Ubiquitin carboxyl-terminal hydrolase 11 promotes autophagy by de-ubiquitinating and stabilizing Beclin-1

Autophagy is a major degradation process that degrades and recycles cytoplasmic materials through lysosome for maintaining cellular homeostasis. Dysregulated autophagy is linked with numerous human diseases including cancer. Autophagy marker protein B-cell lymphoma-2 interacting protein 1 (Beclin-1) is essential for autophagosome initiation and maturation. Recently, Ubiquitin carboxyl-terminal hydrolase 11 (USP11) has been reported to promote or inhibit autophagy without identification of any direct target. Here through biochemical reaction in vitro, we demonstrate that USP11 directly interacts with Beclin-1. Both in vitro and in vivo de-ubiquitination assays revealed that USP11 de-ubiquitinates Beclin-1. USP11-mediated de-ubiquitination stabilized Beclin-1 and enhanced the formation of the autophagy-specific class III phosphatidylinositol 3-kinase complexes 1 and 2, thereby promoting autophagy. Together, our results demonstrated that USP11 promotes autophagy under unperturbed conditions by de-ubiquitinating and stabilizing Beclin-1 which may serve as a therapeutic target for autophagy-related diseases.


Introduction
Autophagy is an evolutionarily conserved cellular recycling process that degrades cytoplasmic proteins, macromolecules, and unwanted organelles through lysosomal degradation to maintain metabolic homeostasis (Rabinowitz & White, 2010;Yang & Klionsky, 2020). In response to different stressors, such as nutrient deprivation and growth factor depletion, autophagy is activated, facilitating cell tolerance of the microenvironment, which prevents cell damage and promotes survival (White & DiPaola, 2009). In mammalian cells, there are three types of autophagy, namely, chaperonemediated autophagy (CMA), microautophagy, and macroautophagy (Yang & Klionsky, 2010). In the CMA pathway, proteins containing a KFERQ-like motif are recognized by the chaperone HSC70 and cochaperons and delivered to the lysosome for degradation (Dong et al., 2021;Kaushik & Cuervo, 2018;Yang et al., 2019). In microautophagy, the lysosomal membrane invaginates long-lived proteins into the lysosome for degradation through membrane fission (Schuck, 2020). Macroautophagy has been studied extensively and differs from CMA and microautophagy. In the macroautophagy pathway, autophagic cargos need to be packaged into double-membrane vesicles and subsequently transported to the lysosome (Dikic & Elazar, 2018). In brief, the macroautophagy pathway comprises five stages: phagophore initiation and nucleation, expansion, maturation, fusion with the lysosome, and degradation.
In this study, we aimed to clarify if USP11 promotes or suppresses autophagy by identification of its direct target(s). We found that USP11 promotes autophagy by directly binding with and de-ubiquitinating Beclin-1, which leads to Beclin-1 stabilization and PI3KC3-1/2 complex formation.

USP11 promotes autophagy in a catalytic activity-dependent manner
Given that the studies before failed to demonstrate if restoring USP11 expression and/or the catalytic activity of USP11 can erase the USP11 depletion-associated autophagic phenotypes, we stably expressed a short hairpin RNA (shRNA) construct targeting the coding region of the USP11 gene in HeLa cells. The resulting USP11 knockdown led to an accumulation of autophagy receptor P62, and there was a decrease in the LC3-II/I ratio on immunoblotting analysis (Fig. 1a) and decreased LC3-II puncta formation on immunocytochemical analysis (Fig. 1b). Re-expression of the shRNA-resistant form of wild type HA-tagged USP11 (HA-USP11), but not the catalytically inactive mutant HA-USP11 (C318S), restored the USP11 depletion-induced changes to P62 levels, the LC3-II/I ratio, and LC3-II puncta formation (Figs. 1a and 1b). Conversely, overexpression of wild type HA-USP11, but not the HA-USP11 (C318S) mutant, increased the LC3-II/I ratio while decreasing P62 protein levels (Fig. 1c). Taken together, our results demonstrated that USP11 promotes autophagy in an enzymatic activitydependent manner.
Reportedly, Beclin-1 ubiquitination is a target for proteasomal degradation (Han et al., 2018;Pei et al., 2017). We thus speculated that the USP11-mediated de-ubiquitination of Beclin-1 stabilizes the protein. Indeed, when we inhibited USP11 expression in HeLa cells by shRNA treatment, Beclin-1 protein levels reduced, and this reduction was rescued by the re-expression of wild type HA-USP11, but not the catalytically inactive mutant HA-USP11 (C318S) (Fig. 3e). This USP11-depletion-induced reduction of Beclin-1 protein levels was also restored by pretreating the cells with MG-132-a potent cell-permeable proteasome inhibitor (Fig. 3f). We also found that the USP11-knockdown-induced and vector control (VEC) HeLa cells and HeLa cells treated with short hairpin RNA (shRNA) to USP11 (shUSP11). P62 protein levels were normalized to actin. b USP11 depletion decreases LC3 puncta formation. LC3 puncta were measured by immunofluorescence assay and further quantified by ImageJ software. A two-tailed Student's t test was used to determine the statistical significance of group differences. *p < 0.05, **p < 0.01, ****p < 0.001. c USP11 overexpression promotes autophagy. Western blot showing the expression of HAtagged wild type and enzymatic activity defective mutant (C318S) USP11, P62, and LC3 in normal HeLa cells. P62 protein levels were normalized to actin reduction in Beclin-1 protein levels occurred both under unperturbed and glucose-starvation-induced autophagy conditions (Fig. 3g). Our findings therefore indicate that Beclin-1 is a substrate of USP11, and USP11-mediated de-ubiquitination stabilizes Beclin-1.

USP11 promotes the formation of autophagy-specific PI3KC3 complexes
The scaffolding protein Beclin-1 brings in one of two positive regulators, ATG14 or UVRAG, to serve as the Western blot showing the endogenous USP11 in the anti-Beclin-1 immunoprecipitates but not the anti-IgG (negative control) immunoprecipitates from HEK293T cells. b Ectopic tagged USP11 interacts with Beclin-1. Western blot showing the HA-Beclin-1 in the anti-FLAG immunoprecipitates from HEK293T cells overexpressing HA-Beclin-1 and with or without FLAG-USP11. c USP11 directly interacts with Beclin-1. Western blot showing the bacterial produced His-Beclin-1 in the anti-GST immunoprecipitates that containing GST-USP11 but not the GST (negative control) group. d,e. Starvation stimulates USP11 interaction with Beclin-1. Before harvest, cell culture medium was removed and the cells were washed with PBS for 3 times and then emersed in fresh medium without glucose. Total cell lysates were extracted and subjected to immunoprecipitation with anti-FLAG (d) or anti-Beclin-1 (e) antibody followed by immunoblotting with antibodies as indicated. f Data were collected from GEPIA database (http:// gepia. cancer-pku. cn/); 15 types of tumor (33 types of tumor in total) exhibited gene correlation (R-value > 3, p value < 0.05) between Beclin-1 and USP11 fourth subunit in a mutually exclusive manner to form PI3KC3-C1 and PI3KC3-C2, respectively. We found that the depletion of USP11 expression in HeLa cells decreased Beclin-1 interactions with VPS15, VPS34, ATG14, and UVRAG (Fig. 4a), and this decrease was rescued by the re-expression of HA-USP11, but not that of the catalytically inactive mutant HA-USP11 (C318S) (Fig. 4a). Conversely, overexpressing HA-USP11, but not HA-USP11 (C318S), in HEK293T cells increased the amount of Beclin-1-VPS34-VPS15-ATG14/UVRAG complex formed (Fig. 4b). Taken together, our work has uncovered the direct role of USP11 in autophagy, which involves its deubiquitination of Beclin-1 to promote PI3KC3 complex formation.

Discussion
It was reported that, without rescue experiments, the inhibition of USP10 or USP13 expression by three independent siRNA oligos destabilized Beclin-1, whereas overexpressing USP10 or USP13, but not the catalytically inactive mutant, reduced Beclin-1 ubiquitination in vivo (Liu et al., 2011). While this manuscript was under preparation, another group also found evidence that USP11 de-ubiquitinates and stabilizes Beclin-1 to promote autophagy and subsequent autophagy-induced ferroptosis (Rong et al., 2021). These studies, along with previously mentioned reports of autophagy modulation by USP11, failed to provide in vitro evidence of a direct interaction between USP11 and Beclin-1 HeLa cell that reconstituted with HA-tagged vector control, wild type and enzymatic activity defective mutant (C318S) USP11. f MG132 treatment blocks Beclin-1 degradation in USP11-depleted cells. Cells were treated with 50 μM MG132 for 4 h and harvested for western blot detection. g USP11-depletion leads to Beclin-1 degradation. shNC or shUSP11 HeLa cells were treated with or without glucose starvation. Total cell lysates were harvested, followed by immunoblotting with indicated antibodies or positive in vitro de-ubiquitination assays of USP11/ Beclin-1 (Basic et al., 2021;Qiao et al., 2021;Sun et al., 2021). Thus, these studies could not exclude the possibility that USP11-mediated autophagy is executed through its interacting de-ubiquitinases such as USP4 (L. Zhang et al., 2012), USP7 (Georges et al., 2018), USP15 (Sowa et al., 2009), and OTUD5 (Sowa et al., 2009). In this study, we have provided strong evidence to support the hypothesis that USP11 promotes autophagy by directly de-ubiquitinating and stabilizing Beclin-1. These lines of evidence include results that show (1) USP11 directly interacts with Beclin-1; (2) USP11 de-ubiquitinates Beclin-1 in vitro; and (3) the USP11-mediated de-ubiquitination/stabilization of Beclin-1 and formation of the pro-autophagy PI3KC3 complexes are rescued by the re-expression of wild type USP11, but not the catalytically inactive mutant.
Upregulated USP11 expression has been reported in patient cohorts of gastric cancer , breast cancer (Bayraktar et al., 2013;Zhou et al., 2017), estrogen receptor alpha-positive breast cancer (Dwane et al., 2020), ovarian cancer , hepatocellular carcinoma (Zhang et al., 2018), and colorectal cancer . In addition, high USP11 expression correlated with shorter survival times in patient cohorts of breast cancer (Bayraktar et al., 2013;Garcia et al., 2018;Zhou et al., 2017), estrogen receptor alpha-positive breast cancer (Dwane et al., 2020), ovarian cancer , hepatocellular carcinoma (Zhang et al., 2018), and colorectal cancer . We also investigated the relationship between USP11 and Beclin-1 gene expression in 33 types of TCGA tumors from GEPIA database, and there was a positive correlation between the genes in 15 of the tumor types, each exhibiting R values greater than 3. These results suggest that USP11 may functionally correlate with Beclin-1 in tumor cells.
Several studies have demonstrated that autophagy acts to promote tumor survival and growth in advanced cancers (Dikic & Elazar, 2018), and after exploring the GEPIA database (http:// gepia. cancer-pku. cn/), we discovered that high Beclin-1 expression correlates with shorter survival times in patient cohorts of ovarian cancer, kidney chromophobe, and lower grade brain glioma (Fig. S1). This suggests that autophagy may be linked to the poor prognosis following some tumor therapeutics. Moreover, the USP11mediated stabilization of VCP activated autophagy and conferred colorectal cancer resistance to fluorouracil treatment . Taken together, we demonstrate that Beclin-1 is a direct target for USP11 and USP11-mediated complexes. a USP11-depletion decreases Beclin-1 interaction with other subunits of autophagy-specific class III phosphatidylinositol 3-kinase complexes (PI3KC3). Western blot showing the VPS15, VPS34, ATG14 and UVRAG in the anti-FLAG immunoprecipitates from shUSP11 HeLa cell that reconstituted with HA-tagged vector control, wild type, enzymatic activity defective mutant (C318S) USP11 and with or without FLAG-Beclin-1. b USP11-overexpression promotes Beclin-1 interaction with other subunits of PI3KC3. Western blot showing the VPS15, VPS34, ATG14 and UVRAG in the anti-FLAG immunoprecipitates from HEK293T cells that overexpressing HA-tagged vector control, wild type, enzymatic activity defective mutant (C318S) USP11 and with or without FLAG-Beclin-1 de-ubiquitination and stabilization of Beclin-1 leads to assembling of PI3KC3-C1 and PI3KC3-C2 complex, ultimately promoting autophagy. Therefore, targeting USP11 for inhibition could serve as a therapeutic strategy for autophagy-related diseases.

Cell culture
Human HEK293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in an atmosphere containing 5% CO 2 at 37 ℃.

Glucose starvation
For starvation induction, glucose-free medium (11,966-025, Gibco) were used. Before harvested, cultured cells were quickly washed with PBS for 3 times and added with glucose-free medium for indicated time.

Undenatured co-immunoprecipitation
Cells were harvested and lysed with lysis buffer (150 mM NaCl; 50 mM Tris, pH 7.5; 0.5% NP-40; 5 mM EDTA) at 4 ℃ for 30 min. Further the lysates were centrifuge with 13000g to collect the supernatant for immunoprecipitation with antibody at 4 °C overnight. Then, protein A/G magnetic beads were added to the cell lysates and incubated at 4 °C for 1 h. The beads were washed with cold lysis buffer three times, for 5 min each time, on a rotating mixer.

Immunofluorescence
HeLa cells were washed with PBS three times and fixed with 4% paraformaldehyde at room temperature for 10 min. Then the cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min. After permeabilization, the cells were blocked with 2% BSA in PBST at room temperature for 30 min and further incubated with the primary antibody overnight followed by the secondary antibody for 1 h. After washing with PBST three times, DAPI was added, and the coverslips (CIT-OGLAS) were covered with antifade. And images were captured with OLYMPUS fluorescence microscope (CKX53).

Western blot
Total protein from cells was extracted with 150 mM NaCl lysis buffer as described earlier. Samples were separated using SDS-PAGE and transferred to PVDF membranes. After skim milk blocking, the membranes were incubated with primary antibody at 4 °C overnight. The membranes were incubated with secondary antibodies for 1 h at room temperature, then visualized using electro chemiluminescence detection reagents (SuperSignal™ West Pico PLUS, Thermo, lot#34,580).

Gene correlation
Gene correlation between USP11 and Beclin-1 in tumors was analyzed online by GEPIA (http:// gepia. cancer-pku. cn). This website performs pair-wise gene expression correlation analysis for given sets of TCGA expression data, using Pearson method. Cases that p-Value < 0.05, R-Value > 0.3 were selected as convincible correlation between USP11 and Beclin-1.

Kaplan-Meier survival analysis
The survival analysis is done online (http:// gepia. cancerpku. cn) to analyze correlations between levels of Beclin-1 with overall survival (OS) in cancers. All survival diagrams shown in the manuscript were analyzed by GEPIA using Log-rank test, a.k.a the Mantel-Cox test.

Statistical analysis
Data were analyzed by Prism 7.0 software (GraphPad Prism). A two-tailed Student's t test was used to determine the statistical significance. All data are presented as the means ± s.d. or means ± s.e.m. as indicated, and a p value of < 0.05 was considered statistically significant.
Funding This work was supported by the National Natural Science Foundation of China (Grant no. 31530016, 32,090,031, 31,761,133,012, and 31,800,683) the National Basic Research Program of China (grant no. 2017YFA0503900), the Shenzhen Science and Technology Innovation Commission (grant no. JCYJ20180507182213033 and JCYJ20170412113009742).
Data availability All data generated or analyzed during this study are included in this manuscript. Materials described in this manuscript are available upon request.
Code availability Not applicable.

Conflicts of interest
The authors declare no potential conflicts of interest.
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