Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1

The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.     

Ubiquitination of human leukocyte antigen (HLA)-DM by different membrane-associated RING-CH (MARCH) protein family E3 ligases targets different endocytic pathways

HLA-DM plays an essential role in the peptide loading of classical class II molecules and is present both at the cell surface and in late endosomal peptide-loading compartments. Trafficking of DM within antigen-presenting cells is complex and is, in part, controlled by a tyrosine-based targeting signal present in the cytoplasmic tail of DMβ. Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα. Ubiquitination of DM by MARCH1 and MARCH9 induced loss of DM molecules from the cell surface by a mechanism that cumulatively involved both direct attachment of ubiquitin chains to DMα and a functional tyrosine-based signal on DMβ. In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ. In the absence of this tyrosine residue, levels of DM remained unchanged irrespective of whether DMα was ubiquitinated by MARCH8. The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.     

MARCH5-mediated quality control on acetylated Mfn1 facilitates mitochondrial homeostasis and cell survival

Mitochondrial dynamics and quality control have a central role in the maintenance of cellular integrity. Mitochondrial ubiquitin ligase membrane-associated RING-CH (MARCH5) regulates mitochondrial dynamics. Here, we show that mitochondrial adaptation to stress is driven by MARCH5-dependent quality control on acetylated Mfn1. Under mitochondrial stress conditions, levels of Mfn1 were elevated twofold and depletion of Mfn1 sensitized these cells to apoptotic death. Interestingly, overexpression of Mfn1 also promoted cell death in these cells, indicating that a fine tuning of Mfn1 levels is necessary for cell survival. MARCH5 binds Mfn1 and the MARCH5-dependent Mfn1 ubiquitylation was significantly elevated under mitochondrial stress conditions along with an increase in acetylated Mfn1. The acetylation-deficient K491R mutant of Mfn1 showed weak interaction with MARCH5 as well as reduced ubiquitylation. Neither was observed in the acetylation mimetic K491Q mutant. In addition, MARCH5-knockout mouse embryonic fibroblast and MARCH5^H43W-expressing HeLa cells lacking ubiquitin ligase activity experienced rapid cell death upon mitochondrial stress. Taken together, a fine balance of Mfn1 levels is maintained by MARCH5-mediated quality control on acetylated Mfn1, which is crucial for cell survival under mitochondria stress conditions.     

NIRF, a novel ubiquitin ligase, interacts with hepatitis B virus core protein and promotes its degradation

Hepatitis B virus (HBV) core protein (HBc) is a major component of viral nucleocapsid and a multifunctional protein involved in viral maturation and release. It is unstable and present in cells at low level because of K96 lysine residue, which is a ubiquitin acceptor site. Np95/ICBP90-like RING finger protein (NIRF) has auto-ubiquitination activity which is the hallmark of a ubiquitin ligase. In the present study, ubiquitin ligase, NIRF, binds to HBc and leads to the proteasome-mediated degradation of HBc in vivo. NIRF down-regulates HBc protein level, resulting in the decrease of the amount of HBV particles in supernatant of HepG2.2.15 cells. However knockdown of NIRF significantly increases endogenous HBc protein level, leading to HBV release. The results reveal that NIRF interacts with HBc and promotes the degradation of HBc in vivo. The pathway of NIRF-mediated ubiquitin–proteasome affects the release of HBV particles by controlling the amounts of HBc. It indicates that NIRF may participate in the maturation of HBV.     

YopJ targets TRAF proteins to inhibit TLR-mediated NF-kappaB, MAPK and IRF3 signal transduction

The Yersinia pestis virulence factor YopJ is a potent inhibitor of the NF-kappaB and MAPK signalling pathways, however, its molecular mechanism and relevance to pathogenesis are the subject of much debate. In this report, we characterize the effects of this type III effector protein on bone fide signalling events downstream of Toll-like receptors (TLRs), critical sensors in innate immunity. YopJ inhibited TLR-mediated NF-kappaB and MAP kinase activation, as suggested by previous studies. In addition, induction of the TLR-mediated interferon response was blocked by YopJ, indicating that YopJ also inhibits IRF3 signalling. Examination of the NF-kappaB signalling pathway in detail suggested that YopJ acts at the level of TAK1 (MAP3K7) activation. Further studies revealed a YopJ-dependent decrease in the ubiquitination of TRAF3 and TRAF6. These data support the hypothesis that YopJ is a deubiquitinating protease that acts on TRAF proteins to prevent or remove the K63-polymerized ubiquitin conjugates required for signal transduction. Our data do not directly address the alternative hypothesis that YopJ is an acetyltransferase that acts on the activation loop of IKK and MKK proteins, but support the conclusion that the critical function of YopJ is to deubiquinate TRAF proteins.     

Reduced secretion of YopJ by Yersinia limits in vivo cell death but enhances bacterial virulence

Numerous microbial pathogens modulate or interfere with cell death pathways in cultured cells. However, the precise role of host cell death during in vivo infection remains poorly understood. Macrophages infected by pathogenic species of Yersinia typically undergo an apoptotic cell death. This is due to the activity of a Type III secreted effector protein, designated YopJ in Y. pseudotuberculosis and Y. pestis, and YopP in the closely related Y. enterocolitica. It has recently been reported that Y. enterocolitica YopP shows intrinsically greater capacity for being secreted than Y. pestis YopJ, and that this correlates with enhanced cytotoxicity observed for high virulence serotypes of Y. enterocolitica. The enzymatic activity and secretory capacity of YopP from different Y. enterocolitica serotypes have been shown to be variable. However, the underlying basis for differential secretion of YopJ/YopP, and whether reduced secretion of YopJ by Y. pestis plays a role in pathogenesis during in vivo infection, is not currently known. It has also been reported that similar to macrophages, Y. enterocolitica infection of dendritic cells leads to YopP-dependent cell death. We demonstrate here that in contrast to Y. enterocolitica, Y. pseudotuberculosis infection of bone marrow-derived dendritic cells does not lead to increased cell death. However, death of Y. pseudotuberculosis-infected dendritic cells is enhanced by ectopic expression of YopP in place of YopJ. We further show that polymorphisms at the N-terminus of the YopP/YopJ proteins are responsible for their differential secretion, translocation, and consequent cytotoxicity. Mutation of two amino acids in YopJ markedly enhanced both translocation and cytotoxicity. Surprisingly, expression of YopP or a hypersecreted mutant of YopJ in Y. pseudotuberculosis resulted in its attenuation in oral mouse infection. Complete absence of YopJ also resulted in attenuation of virulence, in accordance with previous observations. These findings suggest that control of cytotoxicity is an important virulence property for Y. pseudotuberculosis, and that intermediate levels of YopJ-mediated cytotoxicity are necessary for maximal systemic virulence of this bacterial pathogen.     

DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell-cell contact

PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.     

Mitochondrial E3 ligase MARCH5 regulates FUNDC1 to fine‐tune hypoxic mitophagy

Mitophagy is an essential process for mitochondrial quality control and turnover. It is activated by two distinct pathways, one dependent on ubiquitin and the other dependent on receptors including FUNDC1. It is not clear whether these pathways coordinate to mediate mitophagy in response to stresses, or how mitophagy receptors sense stress signals to activate mitophagy. We find that the mitochondrial E3 ligase MARCH5, but not Parkin, plays a role in regulating hypoxia‐induced mitophagy by ubiquitylating and degrading FUNDC1. MARCH5 directly interacts with FUNDC1 to mediate its ubiquitylation at lysine 119 for subsequent degradation. Degradation of FUNDC1 by MARCH5 expression desensitizes mitochondria to hypoxia‐induced mitophagy, whereas knockdown of endogenous MARCH5 significantly inhibits FUNDC1 degradation and enhances mitochondrial sensitivity toward mitophagy‐inducing stresses. Our findings reveal a feedback regulatory mechanism to control the protein levels of a mitochondrial receptor to fine‐tune mitochondrial quality.     

Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase

Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximately PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.     

Regulation of the Mdm2–p53 pathway by the ubiquitin E3 ligase MARCH7

The tumor suppressor p53 plays a prominent role in the protection against cancer. The activity of p53 is mainly controlled by the ubiquitin E3 ligase Mdm2, which targets p53 for proteasomal degradation. However, the regulation of Mdm2 remains not well understood. Here, we show that MARCH7, a RING domain‐containing ubiquitin E3 ligase, physically interacts with Mdm2 and is essential for maintaining the stability of Mdm2. MARCH7 catalyzes Lys⁶³‐linked polyubiquitination of Mdm2, which impedes Mdm2 autoubiquitination and degradation, thereby leading to the stabilization of Mdm2. MARCH7 also promotes Mdm2‐dependent polyubiquitination and degradation of p53. Furthermore, MARCH7 is able to regulate cell proliferation, DNA damage‐induced apoptosis, and tumorigenesis via a p53‐dependent mechanism. These findings uncover a novel mechanism for the regulation of Mdm2 and reveal MARCH7 as an important regulator of the Mdm2–p53 pathway.     

MARCH7 E3 ubiquitin ligase is highly expressed in developing spermatids of rats and its possible involvement in head and tail formation

Spermatogenesis is a highly complicated metamorphosis process of male germ cells. Recent studies have provided evidence that the ubiquitin–proteasome system plays an important role in sperm head shaping, but the underlying mechanism is less understood. In this study, we localized membrane-associated RING-CH (MARCH)7, an E3 ubiquitin ligase, in rat testis. Northern blot analysis showed that March7 mRNA is expressed ubiquitously but highly in the testis and ovary. In situ hybridization of rat testis demonstrated that March7 mRNA is expressed weakly in spermatogonia and its level is gradually increased as they develop. Immunohistochemical analysis detected MARCH7 protein expression in spermiogenic cells from late round spermatids to elongated spermatids and in epididymal spermatozoa. Moreover, MARCH7 was found to be localized to the caudal end of the developing acrosome of late round and elongating spermatids, colocalizing with β-actin, a component of the acroplaxome. In addition, MARCH7 was also detected in the developing flagella and its expression levels were prominent in elongated spermatids. We also showed that MARCH7 catalyzes lysine 48 (K48)-linked ubiquitination. Immunolocalization studies revealed that K48-linked ubiquitin chains were detected in the heads of elongating spermatids and in the acrosome/acroplaxome, neck, midpiece and cytoplasmic lobes of elongated spermatids. These results suggest that MARCH7 is involved in spermiogenesis by regulating the structural and functional integrity of the head and tail of developing spermatids.     

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.     

MARCH5-FUNDC1 axis fine-tunes hypoxia-induced mitophagy

Mitophagy is responsible for removal of damaged mitochondria and is therefore a fundamental process in mitochondrial quality control. Both ubiquitin-dependent and receptor-dependent pathways are considered to mediate mitophagy. These distinct mechanisms may be activated in response to distinct mitochondrial stresses. An intriguing question is whether and how crosstalk occurs between the distinct pathways to coordinate mitophagy. We have uncovered a striking piece of evidence to demonstrate that the mitophagy receptor FUNDC1 is a substrate of MARCH5, a mitochondrially localized E3 ubiquitin ligase. In response to hypoxia, MARCH5 degrades redundant FUNDC1 to fine-tune hypoxia-induced mitophagy, whereas ablation of MARCH5 leads to accumulation of FUNDC1 and an exaggerated mitophagic phenotype. Mechanistic studies demonstrate that hypoxic insult enhances the interaction of FUNDC1 with MARCH5, which ubiquitinates FUNDC1 at lysine 119 for subsequent degradation. MARCH5-based ubiquitination and degradation of FUNDC1 circumvents injudicious removal of cellular mitochondria. However, severe hypoxic stress leads to dephosphorylation of FUNDC1, increasing mitophagic flux.     

Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity

The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes.     

Acetylation and phosphorylation of SRSF2 control cell fate decision in response to cisplatin

SRSF2 is a serine/arginine-rich protein belonging to the family of SR proteins that are crucial regulators of constitutive and alternative pre-mRNA splicing. Although it is well known that phosphorylation inside RS domain controls activity of SR proteins, other post-translational modifications regulating SRSF2 functions have not been described to date. In this study, we provide the first evidence that the acetyltransferase Tip60 acetylates SRSF2 on its lysine 52 residue inside the RNA recognition motif, and promotes its proteasomal degradation. We also demonstrate that the deacetylase HDAC6 counters this acetylation and acts as a positive regulator of SRSF2 protein level. In addition, we show that Tip60 downregulates SRSF2 phosphorylation by inhibiting the nuclear translocation of both SRPK1 and SRPK2 kinases. Finally, we demonstrate that this acetylation/phosphorylation signalling network controls SRSF2 accumulation as well as caspase-8 pre-mRNA splicing in response to cisplatin and determines whether cells undergo apoptosis or G(2)/M cell cycle arrest. Taken together, these results unravel lysine acetylation as a crucial post-translational modification regulating SRSF2 protein level and activity in response to genotoxic stress.