Characterization of the Deubiquitinating Activity of USP19 and Its Role in Endoplasmic Reticulum-associated Degradation

Deubiquitinating enzymes (DUBs) regulate various cellular processes ranging from protein degradation to cellular signaling. USP19, the only DUB containing a carboxyl-terminal transmembrane domain, was proposed to function in endoplasmic reticulum-associated degradation (ERAD). Here we characterize the function and regulation of USP19. We identify Hsp90 as a specific partner that binds the catalytic domain of USP19 to promote substrate association. Intriguingly, although overexpressed USP19 interacts with Derlin-1 and other ERAD machinery factors in the membrane, endogenous USP19 is mostly in the cytosol where it binds Hsp90. Accordingly, we detect neither interaction of endogenous USP19 with Derlin-1 nor significant effect on ERAD by USP19 depletion. The USP19 transmembrane domain appears to be partially stabilized in the cytosol by an interaction with its own catalytic domain, resulting in auto-inhibition of its deubiquitinating activity. These results clarify the role of USP19 in ERAD and suggest a novel DUB regulation that involves chaperone association and membrane integration. Moreover, our study indicates that the localization of tail-anchored membrane proteins can be subject to regulation in cells.     

The ubiquitin E3 ligase MARCH7 is differentially regulated by the deubiquitylating enzymes USP7 and USP9X

Protein modification by one or more ubiquitin chains serves a critical signalling function across a wide range of cellular processes. Specificity within this system is conferred by ubiquitin E3 ligases, which target the substrates. Their activity is balanced by deubiquitylating enzymes (DUBs), which remove ubiquitin from both substrates and ligases. The RING-CH ligases were initially identified as viral immunoevasins involved in the downregulation of immunoreceptors. Their cellular orthologues, the Membrane-Associated RING-CH (MARCH) family represent a subgroup of the classical RING genes. Unlike their viral counterparts, the cellular RING-CH proteins appear highly regulated, and one of these in particular, MARCH7, was of interest because of a potential role in neuronal development and lymphocyte proliferation. Difficulties in detection and expression of this orphan ligase lead us to search for cellular cofactors involved in MARCH7 stability. In this study, we show that MARCH7 readily undergoes autoubiquitylation and associates with two deubiquitylating enzymes – ubiquitin-specific protease (USP)9X in the cytosol and USP7 in the nucleus. Exogenous expression and short interfering RNA depletion experiments demonstrate that MARCH7 can be stabilized by both USP9X and USP7, which deubiquitylate MARCH7 in the cytosol and nucleus, respectively. We therefore demonstrate compartment-specific regulation of this E3 ligase through recruitment of site-specific DUBs.     

The ER‐resident ubiquitin‐specific protease 19 participates in the UPR and rescues ERAD substrates

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic‐reticulum‐associated degradation (ERAD). Although the involvement of membrane‐associated ubiquitin‐conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening a database of human deubiquitinating enzymes (DUBs), we have identified a putative transmembrane domain in ubiquitin‐specific protease (USP)19. We show that USP19 is a tail‐anchored ubiquitin‐specific protease localized to the ER and is a target of the unfolded protein response. USP19 rescues the ERAD substrates cystic fibrosis transmembrane conductance regulator (CFTR)ΔF508 and T‐cell receptor‐α (TCRα) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRα but not CFTRΔF508, suggesting that USP19 might also exert a non‐catalytic function on specific ERAD substrates. Thus, USP19 is the first example of a membrane‐anchored DUB involved in the turnover of ERAD substrates.     

USP2 is an SKP2 deubiquitylase that stabilizes both SKP2 and its substrates

The stability of a protein is regulated by a balance between its ubiquitylation and deubiquitylation. S-phase kinase-associated protein 2 (SKP2) is an oncogenic F-box protein that recognizes tumor suppressor substrates for targeted ubiquitylation by the E3 ligase SKP1-Cullin1-F-box and degradation by proteasome. SKP2 is itself ubiquitylated by the E3 ligases APC/C^CDH1 and SCF^FBXW2, and deubiquitylated by deubiquitylases (DUBs) USP10 and USP13. Given the biological significance of SKP2, it is likely that the other E3s or DUBs may also regulate its stability. Here, we report the identification and characterization of USP2 as a new DUB. We first screened a panel of DUBs and found that both USP2 and USP21 bound to endogenous SKP2, but only USP2 deubiquitylated and stabilized SKP2 protein. USP2 inactivation via siRNA knockdown or small-molecule inhibitor treatment remarkably shortened SKP2 protein half-life by enhancing its ubiquitylation and subsequent degradation. Unexpectedly, USP2-stabilized SKP2 did not destabilize its substrates p21 and p27. Mechanistically, USP2 bound to SKP2 via the leucine-rich repeat substrate-binding domain on SKP2 to disrupt the SKP2-substrate binding, leading to stabilization of both SKP2 and these substrates. Biologically, growth suppression induced by USP2 knockdown or USP2 inhibitor is partially mediated via modulation of SKP2 and its substrates. Our study revealed a new mechanism of the cross-talk among the E3–DUB substrates and its potential implication in targeting the USP2–SKP2 axis for cancer therapy.     

Deubiquitinating enzyme USP36 contains the PEST motif and is polyubiquitinated

The ubiquitin-mediated protein degradation pathway has been emphasized for the regulation of numerous cellular mechanisms and the significance of deubiquitination, mediated by deubiquitinating (DUB) enzymes, has been emerging as an essential regulatory step to control these cellular mechanisms. Previously, we demonstrated a human DUB enzyme, HeLa DUB-1, expressed in human ovarian cancer cells. Here, we report human USP36, which has the extension of the C-terminal region of HeLa DUB-1 and has conserved amino acid domains as previously shown in other DUBs. Human USP36, encoding a DUB enzyme, was isolated from ovarian cancer cells using RT-PCR and characterized. We identified DUB enzyme activity of USP36 by analyzing its capability to cleave the ubiquitin. Interestingly, structural and immunoprecipitation analyses revealed for the first time that USP36 contains the PEST motif and is polyubiquitinated.     

A deubiquitinase negatively regulates retro-translocation of nonubiquitinated substrates

Endoplasmic reticulum (ER) membrane–bound E3 ubiquitin ligases promote ER-associated degradation (ERAD) by ubiquitinating a retro-translocated substrate that reaches the cytosol from the ER, targeting it to the proteasome for destruction. Recent findings implicate ERAD-associated deubiquitinases (DUBs) as positive and negative regulators during ERAD, reflecting the different consequences of deubiquitinating a substrate prior to proteasomal degradation. These observations raise the question of whether a DUB can control the fate of a nonubiquitinated ERAD substrate. In this study, we probed the role of the ERAD-associated DUB, YOD1, during retro-translocation of the nonubiquitinated cholera toxin A1 (CTA1) peptide, a critical intoxication step. Through combining knockdown, overexpression, and binding studies, we demonstrated that YOD1 negatively controls CTA1 retro-translocation, likely by deubiquitinating and inactivating ubiquitinated ERAD components that normally promote toxin retro-translocation. YOD1 also antagonizes the proteasomal degradation of nonglycosylated pro-α factor, a postulated nonubiquitinated yeast ERAD substrate, in mammalian cells. Our findings reveal that a cytosolic DUB exerts a negative function during retro-translocation of nonubiquitinated substrates, potentially by acting on elements of the ERAD machinery.     

Regulation of the Polycomb protein RING1B ubiquitination by USP7

The E3 ubiquitin ligase RING1B plays an important role in Polycomb-mediated gene silencing by monoubiquitinating histone H2A. Both the activity and stability of RING1B are controlled by ubiquitination in two distinct manners. Self ubiquitination of RING1B generates K6, K27 and K48-based mixed polyubiquitin chain, and is required for its activity as a ligase. On the other hand, its proteasomal degradation is mediated by another ligase; E6-AP catalyzes the formation of K48-based chains. Since these two modes of ubiquitination target the same lysine residues and are therefore mutually exclusive, an important mode of regulation of RING1B should be at the level of deubiquitination. Here we identify USP7 as a deubiquitinating enzyme that regulates the ubiquitination state of RING1B. RING1B interacts with USP7, which is mediated in part by its RING domain. In addition, USP7 was found in a complex with other Polycomb proteins, suggesting a broad role in regulating these complexes. Although, USP7 directly and specifically deubiquitinates RING1B in vitro and in vivo, it does not discriminate between the activating and proteolysis-targeting modes of ubiquitination, and therefore has a stabilizing effect on RING1B.     

The E3 Ubiquitin Ligase MARCH6 Degrades Squalene Monooxygenase and Affects 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase and the Cholesterol Synthesis Pathway

The mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.     

Ubiquitin-specific peptidase 8 regulates the trafficking and stability of the human organic anion transporter 1

BackgroundOrganic anion transporter 1 (OAT1) plays a vital role in avoiding the potential toxicity of various anionic drugs through the involvement of kidney elimination. We previously demonstrated that ubiquitin conjugation to OAT1 led to OAT1 internalization from cell surface, followed by degradation. Ubiquitination is a dynamic process, where deubiquitination is catalyzed by a class of ubiquitin-specific peptidases.MethodsThe role of ubiquitin-specific peptidase 8 (USP8) in hOAT1 function, expression and ubiquitination was assessed by conducting transporter uptake assay, biotinylation assay and ubiquitination assay.ResultsWe demonstrated that USP8 overexpression in hOAT1-expressing cells led to an increased hOAT1 transporter activity and expression, which correlated well with a reduced hOAT1 ubiquitination. Such phenomenon was not observed in inactive USP8 mutant-transfected cells. In addition, the knockdown of endogenous USP8 by USP8-specific siRNA resulted in an increased hOAT1 ubiquitination, which correlated well with a decrease in hOAT1 expression and transport activity. Biotinylation experiments demonstrated that USP8-induced increase in hOAT1 expression and transport activity occurred through a deceleration of the rates of hOAT1 internalization and degradation.ConclusionsThese results indicated the regulatory role of USP8 in OAT1 function, expression, trafficking, and stability.General significanceUSP8 could be a new target for modulating OAT1-mediated drug transport.     

USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15

Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.     

The deubiquitinating enzyme USP10 regulates the endocytic recycling of CFTR in airway epithelial cells

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a cyclic AMP-regulated chloride channel that plays an important role in regulating the volume of the lung airway surface liquid, and thereby mucociliary clearance and elimination of pathogens from the lung. In epithelial cells, cell surface CFTR abundance is determined in part by regulating both CFTR endocytosis from the apical plasma membrane and recycling back to the plasma membrane. We recently reported, using an activity-based chemical screen to identify active deubiquitinating enzymes (DUBs) in human airway epithelial cells, that Ubiquitin Specific Protease-10 (USP10) is located and active in the early endosomal compartment and regulates the deubiquitination of CFTR and thereby promotes its endocytic recycling. siRNA-mediated knockdown of USP10 increased the multi-ubiquitination and lysosomal degradation of CFTR and decreased the endocytic recycling and the half-life of CFTR in the apical membrane, as well as CFTR-mediated chloride secretion. Over-expression of wild-type USP10 reduced CFTR multi-ubiquitination and degradation, while over-expression of a dominant-negative USP10 promoted increased multi-ubiquitination and lysosomal degradation of CFTR. In the current study, we show localization and activity of USP10 in the early endosomal compartment of primary bronchial epithelial cells, as well as an interaction between CFTR and USP10 in this compartment. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes, thereby enhancing the endocytic recycling and cell surface expression of CFTR.     

Role of Derlin-1 protein in proteostasis regulation of ATP-sensitive potassium channels

ATP-sensitive potassium (K(ATP)) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2 regulate insulin secretion by linking glucose metabolism with membrane potential. The number of K(ATP) channels in the plasma membrane affects the sensitivity of β-cells to glucose. Aberrant surface channel expression leads to insulin secretion disease. Previously, we have shown that K(ATP) channel proteins undergo endoplasmic reticulum (ER)-associated degradation (ERAD) via the ubiquitin-proteasome pathway, and inhibition of proteasome function results in an increase in channel surface expression. Here, we investigated whether Derlin-1, a protein involved in retrotranslocation of misfolded or misassembled proteins across the ER membrane for degradation by cytosolic proteasomes, plays a role in ERAD and, in turn, biogenesis efficiency of K(ATP) channels. We show that both SUR1 and Kir6.2 form a complex with Derlin-1 and an associated AAA-ATPase, p97. Overexpression of Derlin-1 led to a decrease in the biogenesis efficiency and surface expression of K(ATP) channels. Conversely, knockdown of Derlin-1 by RNA interference resulted in increased processing of SUR1 and a corresponding increase in surface expression of K(ATP) channels. Importantly, knockdown of Derlin-1 increased the abundance of disease-causing misfolded SUR1 or Kir6.2 proteins and even partially rescued surface expression in a mutant channel. We conclude that Derlin-1, by being involved in ERAD of SUR1 and Kir6.2, has a role in modulating the biogenesis efficiency and surface expression of K(ATP) channels. The results suggest that physiological or pathological changes in Derlin-1 expression levels may affect glucose-stimulated insulin secretion by altering surface expression of K(ATP) channels.     

Noxa controls Mule-dependent Mcl-1 ubiquitination through the regulation of the Mcl-1/USP9X interaction

The level of the Mcl-1 pro-survival protein is highly regulated, and the down-regulation of Mcl-1 expression favors the apoptotic process. Mcl-1 physically interacts with different BH3-only proteins; particularly, Noxa is involved in the modulation of Mcl-1 expression. In this study, we demonstrated that Noxa triggers the degradation of Mcl-1 at the mitochondria according to the exclusive location of Noxa at this compartment. The Noxa-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Because the USP9X deubiquitinase was recently demonstrated to be involved in Mcl-1 protein turnover by preventing its degradation through the removal of conjugated ubiquitin, we investigated whether Noxa affected the deubiquitination process. Interestingly, Noxa over-expression caused a decrease in the USP9X/Mcl-1 interaction associated with an increase in the Mcl-1 polyubiquitinated forms. Additionally, Noxa over-expression triggered an increase in the Mule/Mcl-1 interaction in parallel with the decrease in Mule/USP9X complex formation. Taken together, these modifications result in the degradation of Mcl-1 by the proteasome machinery. The implication of Noxa in the regulation of Mcl-1 proteasomal degradation adds complexity to this process, which is governed by multiple interactions.     

A Pseudomonas aeruginosa toxin that hijacks the host ubiquitin proteolytic system

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.     

Ubiquitin-Specific Proteases 25 Negatively Regulates Virus-Induced Type I Interferon Signaling

Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-β, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), as well as the phosphorylation of IRF3 and NF-κB subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-β. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.