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

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100405

ISSN

1083-351X

Autores

Rui Jia, Juan S. Bonifacino,

Tópico(s)

Studies on Chitinases and Chitosanases

Resumo

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. Lim J.H. Jung C.R. Ubiquitination of MAP1LC3B by pVHL is associated with autophagy and cell death in renal cell carcinoma.Cell Death Dis. 2019; 10: 279Crossref PubMed Scopus (12) Google Scholar). Both monoubiquitination and polyubiquitination lead to proteasomal degradation of LC3B, with consequent decrease 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, 13Kang H.M. Noh K.H. Chang T.K. Park D. Cho H.S. Lim J.H. Jung C.R. Ubiquitination of MAP1LC3B by pVHL is associated with autophagy and cell death in renal cell carcinoma.Cell Death Dis. 2019; 10: 279Crossref PubMed Scopus (12) Google Scholar). In general, protein ubiquitination is reversed by Ub removal catalyzed by a family of isopeptidases known as deubiquitinating enzymes (DUBs) (14Clague M.J. Urbé S. Komander D. Breaking the chains: deubiquitylating enzyme specificity begets function.Nat. Rev. Mol. Cell Biol. 2019; 20: 338-352Crossref PubMed Scopus (158) Google Scholar). However, this has not yet been shown to be the case for ubiquitinated LC3B. Herein, we report the results of a CRISPR-Cas9 KO screen that identifies USP10 as a DUB that deubiquitinates LC3B, thus increasing LC3B levels and autophagic activity. To identify potential LC3B DUBs, we conducted a CRISPR-Cas9 KO screen using a human H4 neuroglioma cell line that expresses LC3B endogenously tagged with tandem GFP-mCherry (H4-tfLC3B cells) (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). These cells were mutagenized with a lentiviral CRISPR-Cas9 KO library targeting 661 ubiquitination-related genes (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) (Fig. 1A). The screen was based on the hypothesis that depletion of specific DUBs would increase LC3B ubiquitination and degradation, thus decreasing GFP and mCherry fluorescence signals. Mutagenized H4-tfLC3B cells exhibiting low GFP-mCherry fluorescence were collected by fluorescence-activated cell sorting (FACS). These cells were then propagated and subjected to two more rounds of sorting and propagation, until the population of low GFP-mCherry cells was enriched to 90.8% (Fig. 1, A and B). The abundance of every single-guide RNA (sgRNA) in sorted cells relative to unsorted cells was determined by next-generation sequencing (NGS) and the MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout) algorithm (15Li W. Xu H. Xiao T. Cong L. Love M.I. Zhang F. Irizarry R.A. Liu J.S. Brown M. Liu X.S. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.Genome Biol. 2014; 15: 554Crossref PubMed Scopus (542) Google Scholar) (Fig. 1C, Tables S1 and S2). The top-scoring DUB in this screen (ranked number 9 in order of significance) was USP10. The other proteins among the top ten hits were the immunoproteasome subunit beta type-10 (PSMB10) and components of several E3 ubiquitin ligases (Table S2); because the aim of the present study was to identify a putative LC3B-deubiquitinating enzyme, the functional significance of these other proteins was not investigated. To confirm a role of USP10 in the regulation of LC3B levels, we silenced USP10 expression by transfecting H4 cells with USP10 siRNA. We observed that USP10 silencing decreased the endogenous levels of both the cytosolic LC3B-I and membrane-bound LC3B-II forms by ∼2–2.5-fold (Fig. 2, A and B). Similar decreases in LC3B-I and LC3B-II levels were observed upon silencing of USP10 in the HeLa human cervical carcinoma and HEK293T human embryonic kidney cell lines (Fig. S1, A–C). Furthermore, CRISPR-Cas9 KO of USP10 in H4 cells resulted in approximately twofold decreases in both LC3B-I and LC3B-II endogenous levels (Fig. 2, C and D). Stable expression of MYC-USP10 in USP10-KO cells restored levels of LC3B-I and LC3B-II to those in WT cells (Fig. 2, E and F). Analyses of other Atg8-family members showed that USP10 KO in H4 cells also reduced the levels of LC3A, but not GABARAP and GABARAPL1 (Fig. S2, A–E). The effect of USP10 KO on the levels of LC3C and GABARAPL2 could not be examined because of lack of suitable antibodies for immunoblotting. Finally, we observed that transfection of USP10-KO H4 cells with siRNA to UBA6, the Ub-activating enzyme that participates in LC3B ubiquitination (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), increased the levels of both LC3B-I and LC3B-II (Fig. S3, A and B). These results thus demonstrated that USP10 depletion reduced the levels of endogenous LC3B, an effect that was opposite to that caused by depletion of the ubiquitinating 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 can be turned over by both lysosomal (16Tanida I. Minematsu-Ikeguchi N. Ueno T. Kominami E. Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy.Autophagy. 2005; 1: 84-91Crossref PubMed Scopus (852) 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, 13Kang H.M. Noh K.H. Chang T.K. Park D. Cho H.S. Lim J.H. Jung C.R. Ubiquitination of MAP1LC3B by pVHL is associated with autophagy and cell death in renal cell carcinoma.Cell Death Dis. 2019; 10: 279Crossref PubMed Scopus (12) Google Scholar). To examine which pathway was responsible for the reduced levels of LC3B in USP10-deficient cells, we tested the effect of specific inhibitors. LC3B-I is converted to LC3B-II during autophagosome formation, and LC3B-II is eventually degraded, together with autophagy receptors and substrates, upon fusion of autophagosomes with lysosomes (16Tanida I. Minematsu-Ikeguchi N. Ueno T. Kominami E. Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy.Autophagy. 2005; 1: 84-91Crossref PubMed Scopus (852) Google Scholar, 17Pankiv S. Clausen T.H. Lamark T. Brech A. Bruun J.A. Outzen H. Øvervatn A. Bjørkøy G. Johansen T. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy.J. Biol. Chem. 2007; 282: 24131-24145Abstract Full Text Full Text PDF PubMed Scopus (2902) Google Scholar). Inhibition of lysosomal degradation by treatment with the v-ATPase inhibitor bafilomycin A1 dramatically increased the levels of LC3B-II in both WT and USP10-KO H4 cells, indicative of the accumulation of undegraded LC3B-II in lysosomes in both cell lines (i.e., a measure of "autophagic flux") (Fig. 3, A–C). Importantly, the levels of LC3B-II under these conditions were still lower in USP10-KO cells than in WT cells (Fig. 3, A–C). Furthermore, autophagy induction by removal of amino acids and serum from the medium (i.e., "starvation") decreased the levels of LC3B-II in both WT and USP10-KO cells due to increased LC3B consumption (Fig. 3, A–C); however, the levels of LC3B-II remained lower in USP10-KO relative to WT cells (Fig. 3, A–C). Finally, combined treatment with bafilomycin A1 and nutrient starvation resulted in greater accumulation of LC3B-II in both WT and USP10-KO cells, but, again, LC3B-II levels were lower in USP10-KO than in WT cells (Fig. 3, A–C). These findings indicated that the decreased levels of LC3B in USP10-deficient cells were not due to increased lysosomal/autophagic degradation, but rather preceded the engagement of LC3B in autophagy. To determine if the reduced levels of LC3B in USP10-KO cells were due to increased proteasomal degradation, we incubated WT and USP10-KO cells for 6 h with 20 or 50 μM of the proteasome inhibitor MG132. We observed that treatment with either concentration of MG132 increased the amounts of LC3B-I and LC3B-II to similar levels in both WT and USP10-KO cells (Fig. 3, D–F), indicating that differences in LC3B levels between untreated WT and USP10-KO cells were due to proteasomal degradation. It is worth noting that MG132-treated cells accumulated unconjugated LC3B instead of monoubiquitinated LC3B (Fig. 3D), a fact that could be explained by removal of the Ub moiety by proteasomal DUBs (18Lee M.J. Lee B.H. Hanna J. King R.W. Finley D. Trimming of ubiquitin chains by proteasome-associated deubiquitinating enzymes.Mol. Cell Proteomics. 2011; 10 (R110.003871)Abstract Full Text Full Text PDF Scopus (170) Google Scholar). For further proof that the increase in LC3B levels caused by treatment with MG132 was not due to inhibition of autophagy, we examined the effect of knocking out ATG7, an E1-like enzyme that participates in lipidation (i.e., conversion of LC3B-I to LC3B-II) and eventual 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 observed that incubation with 10 μM MG132 caused similar fold increases in LC3B-I levels in both WT and ATG7-KO cells (Fig. S4, A–C), confirming that the effect of MG132 was independent of autophagy. Next, we examined if USP10 altered the ubiquitination state of LC3B. To this end, we cotransfected WT or USP10-KO cells with plasmids encoding HA-tagged Ub (HA-Ub) and either FLAG-tagged LC3B (FLAG-LC3B) or FLAG-LC3B having a mutation in the ubiquitin-acceptor Lys-51 to Arg (FLAG-LC3B-K51R) (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). Cells were incubated with MG132 prior to harvesting. Cell extracts were subjected to immunoprecipitation with antibody to the FLAG epitope followed by immunoblotting with antibody to the HA epitope. We observed that FLAG-LC3B, but not FLAG-LC3B-K51R, was modified with HA-Ub in WT cells (Fig. 4, A and B), as previously reported (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). Importantly, FLAG-LC3B ubiquitination was increased in USP10-KO cells (Fig. 4, A and B). In addition, we found that ubiquitination of FLAG-LC3A and FLAG-LC3C was also increased in USP10-KO cells (Fig. S5). GABARAP, GABARAPL1, and GABARAPL2 are not ubiquitinated (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 were therefore not included in these analyses. These results thus indicated that USP10 reduces the ubiquitination of not only LC3B, but also LC3A and LC3C. We also used an in vitro assay to test the effect of treating HA-Ub-conjugated FLAG-LC3B with recombinant His6-tagged USP10 (His6-USP10) or His6-tagged USP13 (His6-USP13) (specificity control). We observed that addition of His6-USP10, but not His6-USP13, reduced the amount of HA-Ub-conjugated FLAG-LC3B (Fig. 4, C and D), consistent with USP10 specifically catalyzing the deubiquitination of LC3B. Finally, we found that GFP-tagged LC3B (GFP-LC3B) co-immunoprecipitated with FLAG-USP10 but not FLAG-USP13 (Fig. 4E). Taken together, the above experiments demonstrated that USP10 specifically deubiquitinates LC3B both in vivo and in vitro. The degradation of selected cargos by autophagy is achieved through recognition by various autophagy receptors, which are eventually degraded in autolysosomes together with their 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). To investigate the effect of USP10 KO on autophagy-receptor degradation, we incubated WT and USP10-KO H4 cells with the translation inhibitor cycloheximide (CHX) for 0, 2, 4, and 6 h in complete medium (Fig. 5, A-C) or starvation medium (Fig. S6, A–C) and determined the endogenous levels of the autophagy receptors SQSTM1 and NBR1 by immunoblotting (Figs. 5, A–C and S6, A–C). We observed that, in complete medium, the steady-state levels of SQSTM1 and NBR1 at time 0 were similar in WT and USP10-KO cells, suggesting that the lower level of LC3B in USP10-KO cells was sufficient to maintain basal autophagic activity. Treatment with cycloheximide caused a time-dependent decrease in the levels of SQSTM1 and NBR1 in both WT and USP10-KO cells, but the decrease was slower in USP10-KO cells (Fig. 5, A-C). Similar observations were made by CHX treatment in starvation medium (Fig. S6, A–C). These experiments indicated that the lower levels of LC3B in USP10-KO cells limit the availability of LC3B for recruitment of SQSTM1 and NBR1 to autophagosomes and their eventual degradation in autolysosomes under conditions of protein synthesis inhibition. Aggresome-like induced structures (ALIS) are protein aggregates that can be induced under conditions of stress, such as premature translation termination by treatment with puromycin (19Szeto J. Kaniuk N.A. Canadien V. Nisman R. Mizushima N. Yoshimori T. Bazett-Jones D.P. Brumell J.H. ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy.Autophagy. 2006; 2: 189-199Crossref PubMed Scopus (157) Google Scholar). Puromycin-induced ALIS are usually modified by ubiquitination, selectively recognized by autophagy receptors such as SQSTM1 and NBR1, and subsequently degraded by autophagy (19Szeto J. Kaniuk N.A. Canadien V. Nisman R. Mizushima N. Yoshimori T. Bazett-Jones D.P. Brumell J.H. ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy.Autophagy. 2006; 2: 189-199Crossref PubMed Scopus (157) Google Scholar, 20Liu X.D. Ko S. Xu Y. Fattah E.A. Xiang Q. Jagannath C. Ishii T. Komatsu M. Eissa N.T. Transient aggregation of ubiquitinated proteins is a cytosolic unfolded protein response to inflammation and endoplasmic reticulum stress.J. Biol. Chem. 2012; 287: 19687-19698Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To examine the effect of USP10 KO on the accumulation of ALIS, we incubated WT and USP10-KO H4 cells with 5 μg/ml puromycin for 2 and 3 h and visualized the distribution of endogenous Ub and SQSTM1 by immunofluorescence microscopy. We observed that, under control conditions, neither WT nor USP10-KO H4 cells showed any ALIS (Fig. 5D). After 2 h of incubation with puromycin, however, 15.5% of WT cells and 27.2% in USP10-KO cells showed ALIS accumulation (Fig. 5, D and E). These percentages increased to 20.2% of WT and 33.5% of USP10-KO cells, respectively, after 3 h puromycin incubation (Fig. 5, D and E). These observations indicated that USP10 KO increased ALIS accumulation, consistent with an impaired autophagic response to the formation of abnormal protein aggregates. Taken together, the results presented here indicate that USP10 is required for maintenance of higher steady-state levels of LC3B by deubiquitinating LC3B and reducing its targeting for proteasomal degradation. Higher LC3B levels enable increased autophagy flux under both basal and starvation conditions. This ability is not necessary for maintaining normal levels of the autophagy receptors SQSTM1 and NBR1 and for clearance of protein aggregates in untreated cells, but becomes important for these functions upon inhibition of translation by treatment with cycloheximide or puromycin. Hence, USP10 enables an enhanced autophagic response under stress conditions. This function of USP10 counters that of UBA6, BIRC6, and VHL, which mediate ubiquitination and proteasomal degradation of LC3B, thus decreasing stress-induced 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, 13Kang H.M. Noh K.H. Chang T.K. Park D. Cho H.S. Lim J.H. Jung C.R. Ubiquitination of MAP1LC3B by pVHL is associated with autophagy and cell death in renal cell carcinoma.Cell Death Dis. 2019; 10: 279Crossref PubMed Scopus (12) Google Scholar) (Fig. 5F). Although most of our studies were done on LC3B, we also showed that USP10 (this study), UBA6, and BIRC6 (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) control the ubiquitination and/or levels of LC3A and LC3C, suggesting that the same regulatory mechanism acts on the entire LC3 subset of the Atg8 family. In contrast, the GABARAP subset is not subject to this type of regulation. USP10 has also been shown to enhance autophagy by d

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