TRIM8 regulated autophagy modulates the level of cleaved Caspase-3 subunit to inhibit genotoxic stress induced cell death

In cancer patients, treatment modalities like chemotherapy and radiation exert their anticancer effects by inducing DNA damage. The cancer cells can survive under genotoxic stress by inducing DNA damage response (DDR) or can undergo cell death. The process of autophagy is emerging as crucial regulator of cell survival during different stress conditions. Post translational modification through ubiquitin plays an essential role in DDR during genotoxic stress conditions. Ubiquitin ligases regulate autophagy and cell death pathways however their role during genotoxic stress conditions is not understood. In the current study we identified TRIM8, RING E3 Ligase, as a novel regulator of autophagy during DDR. TRIM8 regulates lysosomal biogenesis and autophagy flux. The turnover of TRIM8 is high and is stabilized during genotoxic stress conditions. TRIM8 regulated autophagy is essential for its cytoprotective role during genotoxic stress induced cell death. TRIM8 stabilizes the turnover of XIAP during genotoxic stress and forms complex with XIAP and caspase-3 to inhibit its activation in presence of etoposide. TRIM8 mediated autophagy promotes degradation of cleaved caspase-3 subunits. This study described TRIM8, as a novel regulator of DDR-autophagy crosstalk, which may play role in survival of cancer cells in presence of genotoxic agents.     

The prolific ATL family of RING-H2 ubiquitin ligases

An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes.     

RING for destruction?

Ubiquitination targets proteins for degradation and is a potent regulator of cellular protein function. Recent results implicate the RING finger domain in specific ubiquitination events; it is possible that all RING proteins act as E3 ubiquitin protein ligases, with implications for a variety of biological areas.     

Arabidopsis CAND1, an unmodified CUL1-interacting protein, is involved in multiple developmental pathways controlled by ubiquitin/proteasome-mediated protein Degradation

Ubiquitin/proteasome-mediated protein degradation controls various developmental pathways in eukaryotes. Cullin-containing complexes are both versatile and abundant groups of RING family ubiquitin E3 ligases, whose activities are subject to control by RUB/Nedd8 (for related to ubiquitin/neural precursor cell-expressed developmentally downregulated 8) modification of their cullin subunits. Here, we report the identification of an Arabidopsis thaliana counterpart of human CAND1 (cullin-associated and neddylation-dissociated) and demonstrate that it can preferentially interact with unmodified CUL1. The Arabidopsis cand1-1 null mutant displays distinct phenotypes, including late flowering, aerial rosettes, floral organ defects, low fertility, dwarfism, loss of apical dominance, and altered responses to multiple plant hormones. Molecular analyses show that many of these defects are because of compromised activity of CUL1-containing ubiquitin E3 ligases, indicating that CAND1 is required for their optimal activity. Furthermore, the cand1-1 mutant displays a partial constitutive photomorphogenic phenotype and has defects in HY5 degradation in the absence of light, a process mediated by a different RING family E3, COP1. Thus, our data provides genetic support for a critical role of CAND1 in regulating various ubiquitin E3 ligases and their targeted cellular and developmental pathways.     

A systematic search for endoplasmic reticulum (ER) membrane-associated RING finger proteins identifies Nixin/ZNRF4 as a regulator of calnexin stability and ER homeostasis

To identify novel regulators of endoplasmic reticulum (ER)-linked protein degradation and ER function, we determined the entire inventory of membrane-spanning RING finger E3 ubiquitin ligases localized to the ER. We identified 24 ER membrane-anchored ubiquitin ligases and found Nixin/ZNRF4 to be central for the regulation of calnexin turnover. Ectopic expression of wild type Nixin induced a dramatic down-regulation of the ER-localized chaperone calnexin that was prevented by inactivation of the Nixin RING domain. Importantly, Nixin physically interacts with calnexin in a glycosylation-independent manner, induces calnexin ubiquitination, and p97-dependent degradation, indicating an ER-associated degradation-like mechanism of calnexin turnover.     

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase

Tripartite motif (TRIM) proteins comprise a large family of RING‐type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled‐coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher‐order oligomerization of the basal coiled‐coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2–ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.     

Post-translational regulation of the cellular levels of DAPK

Death associated protein kinase (DAPK) is a large, multi-domain ser/thr kinase whose activities converge upon multiple signaling pathways that regulate autophagy, caspase-dependent cell death, cell adhesion and migration. The cellular levels of DAPK are post-translationally regulated by the combined activities of two degradation systems, including the ubiquitin proteasome and an extra-lysosomal proteolysis pathway. At least three distinct E3 ubiquitin ligases target DAPK, including mindbomb1, the chaperone dependent ligase, CHIP (carboxy terminus of Hsp70-interacting protein) and a cullin RING ligase complex, KLHL20-Cul3-RBX1. In addition, it appears that the cellular levels of DAPK are also regulated by an extra-lysosomal protease, cathepsin B. While protein quality control and recycling clearly benefit cells by removal of misfolded or toxic proteins and recycling of their components, the finding that multiple surveillance systems target DAPK suggests that these protein degradation systems also act to fine tune DAPK expression levels in response to specific signaling pathways.     

Control of the olive fruit fly using genetics-enhanced sterile insect technique

Ubiquitin signaling pathways rely on E3 ligases for effecting the final transfer of ubiquitin from E2 ubiquitin conjugating enzymes to a protein target. Here we re-evaluate the hybrid RING/HECT mechanism used by the E3 family RING-between-RINGs (RBRs) to transfer ubiquitin to substrates. We place RBRs into the context of current knowledge of HECT and RING E3s. Although not as abundant as the other types of E3s (there are only slightly more than a dozen RBR E3s in the human genome), RBRs are conserved in all eukaryotes and play important roles in biology. Re-evaluation of RBR ligases as RING/HECT E3s provokes new questions and challenges the field.     

LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP-the catalytic subunit of LUBAC-is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.     

Bacteria-host relationship: ubiquitin ligases as weapons of invasion

Eukaryotic cells utilize the ubiquitin (Ub) system for maintaining a balanced functioning of cellular pathways. Although the Ub system is exclusive to eukaryotes, prokaryotic bacteria have developed an armory of Ub ligase enzymes that are capable of employing the Ub systems of various hosts, ranging from plant to animal cells. These enzymes have been acquired through the evolution and can be classified into three main classes, RING (really interesting new gene), HECT (homologous to the E6-AP carboxyl terminus) and NEL (novel E3 ligases). In this review we describe the roles played by different classes of bacterial Ub ligases in infection and pathogenicity. We also provide an overview of the different mechanisms by which bacteria mimic specific components of the host Ub system and outline the gaps in our current understanding of their functions. Additionally, we discuss approaches and experimental tools for validating this class of enzymes as potential novel antibacterial therapy targets.     

Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.     

Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.     

New classes of E3 ligases illuminated by chemical probes

Specificity in the ubiquitin system depends on E3 ligases, largely belonging to a handful of families discovered more than a decade ago. However, the last two years brought a quantum leap in the identification and/or mechanistic characterization of eukaryotic ubiquitin ligases, in part through implementation of activity-based chemical probes and cryo-EM. Here, we survey recent discoveries of RING-Cys-Relay, RZ-finger, and neddylated cullin–RING–ARIH RBR E3–E3 ubiquitin ligase mechanisms. These ligases transfer ubiquitin through unprecedented mechanisms—via novel catalytic domains or domain combinations—and collectively modify unconventional amino acids, non-proteinaceous bacterial lipid targets, and structurally-diverse substrates recruited to numerous cullin–RING ligases. We anticipate major expansion of the types, features, and mechanisms of E3 ligases will emerge from such chemical and structural approaches in the coming years.     

Structural aspects of multi-domain RING/Ubox E3 ligases in DNA repair

Ubiquitin conjugation plays critical roles in virtually all DNA repair pathways. This review provides an overview of the known multi-domain RING/Ubox E3 ligases and their domain structures. An analysis of known RING/Ubox X-ray and NMR structures leads to a discussion of the effects of dimerization. Structural and mechanistic data relating to the E3 ligase preferences for E2 interaction and chain-type specificity are reviewed and the role of the E3 ligases in regulation of the repair pathways is discussed.     

Arabidopsis RING Peroxins are E3 Ubiquitin Ligases that Interact with Two Homologous Ubiquitin Receptor Proteins

Peroxisomes are essential eukaryotic organelles that mediate various metabolic processes. Peroxisome import depends on a group of peroxisome biogenesis factors called peroxins, many of which are evolutionarily conserved. PEX2, PEX10, and PEX12 are three RING-finger-domain-containing integral membrane peroxins crucial for protein import. In yeast (Saccharomyces cerevisae), RING peroxins act as E3 ligases, facilitating the recycling of the peroxisome import receptor protein PEX5 through ubiquitination. In plants, RING peroxins are essential to plant vitality. To elucidate the mode of action of the plant RING peroxins, we employed in vitro assays to show that the Arabidopsis RING peroxins also have E3 ligase activities. We also identified a PEX2-interacting protein, DSK2b, which is a member of the ubiquitin receptor family known to function as shuttle factors ferrying polyubiquitinated substrates to the proteasome for degradation. DSK2b and its tandem duplicate DSK2a are localized in the cytosol and the nucleus, and both interact with the RING domain of PEX2 and PEX12. DSK2 artificial microRNA lines did not display obvious defects in plant growth or peroxisomal processes, indicating functional redundancies among Arabidopsis ubiquitin receptor proteins. Our results suggest that Arabidopsis RING peroxins can function as E3 ligases and act together with the ubiquitin receptor protein DSK2 in the peroxisomal membrane-associated protein degradation system.     

Structural modeling of protein ensembles between E3 RING ligases and SARS-CoV-2: The role of zinc binding domains

BackgroundThe ubiquitin system is a modification process with many different cellular functions including immune signaling and antiviral functions. E3 ubiquitin ligases are enzymes that recruit an E2 ubiquitin-conjugating enzyme bound to ubiquitin in order to catalyze the transfer of ubiquitin from the E2 to a protein substrate. The RING E3s, the most abundant type of ubiquitin ligases, are characterized by a zinc (II)-binding domain called RING (Really Interesting New Gene). Viral replication requires modifying and hijacking key cellular pathways within host cells such as cellular ubiquitination. There are well-established examples where a viral proteins bind to RING E3s, redirecting them to degrade otherwise long-lived host proteins or inhibiting E3’s ubiquitination activity. Recently, three binary interactions between SARS-CoV-2 proteins and innate human immune signaling Ε3 RING ligases: NSP15-RNF41, ORF3a-TRIM59 and NSP9-MIB1 have been experimentally established.MethodsIn this work, we have investigated the mode of the previous experimentally supported NSP15-RNF41, ORF3a,-TRIM59 and NSP9-MIB1 binary interactions by in silico methodologies intending to provide structural insights of E3-virus interplay that can help identify potential inhibitors that could block SARS-CoV-2 infection of immune cells.ConclusionIn silico methodologies have shown that the above human E3 ligases interact with viral partners through their Zn(II) binding domains. This RING mediated formation of stable SARS-CoV-2-E3 complexes indicates a critical structural role of RING domains in immune system disruption by SARS-CoV-2-infection.Data AvailabilityThe data used to support the findings of this research are included within the article and are labeled with references.     

RNF185 Is a Novel E3 Ligase of Endoplasmic Reticulum-associated Degradation (ERAD) That Targets Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

In the endoplasmic reticulum (ER), misfolded or improperly assembled proteins are exported to the cytoplasm and degraded by the ubiquitin-proteasome pathway through a process called ER-associated degradation (ERAD). ER-associated E3 ligases, which coordinate substrate recognition, export, and proteasome targeting, are key components of ERAD. Cystic fibrosis transmembrane conductance regulator (CFTR) is one ERAD substrate targeted to co-translational degradation by the E3 ligase RNF5/RMA1. RNF185 is a RING domain-containing polypeptide homologous to RNF5. We show that RNF185 controls the stability of CFTR and of the CFTRΔF508 mutant in a RING- and proteasome-dependent manner but does not control that of other classical ERAD model substrates. Reciprocally, its silencing stabilizes CFTR proteins. Turnover analyses indicate that, as RNF5, RNF185 targets CFTR to co-translational degradation. Importantly, however, simultaneous depletion of RNF5 and RNF185 profoundly blocks CFTRΔF508 degradation not only during translation but also after synthesis is complete. Our data thus identify RNF185 and RNF5 as a novel E3 ligase module that is central to the control of CFTR degradation.     

Signalling pathways that mediate skeletal muscle hypertrophy and atrophy

Atrophy of skeletal muscle is a serious consequence of numerous diseases, including cancer and AIDS. Successful treatments for skeletal muscle atrophy could either block protein degradation pathways activated during atrophy or stimulate protein synthesis pathways induced during skeletal muscle hypertrophy. This perspective will focus on the signalling pathways that control skeletal muscle atrophy and hypertrophy, including the recently identified ubiquitin ligases muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), as a basis to develop targets for pharmacologic intervention in muscle disease.