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

https://doi.org/10.1016/j.bbrc.2018.02.140Get rights and content

Highlights

  • UCHL1 interacts with LC3 and inhibits autophagosome formation.

  • UCHL1 Parkinson mutant I93 M abolishes to suppress autophagosome formation.

  • UCHL1 inhibitors LDN-57444 and NSC632839 promote autophagosome formation in cells.

Abstract

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.

Introduction

Autophagy-lysosome pathway is essential for cellular health and its dysfunction is associated with various diseases [[1], [2], [3], [4]]. Autophagy is triggered by either environmental nutrition deprivation or internal protein aggregation. One well-studied type macroautophagy is a multistep process involves initiation and elongation of phagophore, fusion of autophagosome with lysosome and degradation of substrates [5,6]. It starts with the ULK1 complex to phosphorylate the downstream effectors such as AMBRA1 and Atg14 and induce the formation of phagophore [5]. LC3-I is then covalently conjugated to phosphatidylethanolamine (PE) on the phagophore to form LC-II executed by the ATG16L1/ATG5-ATG12 complex [7]. LC3-II is a widely used hallmark of autophagosome. p62/SQSTM1 is an adaptor protein that bridges LC3-II to the ubiquitinated substrates and reduction of p62 is usually linked to the activation of autophagy [8]. The phagophore then engulfs substrates and encloses itself to become the autophagosome. After fusion with lysosome, the whole compartment is degraded by the hydrolases in the lysosome.

Protein ubiquitination is a reversible post-translational modification process that regulates many vital signaling pathways. Ubiquitin conjugated on substrates can be removed by DUBs to reverse the functional effects of ubiquitination [9]. It has now been well recognized that autophagy substrates are ubiquitinated and some DUBs have been demonstrated to function in the pathway [10]. USP10, USP13 and A20 have been shown to de-ubiqutinate Beclin1 to negatively regulate autophagy [11,12]. In contrast, other report showed that USP10 de-ubiquitinates AMPKα to positively regulate autophagy [13]. USP8 specifically removes lys6-linked polyubiquitin chains from Parkin, and promotes the autophagic degradation of dysfunctional mitochondria [14]. Whereas USP30 negatively regulates mitophagy by de-ubiquitinating PINK, a kinase on mitochondria membrane [15]. USP33 de-ubiquitinates mono-ubiquitinated RALB, which allows RALB to associate with Beclin1-containing complexes to positively induce autophagy [16]. However, the detail mechanism of the majority of the DUBs is still unidentified.

UCHL1 is a DUB that has been widely studied for its relation to Parkinson's disease (PD) [17,18]. UCHL1 I93 M was reported in a familial autosomal dominant PD [19]. Sporadic reports suggested its involvement in autophagy. One study showed that UCHL1 suppresses autophagic degradation of p21WAF1/Cip1 in cardiac fibroblasts via increasing mTOR activity [20]. Recently, it was reported that UCHL1 interacts with RPTOR in cells, which is not affected by growth factor signaling [21]. UCHL1 is also a key regulator of chaperone mediated autophagy, responsible for removing Parkinson's disease related alpha-synuclein [22]. UCHL1 inhibition by LDN activates autophagic pathway in both an alpha-synuclein overexpressed oligodendroglial cell line [23] and an alpha-synuclein transgenic mouse model [24]. However, the mechanism of how UCHL1 regulating autophagy is still a mystery.

To explore the detail function of DUBs in autophagy, we did a genome wide screen of 55 human DUBs and found a certain number of them affect autophagy in diverse aspects. One of them, UCHL1, interacts with LC3 and inhibits autophagosome formation relying on its DUB activity. These findings demonstrated that UCHL1 plays a critical role in regulating autophagy.

Section snippets

Cell lines, plasmids and reagents

293FT and HeLa cells from ATCC were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (Gemini, USA) and 1% Pen/strep (Life, USA) at 37 °C under 5% CO2. mRFP-GFP-LC3 plasmid was provided by Prof Tamotsu Yoshimori (Tokyo Medical and Dental University, Japan). HeLa cells stably expressing mRFP-GFP-LC3 was maintained in complete medium containing 300 ng/μl G418 (Invitrogen CA, USA) [25]. EGFP-LC3 was constructed by inserting EGFP-LC3 fragment into pcDNA3.0. DUBs were either purchased from

UCHL1 inhibits autophagy and is depending on its DUB activity

To assess whether more DUBs might be functioning in autophagy, we overexpressed the available 55 DUBs in an mRFP-GFP-LC3 stably expressed HeLa cell line respectively, and examined LC3 puncta formation. mRFP-GFP-LC3 is an autophagic flux indicator with yellow LC3 puncta in autophagosomes and red LC3 puncta in autolysosomes due to the acidic environment that quenches GFP but not RFP signal [28]. After a wide screen, we found quite a few DUBs could affect LC3 puncta pattern (data not shown). One

Discussion

Autophagy is a dynamic and complicated process which carry out the basal housekeeping role in degradation of damaged or redundant organelles and protein aggregates. Increasing the number of autophagosome or autolysosome is caused by either activation of autophagosome biogenesis or blockage of autophagic flux [32]. In this study, we have used a double fluorescent labeled LC3 detection system, by tracing the LC3 puncta change, to examine the impact of DUBs in autophagy. In this cell line, when

Conflicts of interest

The authors declare that we have no competing interests.

Acknowledgments

We thank Professor Zongping Xia (Zhejiang University) for the DUB constructs, Dr. Li Tan (the Interdisciplinary Research Center of Biology and Chemistry, IRCBC, China) for the DUBs inhibitors, Professor Tamotsu Yoshimori (Tokyo Medical and Dental University, Japan) for mRFP-GFP-LC3 plasmid, and Dr. Xiaoming Li for helping with use of a confocal microscope.

Funding sources

This study was supported by the National Natural Science Foundation of China (No. 31570781) and the Start-up grant from ShanghaiTech University.

Author information

Y. Liu, C. Yan and Y. Chu designed the experiments and wrote the manuscript; C. Yan, H. Huo, C. Yang, T. Zhang, and Y. Chu performed the experiments.

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