3B through increased ubiquitination and proteasomal degradation. In turn, the reduced LC3B levels result in slower degradation of the autophagy receptors SQSTM1 and NBR1 and an increased accumulation of puromycin-induced aggresome-like structures. Taken together, these findings indicate that the levels of LC3B and autophagic activity are controlled through cycles of LC3B ubiquitination and deubiquitination. Components of the autophagy machinery are subject to regulation by various posttranslational modifications. Previous studies showed that monoubiquitination of LC3B catalyzed by the ubiquitin-activating enzyme UBA6 and ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 targets LC3B for proteasomal degradation, thus reducing LC3B levels and autophagic activity under conditions of stress. However, mechanisms capable of counteracting this process are not known. Herein, we report that LC3B ubiquitination is reversed by the action of the deubiquitinating enzyme USP10. We identified USP10 in a CRISPR-Cas9 knockout screen for ubiquitination-related genes that regulate LC3B levels. Biochemical analyses showed that silencing of USP10 reduces the levels of both the LC3B-I and LC3B-II forms of LC3B through increased ubiquitination and proteasomal degradation. In turn, the reduced LC3B levels result in slower degradation of the autophagy receptors SQSTM1 and NBR1 and an increased accumulation of puromycin-induced aggresome-like structures. Taken together, these findings indicate that the levels of LC3B and autophagic activity are controlled through cycles of LC3B ubiquitination and deubiquitination. Autophagy is a cellular process for the lysosomal degradation of cytoplasmic materials (i.e., “cargos”) such as organelles, protein aggregates, and intracellular pathogens (1Bento C.F. Renna M. Ghislat G. Puri C. Ashkenazi A. Vicinanza M. Menzies F.M. Rubinsztein D.C. Mammalian autophagy: How does it Work.Annu. Rev. Biochem. 2016; 85: 685-713Crossref PubMed Scopus (348) Google Scholar, 2Dikic I. Elazar Z. Mechanism and medical implications of mammalian autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 349-364Crossref PubMed Scopus (738) Google Scholar). This process involves engulfment of the cargos into double-membraned vesicles named autophagosomes. The autophagosomes subsequently fuse with lysosomes to form autolysosomes, where the cargos are degraded and the products of degradation recycled (1Bento C.F. Renna M. Ghislat G. Puri C. Ashkenazi A. Vicinanza M. Menzies F.M. Rubinsztein D.C. Mammalian autophagy: How does it Work.Annu. Rev. Biochem. 2016; 85: 685-713Crossref PubMed Scopus (348) Google Scholar, 2Dikic I. Elazar Z. Mechanism and medical implications of mammalian autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 349-364Crossref PubMed Scopus (738) Google Scholar). Autophagy is critical for cellular homeostasis, and autophagic defects underlie the pathogenesis of many human diseases, including neurodegenerative disorders, cardiomyopathy, cancer, type-II diabetes, and immune system disorders (3Levine B. Kroemer G. Biological functions of autophagy genes: a disease Perspective.Cell. 2019; 176: 11-42Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). The mechanism of autophagy is mediated by a core machinery comprising over 30 different proteins (1Bento C.F. Renna M. Ghislat G. Puri C. Ashkenazi A. Vicinanza M. Menzies F.M. Rubinsztein D.C. Mammalian autophagy: How does it Work.Annu. Rev. Biochem. 2016; 85: 685-713Crossref PubMed Scopus (348) Google Scholar, 2Dikic I. Elazar Z. Mechanism and medical implications of mammalian autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 349-364Crossref PubMed Scopus (738) Google Scholar). In addition, many other proteins act as regulators by altering the activities of core machinery components. Among these regulators are enzymes that catalyze posttranslational modifications such as phosphorylation/dephosphorylation, acetylation/deacetylation, ubiquitination/deubiquitination, lipidation/delipidation, and proteolysis (4Wani W.Y. Boyer-Guittaut M. Dodson M. Chatham J. Darley-Usmar V. Zhang J. Regulation of autophagy by protein post-translational modification.Lab Invest. 2015; 95: 14-25Crossref PubMed Scopus (78) Google Scholar, 5Xie Y. Kang R. Sun X. Zhong M. Huang J. Klionsky D.J. Tang D. Posttranslational modification of autophagy-related proteins in macroautophagy.Autophagy. 2015; 11: 28-45Crossref PubMed Scopus (158) Google Scholar). A key target of regulatory modifications is the autophagy protein LC3B (abbreviation for microtubule associated protein 1 light chain 3 beta or MAP1LC3B), the best studied of six human orthologs of yeast Atg8, the others being LC3A, LC3C, GABARAP, GABARAPL1, and GABARAPL2 (1Bento C.F. Renna M. Ghislat G. Puri C. Ashkenazi A. Vicinanza M. Menzies F.M. Rubinsztein D.C. Mammalian autophagy: How does it Work.Annu. Rev. Biochem. 2016; 85: 685-713Crossref PubMed Scopus (348) Google Scholar, 2Dikic I. Elazar Z. Mechanism and medical implications of mammalian autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 349-364Crossref PubMed Scopus (738) Google Scholar). LC3B participates in cargo engulfment, autophagosome maturation, and autophagosome–lysosome fusion (1Bento C.F. Renna M. Ghislat G. Puri C. Ashkenazi A. Vicinanza M. Menzies F.M. Rubinsztein D.C. Mammalian autophagy: How does it Work.Annu. Rev. Biochem. 2016; 85: 685-713Crossref PubMed Scopus (348) Google Scholar, 2Dikic I. Elazar Z. Mechanism and medical implications of mammalian autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 349-364Crossref PubMed Scopus (738) Google Scholar). Upon induction of autophagy, LC3B is converted from a cytosolic LC3B-I form to a membrane-bound LC3B-II form, which is the active species in autophagy (1Bento C.F. Renna M. Ghislat G. Puri C. Ashkenazi A. Vicinanza M. Menzies F.M. Rubinsztein D.C. Mammalian autophagy: How does it Work.Annu. Rev. Biochem. 2016; 85: 685-713Crossref PubMed Scopus (348) Google Scholar, 2Dikic I. Elazar Z. Mechanism and medical implications of mammalian autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 349-364Crossref PubMed Scopus (738) Google Scholar), LC3B is phosphorylated at different amino-acid residues, in some cases with demonstrated consequences on autophagy. For example, phosphorylation of LC3B on Thr-50 by STK3 and STK4 (serine/threonine kinase 3 and 4), PKCζ (protein kinase Cζ), or NEK9 (NIMA-related kinase 9) regulates autophagosome–lysosome fusion and binding of LC3B to several LC3-interacting region (LIR)-containing proteins, such as SQSTM1 (sequestosome-1, also known as p62), NBR1 (neighbor of BRCA1 gene), FYCO1 (FYVE and coiled-coil domain-containing 1), and ATG4 (autophagy gene 4 product) (6Shrestha B.K. Skytte Rasmussen M. Abudu Y.P. Bruun J.A. Larsen K.B. Alemu E.A. Sjøttem E. Lamark T. Johansen T. NIMA-related kinase 9-mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1.J. Biol. Chem. 2020; 295: 1240-1260Abstract Full Text Full Text PDF PubMed Google Scholar). LC3B is also phosphorylated on Thr-6 and Thr-29 by PKC (protein kinase C), but these modifications do not seem to affect autophagy (7Jiang H. Cheng D. Liu W. Peng J. Feng J. Protein kinase C inhibits autophagy and phosphorylates LC3.Biochem. Biophys. Res. Commun. 2010; 395: 471-476Crossref PubMed Scopus (70) Google Scholar). Acetylation of LC3B also participates in autophagy regulation. Acetyl groups are covalently linked to LC3B on Lys-49 and Lys-51 by EP300 (E1A binding protein p300) and CREBBP (CREB binding protein) acetylases (8Lee I.H. Finkel T. Regulation of autophagy by the p300 acetyltransferase.J. Biol. Chem. 2009; 284: 6322-6328Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 9Huang R. Xu Y. Wan W. Shou X. Qian J. You Z. Liu B. Chang C. Zhou T. Lippincott-Schwartz J. Liu W. Deacetylation of nuclear LC3 drives autophagy initiation under starvation.Mol. Cell. 2015; 57: 456-466Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar) and removed from LC3B by the SIRT1 (NAD-dependent sirtuin 1) deacetylase (10Lee I.H. Cao L. Mostoslavsky R. Lombard D.B. Liu J. Bruns N.E. Tsokos M. Alt F.W. Finkel T. A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 3374-3379Crossref PubMed Scopus (943) Google Scholar). Acetylated LC3B mainly accumulates in the nucleus in an inactive form. Deacetylation of LC3B promotes its redistribution to the cytoplasm, where it participates in autophagy (9Huang R. Xu Y. Wan W. Shou X. Qian J. You Z. Liu B. Chang C. Zhou T. Lippincott-Schwartz J. Liu W. Deacetylation of nuclear LC3 drives autophagy initiation under starvation.Mol. Cell. 2015; 57: 456-466Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Accordingly, EP300 knockdown (KD) stimulates starvation-induced autophagy (8Lee I.H. Finkel T. Regulation of autophagy by the p300 acetyltransferase.J. Biol. Chem. 2009; 284: 6322-6328Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), whereas SIRT1 KD results in autophagy defects (10Lee I.H. Cao L. Mostoslavsky R. Lombard D.B. Liu J. Bruns N.E. Tsokos M. Alt F.W. Finkel T. A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 3374-3379Crossref PubMed Scopus (943) Google Scholar). Recently, autophagy was also shown to be regulated by ubiquitination. We and others found that LC3B is monoubiquitinated by the concerted action of the UBA6 E1 ubiquitin (Ub)-activating enzyme and the BIRC6 hybrid E2 ubiquitin-conjugating enzyme/E3 Ub ligase (11Jiang T.X. Zou J.B. Zhu Q.Q. Liu C.H. Wang G.F. Du T.T. Luo Z.Y. Guo F. Zhou L.M. Liu J.J. Zhang W. Shu Y.S. Yu L. Li P. Ronai Z.A. et al.SIP/CacyBP promotes autophagy by regulating levels of BRUCE/Apollon, which stimulates LC3-I degradation.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 13404-13413Crossref PubMed Scopus (13) Google Scholar, 12Jia R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar), and polyubiquitinated by the von Hippel–Lindau (VHL) tumor suppressor E3 Ub ligase (13Kang H.M. Noh K.H. Chang T.K. Park D. Cho H.S. of by is associated with autophagy and in 2019; PubMed Scopus Google Scholar). monoubiquitination and to proteasomal degradation of with in autophagic activity (11Jiang T.X. Zou J.B. Zhu Q.Q. Liu C.H. Wang G.F. Du T.T. Luo Z.Y. Guo F. Zhou L.M. Liu J.J. Zhang W. Shu Y.S. Yu L. Li P. Ronai Z.A. et al.SIP/CacyBP promotes autophagy by regulating levels of BRUCE/Apollon, which stimulates LC3-I degradation.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 13404-13413Crossref PubMed Scopus (13) Google Scholar, 12Jia R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar, H.M. Noh K.H. Chang T.K. Park D. Cho H.S. of by is associated with autophagy and in 2019; PubMed Scopus Google Scholar). In protein ubiquitination is reversed by Ub catalyzed by a of known as deubiquitinating enzymes S. D. the enzyme Rev. Mol. Cell Biol. 2019; PubMed Scopus (158) Google Scholar). However, this not shown to be the for Herein, we report the results of a CRISPR-Cas9 screen that USP10 as a that thus LC3B levels and autophagic LC3B we a CRISPR-Cas9 screen a human that LC3B with R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar). with a CRISPR-Cas9 ubiquitination-related genes R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar) The screen was on the that of LC3B ubiquitination and degradation, thus and by and to of and the of was to A and The of in to was by and the of CRISPR-Cas9 W. Xu H. T. L. Zhang F. Liu J.S. M. Liu of genes from knockout Biol. PubMed Scopus Google Scholar) and The in this screen in of was USP10. The other proteins the the beta and of several E3 ubiquitin the of the was to a the of these other proteins was not a role of USP10 in the regulation of LC3B we USP10 by with USP10 We that USP10 silencing the levels of both the cytosolic LC3B-I and membrane-bound LC3B-II forms by A and in LC3B-I and LC3B-II levels silencing of USP10 in the human and human CRISPR-Cas9 of USP10 in in in both LC3B-I and LC3B-II levels C and of in levels of LC3B-I and LC3B-II to in and of other showed that USP10 in also reduced the levels of LC3A, but not and The of USP10 on the levels of and GABARAPL2 not be of of for we that of with to the enzyme that participates in LC3B ubiquitination R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar), increased the levels of both LC3B-I and LC3B-II A and results thus demonstrated that USP10 reduced the levels of an that was to that by of the enzymes UBA6 or BIRC6 (11Jiang T.X. Zou J.B. Zhu Q.Q. Liu C.H. Wang G.F. Du T.T. Luo Z.Y. Guo F. Zhou L.M. Liu J.J. Zhang W. Shu Y.S. Yu L. Li P. Ronai Z.A. et al.SIP/CacyBP promotes autophagy by regulating levels of BRUCE/Apollon, which stimulates LC3-I degradation.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 13404-13413Crossref PubMed Scopus (13) Google Scholar, 12Jia R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar). LC3B be over by both lysosomal I. T. E. but not a cellular of LC3 is a for PubMed Scopus Google Scholar) and proteasomal degradation (11Jiang T.X. Zou J.B. Zhu Q.Q. Liu C.H. Wang G.F. Du T.T. Luo Z.Y. Guo F. Zhou L.M. Liu J.J. Zhang W. Shu Y.S. Yu L. Li P. Ronai Z.A. et al.SIP/CacyBP promotes autophagy by regulating levels of BRUCE/Apollon, which stimulates LC3-I degradation.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 13404-13413Crossref PubMed Scopus (13) Google Scholar, 12Jia R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar, H.M. Noh K.H. Chang T.K. Park D. Cho H.S. of by is associated with autophagy and in 2019; PubMed Scopus Google Scholar). which was for the reduced levels of LC3B in we the of LC3B-I is converted to LC3B-II autophagosome and LC3B-II is with autophagy receptors and fusion of autophagosomes with lysosomes I. T. E. but not a cellular of LC3 is a for PubMed Scopus Google Scholar, S. Lamark T. A. Bruun J.A. H. A. G. Johansen T. to to degradation of protein by Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of lysosomal degradation by with the increased the levels of LC3B-II in both and of the accumulation of LC3B-II in lysosomes in both (i.e., a of the levels of LC3B-II under these conditions in in autophagy induction by of and from the (i.e., the levels of LC3B-II in both and to increased LC3B the levels of LC3B-II in to with and in accumulation of LC3B-II in both and LC3B-II levels in in findings that the levels of LC3B in not to increased degradation, but the of LC3B in autophagy. the reduced levels of LC3B in to increased proteasomal degradation, we and for with or of the We that with of increased the of LC3B-I and LC3B-II to levels in both and that in LC3B levels and to proteasomal degradation. is that LC3B of monoubiquitinated LC3B a that be by of the Ub by proteasomal J. D. of ubiquitin by deubiquitinating Cell Full Text Full Text PDF Scopus Google Scholar). For that the in LC3B levels by with was not to of autophagy, we the of an enzyme that participates in (i.e., of LC3B-I to and autophagic degradation of LC3B (1Bento C.F. Renna M. Ghislat G. Puri C. Ashkenazi A. Vicinanza M. Menzies F.M. Rubinsztein D.C. Mammalian autophagy: How does it Work.Annu. Rev. Biochem. 2016; 85: 685-713Crossref PubMed Scopus (348) Google Scholar, 2Dikic I. Elazar Z. Mechanism and medical implications of mammalian autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 349-364Crossref PubMed Scopus (738) Google Scholar). We that with in LC3B-I levels in both and that the of was of autophagy. we USP10 the ubiquitination of this we or with Ub and LC3B or a in the Lys-51 to R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar). with to Cell to with to the by with to the We that but not was with in A and as R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar). ubiquitination was increased in A and In addition, we found that ubiquitination of and was also increased in GABARAP, GABARAPL1, and GABARAPL2 are not R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar), and not in these results thus that USP10 reduces the ubiquitination of not but also and We also an in to the of with USP10 or We that of but not reduced the of C and with USP10 the of we found that LC3B with but not Taken together, the demonstrated that USP10 LC3B both in and in The degradation of cargos by autophagy is through by various autophagy which are degraded in with cargos and with LC3B (1Bento C.F. Renna M. Ghislat G. Puri C. Ashkenazi A. Vicinanza M. Menzies F.M. Rubinsztein D.C. Mammalian autophagy: How does it Work.Annu. Rev. Biochem. 2016; 85: 685-713Crossref PubMed Scopus (348) Google Scholar, 2Dikic I. Elazar Z. Mechanism and medical implications of mammalian autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 349-364Crossref PubMed Scopus (738) Google Scholar). the of USP10 on degradation, we and with the for and in or and the levels of the autophagy receptors SQSTM1 and NBR1 by and We in the levels of SQSTM1 and NBR1 at in and that the of LC3B in was to autophagic with a in the levels of SQSTM1 and NBR1 in both and but the was slower in by in that the levels of LC3B in the of LC3B for of SQSTM1 and NBR1 to autophagosomes and degradation in under conditions of protein are protein that be under conditions of such as by with J. V. R. T. are protein for of the and PubMed Scopus Google Scholar). are by by autophagy receptors such as SQSTM1 and and subsequently degraded by autophagy J. V. R. T. are protein for of the and PubMed Scopus Google Scholar, S. Xu Y. E.A. C. T. M. of proteins is a cytosolic protein to and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of USP10 on the accumulation of we and with for and 3 and the of Ub and SQSTM1 by We under showed of with of and in showed accumulation and increased to of and of 3 and that USP10 increased with an autophagic to the of protein Taken together, the results indicate that USP10 is for of levels of LC3B by deubiquitinating LC3B and reducing its for proteasomal degradation. LC3B levels increased autophagy under both and This is not for levels of the autophagy receptors SQSTM1 and NBR1 and for of protein in but for these functions of by with or USP10 an autophagic under This of USP10 that of and which ubiquitination and proteasomal degradation of thus autophagy (11Jiang T.X. Zou J.B. Zhu Q.Q. Liu C.H. Wang G.F. Du T.T. Luo Z.Y. Guo F. Zhou L.M. Liu J.J. Zhang W. Shu Y.S. Yu L. Li P. Ronai Z.A. et al.SIP/CacyBP promotes autophagy by regulating levels of BRUCE/Apollon, which stimulates LC3-I degradation.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 13404-13413Crossref PubMed Scopus (13) Google Scholar, 12Jia R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar, H.M. Noh K.H. Chang T.K. Park D. Cho H.S. of by is associated with autophagy and in 2019; PubMed Scopus Google Scholar) of studies on we also showed that USP10 and BIRC6 R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar) the ubiquitination levels of and LC3C, that the regulatory mechanism on the LC3 of the In the is not subject to this of regulation. USP10 also shown to autophagy by deubiquitinating a of that catalyze the of at both autophagy initiation and autophagosome J. H. M. Xu L. Li Y. Zhang L. Y. Zhang T. T. M. Zhu Z. Wang H. Yu J. Li Y. et the levels of by regulating the activity of USP10 and Full Text Full Text PDF PubMed Scopus Google Scholar). and USP10 are in a mechanism in which which in and to increased levels and activity J. H. M. Xu L. Li Y. Zhang L. Y. Zhang T. T. M. Zhu Z. Wang H. Yu J. Li Y. et the levels of by regulating the activity of USP10 and Full Text Full Text PDF PubMed Scopus Google Scholar). USP10 and the kinase M. X. B. Liu T. Zhang H. Guo W. J.J. J. H. Wang L. Z. and of by Cell. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). USP10 by various of the autophagy results are with of studies that showed that the levels of LC3 proteins autophagy For example, KD of LC3 proteins of SQSTM1 levels and accumulation of polyubiquitinated E. E. R. Elazar Z. The and of LC3 into Cell Sci. 2008; PubMed Scopus Google Scholar, H. E. T. F. V. Elazar Z. LC3 and are both act in autophagosome J. 2010; PubMed Scopus Google Scholar, W. T. Zhang Z. F. J. A. of autophagy-related gene LC3 the of 2015; PubMed Scopus Google Scholar). of LC3 proteins autophagic the of and reducing and Huang LC3 reduces through and autophagic activity in and 2015; PubMed Scopus Google Scholar). In that autophagy as for the of various of that for D.C. P. B. Autophagy as a target for Rev. 11: PubMed Scopus Google Scholar, L. B. Kroemer G. of autophagy: and Rev. PubMed Scopus Google Scholar). is in targets for of autophagy. We that that LC3 levels through of USP10 or of and be for a of autophagy The that USP10 also regulates and J. H. M. Xu L. Li Y. Zhang L. Y. Zhang T. T. M. Zhu Z. Wang H. Yu J. Li Y. et the levels of by regulating the activity of USP10 and Full Text Full Text PDF PubMed Scopus Google Scholar, M. X. B. Liu T. Zhang H. Guo W. J.J. J. H. Wang L. Z. and of by Cell. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar) of USP10 as autophagy Cell and as R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar). R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar), and of in and at of USP10 UBA6 and was with to the was by with and We to the LC3B UBA6 USP10 Ub Ub SQSTM1 SQSTM1 NBR1 and from and from and from and proteins from Biochem. from from from from from from from R. J. Y. Bonifacino J.S. and fusion of lysosomes with PubMed Scopus Google Scholar), R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar) and R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8: e50034Crossref PubMed Scopus (13) Google Scholar) and by for human USP10 from into with an or The of USP10 with was by with and the of USP10 with was by with and The of and with by was by the of with as The of was from with and The of USP10 and by by CRISPR-Cas9 as J. C. R. M. P. Bonifacino J.S. a that regulates Cell. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). The for USP10 and into with the different on a to a at the in was and of the target was by with and The was by and by with and and at to with in the of at the and with and at The with 1 The ubiquitination screen was as R. Bonifacino J.S. 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