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Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract Background Ubiquitylation (ubi) of the intracellular domain of the Notch ligand Delta (Dl) by the E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) on lysines (Ks) is thought to be essential for the its signalling activity. Nevertheless, we have previously shown that DlK2R-HA, a Dl variant where all Ks in its intracellular domain (ICD) are replaced by the structurally similar arginine (R), still possess weak activity if over-expressed. This suggests that ubi is not absolutely required for Dl signalling. However, it is not known whether the residual activity of DlK2R-HA is an effect of over-expression and, if not, whether DlK2R can provide sufficient activity for the whole development of Drosophila. Results To clarify these issues, we generated and analysed Dl attP -DlK2R-HA, a knock-in allele into the Dl locus. Our analysis of this allele reveals that the sole presence of one copy of Dl attP -DlK2R-HA can provide sufficient activity for completion of development. It further indicates that while ubi is required for the full activity of Dl in Mib1-dependent processes, it is not essential for Neur-controlled neural development. We identify three modes of Dl signalling that are either dependent or independent of ubi. Importantly, all modes depend on the presence of the endocytic adapter Epsin. During activation of Dl, direct binding of Epsin appears not to be an essential requirement. In addition, our analysis further reveals that the Ks are required to tune down the cis-inhibitory interaction of Dl with Notch. Conclusions Our results indicate that Dl can activate the Notch pathway without ubi of its ICD. It signals via three modes. Ubi is specifically required for the Mib1-dependent processes and the adjustment of cis-inhibition. In contrast to Mib1, Neur can efficiently activate Dl without ubi. Neur probably acts as an endocytic co-adapter in addition to its role as E3 ligase. Endocytosis, regulated in a ubi-dependent or ubi-independent manner is required for signalling and also suppression of cis-inhibition. The findings clarify the role of ubi of the ligands during Notch signalling.

Background: Ubiquitylation (ubi) of the intracellular domain of the Notch ligand Delta (Dl) by the E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) on lysines (Ks) is thought to be essential for the its signalling activity. Nevertheless, we have previously shown that DlK2R-HA, a Dl variant where all Ks in its intracellular domain (ICD) are replaced by the structurally similar arginine (R), still possess weak activity if over-expressed. This suggests that ubi is not absolutely required for Dl signalling. However, it is not known whether the residual activity of DlK2R-HA is an effect of over-expression and, if not, whether DlK2R can provide sufficient activity for the whole development of Drosophila. Results: To clarify these issues, we generated and analysed DlattP-DlK2R-HA, a knock-in allele into the Dl locus. Our analysis of this allele reveals that the sole presence of one copy of DlattP-DlK2R-HA can provide sufficient activity for completion of development. It further indicates that while ubi is required for the full activity of Dl in Mib1-dependent processes, it is not essential for Neur-controlled neural development. We identify three modes of Dl signalling that are either dependent or independent of ubi. Importantly, all modes depend on the presence of the endocytic adapter Epsin. During activation of Dl, direct binding of Epsin appears not to be an essential requirement. In addition, our analysis further reveals that the Ks are required to tune down the cis-inhibitory interaction of Dl with Notch. Conclusions: Our results indicate that Dl can activate the Notch pathway without ubi of its ICD. It signals via three modes. Ubi is specifically required for the Mib1-dependent processes and the adjustment of cis-inhibition. In contrast to Mib1, Neur can efficiently activate Dl without ubi. Neur probably acts as an endocytic co-adapter in addition to its role as E3 ligase. Endocytosis, regulated in a ubi-dependent or ubi-independent manner is required for signalling and also suppression of cis-inhibition. The findings clarify the role of ubi of the ligands during Notch signalling. [ABSTRACT FROM AUTHOR]

The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC. [ABSTRACT FROM AUTHOR]

Abstract Background Ubiquitylation of the ligands and the receptor plays an important part in the regulation of the activity of the evolutionary conserved Notch signalling pathway. However, its function for activation of Notch is not completely understood, despite the identification of several E3 ligases devoted to the receptor. Results Here we analysed a variant of the Notch receptor where all lysines in its intracellular domain are replaced by arginines. Our analysis of this variant revealed that ubiquitylation of Notch is not essential for its endocytosis. We identified two functions for ubiquitylation of lysines in the Notch receptor. First, it is required for the degradation of free Notch intracellular domain (NICD) in the nucleus, which prevents a prolonged activation of the pathway. More importantly, it is also required for the incorporation of Notch into intraluminal vesicles of maturing endosomes to prevent ligand-independent activation of the pathway from late endosomal compartments. Conclusions The findings clarify the role of lysine-dependent ubiquitylation of the Notch receptor and indicate that Notch is endocytosed by several independent operating mechanisms.

Background: Ubiquitylation of the ligands and the receptor plays an important part in the regulation of the activity of the evolutionary conserved Notch signalling pathway. However, its function for activation of Notch is not completely understood, despite the identification of several E3 ligases devoted to the receptor. Results: Here we analysed a variant of the Notch receptor where all lysines in its intracellular domain are replaced by arginines. Our analysis of this variant revealed that ubiquitylation of Notch is not essential for its endocytosis. We identified two functions for ubiquitylation of lysines in the Notch receptor. First, it is required for the degradation of free Notch intracellular domain (NICD) in the nucleus, which prevents a prolonged activation of the pathway. More importantly, it is also required for the incorporation of Notch into intraluminal vesicles of maturing endosomes to prevent ligand-independent activation of the pathway from late endosomal compartments. Conclusions: The findings clarify the role of lysine-dependent ubiquitylation of the Notch receptor and indicate that Notch is endocytosed by several independent operating mechanisms. [ABSTRACT FROM AUTHOR]

Protein degradation by the Ubiquitin-Proteasome System is one of the main mechanisms of the regulation of cellular proteostasis, and the E3 ligases are the key effectors for the protein recognition and degradation. Many E3 ligases have key roles in cell cycle regulation, acting as checkpoints and checkpoint regulators. One of the many important proteins involved in the regulation of the cell cycle are the members of the Histone Deacetylase (HDAC) family. The importance of zinc dependent HDACs in the regulation of chromatin packing and, therefore, gene expression, has made them targets for the design and synthesis of HDAC inhibitors. However, achieving potency and selectivity has proven to be a challenge due to the homology between the zinc dependent HDACs. PROteolysis TArgeting Chimaera (PROTAC) design has been demonstrated to be a useful strategy to inhibit and selectively degrade protein targets. In this review, we attempt to summarize the E3 ligases that naturally ubiquitinate HDACs, analyze their structure, and list the known ligands that can bind to these E3 ligases and be used for PROTAC design, as well as the already described HDAC-targeted PROTACs.

Germline specification is the first step during sexual and apomictic plant reproduction, and takes place in the nucellus of the ovule, a specialized domain of the reproductive flower tissues. In each case, a sporophytic cell is determined to form the sexual megaspore mother cell (MMC) or an apomictic initial cell (AIC). These differ in their developmental fates: while the MMC undergoes meiosis, the AIC modifies or omits meiosis to form the female gametophyte. Despite great interest in these distinct developmental processes, little is known about their gene regulatory basis. To elucidate the gene regulatory networks underlying germline specification, we conducted tissue-specific transcriptional profiling using laser-assisted microdissection and RNA sequencing to compare the transcriptomes of nucellar tissues between different sexual and apomictic Boechera accessions representing four species and two ploidy levels. This allowed us to distinguish between expression differences caused by genetic background or reproductive mode. Statistical data analysis revealed 45 genes that were significantly differentially expressed, and which potentially play a role for determination of the reproductive mode. Based on annotations, these included F-box genes and E3 ligases that most likely relate to genes previously described as regulators important for germline development. Our findings provide novel insights into the transcriptional basis of sexual and apomictic reproduction. [ABSTRACT FROM AUTHOR]

Tripartite motif (TRIM) proteins are RING E3 ubiquitin ligases defined by a shared domain structure. Several of them are implicated in rare genetic diseases, and mutations in TRIM32 and TRIM-like malin are associated with Limb-Girdle Muscular Dystrophy R8 and Lafora disease, respectively. These two proteins are evolutionary related, share a common ancestor, and both display NHL repeats at their C-terminus. Here, we revmniew the function of these two related E3 ubiquitin ligases discussing their intrinsic and possible common pathophysiological pathways.

Glaucoma represents a group of progressive neurodegenerative diseases characterized by the loss of retinal ganglion cells (RGCs) and their axons with subsequent visual field impairment. The disease develops through largely uncharacterized molecular mechanisms, that are likely to occur in different localized cell types, either in the anterior (e.g., trabecular meshwork cells) or posterior (e.g., Muller glia, retinal ganglion cells) segments of the eye. Genomic and preclinical studies suggest that glaucoma pathogenesis may develop through altered ubiquitin (Ub) signaling. Ubiquitin conjugation, referred to as ubiquitylation, is a major post-synthetic modification catalyzed by E1-E2-E3 enzymes, that profoundly regulates the turnover, trafficking and biological activity of the targeted protein. The development of new technologies, including proteomics workflows, allows the biology of ubiquitin signaling to be described in health and disease. This post-translational modification is emerging as a key role player in neurodegeneration, gaining relevance for novel therapeutic options, such as in the case of Proteolysis Targeting Chimeras technology. Although scientific evidence supports a link between Ub and glaucoma, their relationship is still not well-understood. Therefore, this review provides a detailed research-oriented discussion on current evidence of Ub signaling in glaucoma. A review of genomic and genetic data is provided followed by an in-depth discussion of experimental data on ASB10, parkin and optineurin, which are proteins that play a key role in Ub signaling and have been associated with glaucoma. [ABSTRACT FROM AUTHOR]

ARTS (Sept4_i2) is a pro-apoptotic mitochondrial tumor suppressor protein which binds to and causes degradation of XIAP (X-linked inhibitor of apoptosis). We recently showed that ARTS brings XIAP into close proximity to Bcl-2, creating a complex which enables degradation of both these major anti-apoptotic proteins and promotes apoptosis. The possible therapeutic implications are discussed here.

Ubiquitylation (ubi) by the E3-ligases Mindbomb1 (Mib1) and Neuralized (Neur) is required for activation of the DSL ligands Delta (Dl) and Serrate (Ser) to activate Notch signalling. These ligases transfer ubiquitin to lysines of the ligands' intracellular domains (ICDs), which sends them into an Epsin-dependent endocytic pathway. Here, we have tested the requirement of ubi of Dl for signalling. We found that Dl requires ubi for its full function, but can also signal in two ubi-independent modes, one dependent and one independent of Neur. We identified two neural lateral specification processes where Dl signals in an ubi-independent manner. Neur, which is needed for these processes, was shown to be able to activate Dl in an ubi-independent manner. Our analysis suggests that one important role of DSL protein ubi by Mib1 is their release from cis-inhibitory interactions with Notch, enabling them to trans-activate Notch on adjacent cells.

Ubiquitination is a multifunctional posttranslational modification controlling the activity, subcellular localization and stability of proteins. The E3 ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) is an essential epigenetic factor that recognizes repressive histone marks as well as hemi-methylated DNA and recruits DNA methyltransferase 1. To explore enzymatic functions of UHRF1 beyond epigenetic regulation, we conducted a comprehensive screen in mouse embryonic stem cells to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2. We found differentially ubiquitinated peptides associated with a variety of biological processes such as transcriptional regulation and DNA damage response. Most prominently, we identified PCNA-associated factor 15 (PAF15; also known as Pclaf, Ns5atp9 , KIAA0101 and OEATC-1) as a specific ubiquitination target of UHRF1. Although the function of PAF15 ubiquitination in translesion DNA synthesis is well characterized, the respective E3 ligase had been unknown. We could show that UHRF1 ubiquitinates PAF15 at Lys 15 and Lys 24 and promotes its binding to PCNA during late S-phase. In summary, we identified novel ubiquitination targets that link UHRF1 to transcriptional regulation and DNA damage response. [ABSTRACT FROM AUTHOR]

Metastatic melanoma is the most fatal type of skin cancer. The roles of matrix metalloproteinases (MMPs) have well been established in the onset of melanoma. Basigin (BSG) belongs to the immunoglobulin superfamily and is critical for induction of extracellular MMPs during the onset of various cancers including melanoma. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3-ligase that interacts with BSG and mediates its membrane localization, which leads to MMP expression in melanoma cells. This makes TRAF6 a potential therapeutic target in melanoma. We here conducted protein-protein interaction studies on TRAF6 and BSG to get molecular level insights of the reactions. The structure of human BSG was constructed by protein threading. Molecular-docking method was applied to develop the TRAF6-BSG complex. The refined docked complex was further optimized by molecular dynamics simulations. Results from binding free energy, surface properties, and electrostatic interaction analysis indicate that Lys340 and Glu417 of TRAF6 play as the anchor residues in the protein interaction interface. The current study will be helpful in designing specific modulators of TRAF6 to control melanoma metastasis. [ABSTRACT FROM AUTHOR]

Summary We describe a mechanism by which the anti-apoptotic B cell lymphoma 2 (Bcl-2) protein is downregulated to induce apoptosis. ARTS (Sept4_i2) is a tumor suppressor protein that promotes cell death through specifically antagonizing XIAP (X-linked inhibitor of apoptosis). ARTS and Bcl-2 reside at the outer mitochondrial membrane in living cells. Upon apoptotic induction, ARTS brings XIAP and Bcl-2 into a ternary complex, allowing XIAP to promote ubiquitylation and degradation of Bcl-2. ARTS binding to Bcl-2 involves the BH3 domain of Bcl-2. Lysine 17 in Bcl-2 serves as the main acceptor for ubiquitylation, and a Bcl-2 K17A mutant has increased stability and is more potent in protection against apoptosis. Bcl-2 ubiquitylation is reduced in both XIAP- and Sept4/ARTS-deficient MEFs, demonstrating that XIAP serves as an E3 ligase for Bcl-2 and that ARTS is essential for this process. Collectively, these results suggest a distinct model for the regulation of Bcl-2 by ARTS-mediated degradation. [ABSTRACT FROM AUTHOR]

Lafora disease (LD, OMIM254780) is a rare and fatal form of progressive myoclonus epilepsy (PME). Among PMEs, LD is unique because of the rapid neurological deterioration of the patients and the appearance in brain and peripheral tissues of insoluble glycogen-like (polyglucosan) inclusions, named Lafora bodies (LBs). LD is caused by mutations in the EPM2A gene, encoding the dual phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Laforin and malin form a functional complex that is involved in the regulation of glycogen synthesis. Thus, in the absence of a functional complex glycogen accumulates in LBs. In addition, it has been suggested that the laforin-malin complex participates in alternative physiological pathways, such as intracellular protein degradation, oxidative stress, and the endoplasmic reticulum unfolded protein response. In this work we review the possible cellular functions of laforin and malin with a special focus on their role in the ubiquitination of specific substrates. We also discuss here the pathological consequences of defects in laforin or malin functions, as well as the therapeutic strategies that are being explored for LD.

Die heutigen, auf Inhibitoren basierenden Therapieansätze sind zwangsläufig eingeschränkt durch 1) die Notwendigkeit von hoher systemischer Exposition, um ausreichende In‐vivo‐Effekte zu gewährleisten, 2) mögliche Nebenwirkungen aufgrund von hohen In‐vivo‐Konzentrationen des Wirkstoffs und 3) die Notwendigkeit, an ein aktives Zentrum zu binden, und die damit einhergehende Einschränkung der zugänglichen Zielproteine. Ein alternativer Ansatz nutzt einen neuen, katalytischen Mechanismus, um die Funktion eines Proteins irreversibel zu hemmen: den induzierten Abbau des Proteins durch die Rekrutierung an die zelluläre Maschinerie zur Qualitätskontrolle. Frühere Proteinabbau‐Strategien hatten kein hohes therapeutisches Anwendungspotenzial, doch neue Studien zu niedermolekularen PROTACs (“Proteolysis Targeting Chimeras”) haben gezeigt, dass diese Technik den Abbau von mehreren verschiedenen Proteinklassen überaus effizient vermitteln kann. Zerstörerische Chimären: Der durch niedermolekulare Verbindungen vom Typ der PROTACs („Proteolysis Targeting Chimeras”) ausgelöste Proteinabbau kann eine Alternative zu derzeitigen Inhibitor‐basierten Therapiekonzepten bieten. Neueste Erkenntnisse zur Entwicklung dieser Wirkstoffe werden beschrieben, und die Möglichkeiten und Herausforderungen der neuen Strategie werden diskutiert. [ABSTRACT FROM AUTHOR]

Highlights: [•] E2s and E3s work together to catalyze the transfer of ubiquitin to substrate. [•] The rate of ubiquitin transfer to the substrate governs its proteasomal degradation. [•] Ubiquitinated substrates induce large conformational changes in the proteasome. [Copyright &y& Elsevier]

The Ro52 protein of the Ro/SSA antigen was recently defined as an E3 ligase controlling cytokine production. Autoantibodies from systemic lupus erythematosus (SLE) patients targeting the Ro52-RING domain, containing the E3 ligase activity, have been shown to inhibit the E3 ligase activity of Ro52. The objective of the present study was to investigate correlations between clinical parameters in patients with SLE and levels of Ro/SSA (Ro52 and Ro60) and La/SSB autoantibodies, including autoantibodies directed towards the functional RING and B-box domains of the Ro52 protein. SLE patients (n = 232) were clinically examined and disease activity indices collected concurrently to blood sampling. The samples were analyzed for immunological parameters including autoantibodies.Ro52 autoantibody levels were associated with more variables than the other analyzed antibodies and were significantly associated with several individual items related to sSS and the diagnosis of sSS itself (p = 0.004). Other associated variables were high sedimentation rate (p = 0.0003), levels of immunoglobulins (p = 0.0003), and an inverse correlation with levels of lymphocytes (p = 0.003) and leukocytes (p = 0.01). Antibodies to the RING domain of Ro52, which is the functionally active domain with E3 ligase activity, were significantly correlated with disease activity as measured by the SLAM score.We conclude that autoantibodies against Ro52 and in particular its functional RING domain are important in lupus patients and associated with several clinical and laboratory features of the disease. The impact on disease activity of Ro52-RING specific antibodies was especially noted, and could imply a functional role for these autoantibodies in inhibiting Ro52 activity, which is important for the control of proinflammatory cytokine production, including type 1 interferons. [ABSTRACT FROM AUTHOR]

Abstract: The interferon regulated transcription factor IRF-1 is a tumour suppressor protein that is activated in response to viral infection and cell signalling activated by double stranded DNA lesions. IRF-1 has a short half-life (t 0.5 20–40 min) allowing rapid changes in steady state levels by modulating its rate of degradation and/or synthesis. However, little is known about the pathway(s) leading to IRF-1 protein degradation or what determines the rate of degradation in cells. Here we establish a role for discrete motifs in the enhancer domain of IRF-1 in directing polyubiquitination and degradation. By studying the structure of the enhancer domain as related to its role in the turnover of IRF-1 we have demonstrated that this region is not subject to modification by ubiquitin but rather that it contains both an ubiquitination signal and a distinct degradation signal. Removal of the C-terminal 70 amino acids from IRF-1 inhibits both its degradation and polyubiquitination, whereas removal of the C-terminal 25 amino acids inhibits degradation of the protein but does not prevent its ubiquitination. Furthermore, consistent with the C-terminus being involved in targeting or recognition by an E3-ligase or associated protein(s) the enhancer domain can act in trans to inhibit IRF-1 ubiquitination by endogenous E3-ligase activity. The identification of structural determinants that signals IRF-1 polyubiquitination and which can be uncoupled from IRF-1 degradation lends support to the idea that the degradation of selective substrates can be regulated at multiple steps in the ubiquitin–proteasome system. [Copyright &y& Elsevier]

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are critical for nervous system development and maintenance. GFLs promote survival and growth via activation of the receptor tyrosine kinase (RTK) Ret. In sympathetic neurons, the duration of Ret signaling is governed by how rapidly Ret is degraded after its activation. In an effort to elucidate mechanisms that control the half-life of Ret, we have identified two novel Ret interactors, CD2-associated protein (CD2AP) and Cbl-3. CD2AP, an adaptor molecule involved in the internalization of ubiquitinated RTKs, is associated with Ret under basal, unstimulated conditions in neurons. After Ret activation by GDNF, CD2AP dissociates. Similarly, the E3-ligase Cbl-3 interacts with unphosphorylated Ret and dissociates from Ret after Ret activation. In contrast to their dissociation from autophosphorylated Ret, an interaction between CD2AP and Cbl-3 is induced by GDNF stimulation of sympathetic neurons, suggesting that CD2AP and Cbl-3 dissociate from Ret as a complex. In neurons, the overexpression of CD2AP enhances the degradation of Ret and inhibits GDNF-dependent survival, and gene silencing of CD2AP blocks Ret degradation and promotes GDNF-mediated survival. Surprisingly, Cbl-3 overexpression dramatically stabilizes activated Ret and enhances neuronal survival, even though Cbl-family E3 ligases normally function to trigger RTK downregulation. In combination with CD2AP, however, Cbl-3 promotes Ret degradation rapidly and almost completely blocks survival promotion by GDNF, suggesting that Cbl-3 acts as a switch that is triggered by CD2AP and oscillates between inhibition and promotion of Ret degradation. Consistent with the hypothesis, Cbl-3 silencing in neurons only inhibited Ret degradation and enhanced neuronal survival in combination with CD2AP silencing. CD2AP and Cbl-3, therefore, constitute a checkpoint that controls the extent of Ret downregulation and, thereby, the sensitivity of neurons to GFLs. [ABSTRACT FROM AUTHOR]

Abstract: Duchenne muscular dystrophy (DMD) is one of the most severe X-linked, inherited diseases of childhood, characterized by progressive muscle wasting and weakness as the consequence of mutations in the dystrophin gene. The protein encoded by dystrophin is a huge cytosolic protein that links the intracellular F-actin filaments to the members of the dystrophin–glycoprotein–complex (DGC). Dystrophin deficiency results in the absence or reduction of complex components that are degraded through an unknown pathway. We show here that muscle degeneration in a Caenorhabditis elegans DMD model is efficiently reduced by downregulation of chn-1, encoding the homologue of the human E3/E4 ubiquitylation enzyme CHIP. A deletion mutant of chn-1 delays the cell death of body-wall muscle cells and improves the motility of animals carrying mutations in dystrophin and MyoD. Elimination of chn-1 function in the musculature, but not in the nervous system, is sufficient for this effect, and can be phenocopied by proteasome inhibitor treatment. This suggests a critical role of CHIP/CHN-1-mediated ubiquitylation in the control of muscle wasting and degeneration and identifies a potential new drug target for the treatment of this disease. [Copyright &y& Elsevier]

In the developing Drosophila eye, Rap/Fzr plays a critical role in neural patterning by regulating the timely exit of precursor cells. Rap/Fzr (Retina aberrant in pattern/Fizzy related) is an activator of the E3 Ubiquitin ligase, the APC (Anaphase Promoting Complex-cyclosome) that facilitates the stage specific proteolytic destruction of mitotic regulators, such as cyclins and cyclin-dependent kinases. To identify novel functional roles of Rap/Fzr, we conducted an F1 genetic modifier screen to identify genes which interact with the partial-loss-function mutations in rap/fzr. We screened 2741 single P-element, lethal insertion lines and piggyBac lines on the second and third chromosome for dominant enhancers and suppressors of the rough eye phenotype of rap/fzr. From this screen, we have identified 40 genes that exhibit dosage-sensitive interactions with rap/fzr; of these, 31 have previously characterized cellular functions. Seven of the modifiers identified in this study are regulators of cell cycle progression with previously known interactions with rap/fzr. Among the remaining modifiers, 27 encode proteins involved in other cellular functions not directly related to cell-cycle progression. The newly identified variants fall into at least three groups based on their previously known cellular functions: transcriptional regulation, regulated proteolysis, and signal transduction. These results suggest that, in addition to cell cycle regulation, rap/fzr regulates ubiquitin-ligase-mediated protein degradation in the developing nervous system as well as in other tissues. [ABSTRACT FROM AUTHOR]

Abstract: Cullin-5 (Cul-5), a member of the cullin gene family of scaffold proteins of E3 ubiquitin-ligase complexes, has a role in proteolysis and cell cycle regulation. We recently demonstrated that cul-5 mRNA is ubiquitously expressed in the central nervous system. The present study used quantitative real time polymerase chain reaction and western blotting to measure changes in cul-5 mRNA and Cul-5 protein expression, respectively, in the injured CNS in response to traumatic brain injury (TBI). cul-5 mRNA levels were significantly decreased in the ipsilateral rat cerebral cortex on Days 1 and 7, but not on Day 3 following TBI. In the ipsilateral hippocampus, cul-5 mRNA was significantly reduced on Day 1 after TBI. Cul-5 protein levels were significantly decreased in the ipsilateral rat cerebral cortex on Days 1–7 post-TBI while levels were significantly lower in the ipsilateral hippocampus on Days 3–7 post-TBI. Since Cul-5 is ubiquitously expressed in eukaryotic cells and is linked to proteasome-mediated protein degradation, it may have a role in CNS cell fate determination under conditions of traumatic stress. [Copyright &y& Elsevier]

The plant hormone ethylene regulates growth, development and stress responses. In recent years, various genomic and proteomic approaches have been initiated to understand both the range of ethylene responses in the plant and the mechanism of signal transduction. Transcriptional profiling experiments reveal broad-ranging effects of ethylene upon gene regulation, with up to 7 per cent of the genes examined demonstrating a significant level of response in one study. Both transcriptional and post-transcriptional mechanisms regulate the expression of components within the ethylene signal transduction pathway. The importance of post-transcriptional regulation via the ubiquitin/proteasome-mediated degradation pathway is apparent in studies on the accumulation of ethylene insensitive 3 (EIN3), a key transcription factor in the pathway. Protein complexes also play a role in modulating ethylene signal transduction, with interactions between the ethylene receptors and the Raf-like kinase constitutive triple response-1 (CTR1) being required for ethylene perception at the endoplasmic reticulum. In this paper, recent developments in unravelling the transcriptional and post-transcriptional regulation of the ethylene signalling and response pathways are considered, along with the latest developments in unravelling the biochemical mechanism behind ethylene perception. [ABSTRACT FROM AUTHOR]

S mall u biquitin related mo difier SUMO-1 and its homologs can be conjugated to a large number of cellular proteins. This involves an enzymatic cascade that resembles ubiquitination, and the modification can be reverted by isopeptidases. SUMOylation does not lead to degradation but instead appears to regulate protein/protein interactions, intracellular localization and protects some modified targets from ubiquitin-dependent degradation. Data collected for more than 30 different target proteins point to two cellular processes, nucleocytoplasmic transport and intranuclear targeting, in which SUMO plays an active role. Here we will focus on links between SUMO and nuclear transport. [ABSTRACT FROM AUTHOR]

Protein degradation by the Ubiquitin-Proteasome System is one of the main mechanisms of the regulation of cellular proteostasis, and the E3 ligases are the key effectors for the protein recognition and degradation. Many E3 ligases have key roles in cell cycle regulation, acting as checkpoints and checkpoint regulators. One of the many important proteins involved in the regulation of the cell cycle are the members of the Histone Deacetylase (HDAC) family. The importance of zinc dependent HDACs in the regulation of chromatin packing and, therefore, gene expression, has made them targets for the design and synthesis of HDAC inhibitors. However, achieving potency and selectivity has proven to be a challenge due to the homology between the zinc dependent HDACs. PROteolysis TArgeting Chimaera (PROTAC) design has been demonstrated to be a useful strategy to inhibit and selectively degrade protein targets. In this review, we attempt to summarize the E3 ligases that naturally ubiquitinate HDACs, analyze their structure, and list the known ligands that can bind to these E3 ligases and be used for PROTAC design, as well as the already described HDAC-targeted PROTACs. [ABSTRACT FROM AUTHOR]

Effective maintenance of plant iron (Fe) homoeostasis relies on a network of transcription factors (TFs) that respond to environmental conditions and regulate Fe uptake, translocation, and storage. The iron-related transcription factor 3 (IRO3), as well as haemerythrin motif-containing really interesting new gene (RING) protein and zinc finger protein (HRZ), are major regulators of Fe homeostasis in diploid species like Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.), but remain uncharacterised in hexaploid bread wheat (Triticum aestivum L.). In this study, we have identified, annotated, and characterised three TaIRO3 homoeologs and six TaHRZ1 and TaHRZ2 homoeologs in the bread wheat genome. Protein analysis revealed that TaIRO3 and TaHRZ proteins contain functionally conserved domains for DNA-binding, dimerisation, Fe binding, or polyubiquitination, and phylogenetic analysis revealed clustering of TaIRO3 and TaHRZ proteins with other monocot IRO3 and HRZ proteins, respectively. Quantitative reverse-transcription PCR analysis revealed that all TaIRO3 and TaHRZ homoeologs have unique tissue expression profiles and are upregulated in shoot tissues in response to Fe deficiency. After 24 h of Fe deficiency, the expression of TaHRZ homoeologs was upregulated, while the expression of TaIRO3 homoeologs was unchanged, suggesting that TaHRZ functions upstream of TaIRO3 in the wheat Fe homeostasis TF network. [ABSTRACT FROM AUTHOR]

Tripartite motif (TRIM) proteins are RING E3 ubiquitin ligases defined by a shared domain structure. Several of them are implicated in rare genetic diseases, and mutations in TRIM32 and TRIM-like malin are associated with Limb-Girdle Muscular Dystrophy R8 and Lafora disease, respectively. These two proteins are evolutionary related, share a common ancestor, and both display NHL repeats at their C-terminus. Here, we revmniew the function of these two related E3 ubiquitin ligases discussing their intrinsic and possible common pathophysiological pathways. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GB) is the most malignant primary brain tumor in humans, with an overall survival of approximatively 15 months. The molecular heterogeneity of GB, as well as its rapid progression, invasiveness and the occurrence of drug-resistant cancer stem cells, limits the efficacy of the current treatments. In order to develop an innovative therapeutic strategy, it is mandatory to identify and characterize new molecular players responsible for the GB malignant phenotype. In this study, the RNA-binding ubiquitin ligase MEX3A was selected from a gene expression analysis performed on publicly available datasets, to assess its biological and still-unknown activity in GB tumorigenesis. We find that MEX3A is strongly up-regulated in GB specimens, and this correlates with very low protein levels of RIG-I, a tumor suppressor involved in differentiation, apoptosis and innate immune response. We demonstrate that MEX3A binds RIG-I and induces its ubiquitylation and proteasome-dependent degradation. Further, the genetic depletion of MEX3A leads to an increase of RIG-I protein levels and results in the suppression of GB cell growth. Our findings unveil a novel molecular mechanism involved in GB tumorigenesis and suggest MEX3A and RIG-I as promising therapeutic targets in GB. [ABSTRACT FROM AUTHOR]

In yeast, the main ubiquitin ligase responsible for the sorting of proteins to the lysosomal vacuole is Rsp5, a member of the Nedd4 family of ligases whose distinguishing features are a catalytic homologous to E6AP C terminus (HECT) domain and 3 central WW domains that bind PY motifs in target proteins. Many substrates do not bind Rsp5 directly and instead rely on PY-containing adaptor proteins that interact with Rsp5. Recent studies indicate that the activities of these adaptors are elevated when they undergo ubiquitination, yet the mechanism whereby ubiquitination activates the adaptors and how this process is regulated remain unclear. Here, we report on a mechanism that explains how ubiquitination stimulates adaptor function and how this process can be regulated by the Rsp5-associated deubiquitinase, Ubp2. Our overexpression experiments revealed that several adaptors compete for Rsp5 in vivo. We found that the ability of the adaptors to compete effectively was enhanced by their ubiquitination and diminished by a block of their ubiquitination. Ubiquitination-dependent adaptor activation required a ubiquitin-binding surface within the Rsp5 catalytic HECT domain. Finally, like constitutively ubiquitinated adaptors, a Ubp2 deficiency increased both the adaptor activity and the ability to compete for Rsp5. Our data support a model whereby ubiquitinated Rsp5 adaptors are more active when "locked" onto Rsp5 via its N-lobe ubiquitin-binding surface and less active when they are "unlocked" by Ubp2-mediated deubiquitination. • The Rsp5 substrate adaptor protein Hua1 requires ubiquitination to function • Rsp5 adaptor proteins compete for Rsp5 function more potently when ubiquitinated • Elevated function of ubiquitinated adaptors depends on a Ub-binding site on Rsp5 • Ubp2 activity may unlock ubiquitinated adaptors from Rsp5 The HECT-type Ub ligase Rsp5 uses adaptor proteins to connect to substrates. MacDonald et al. show that adaptor ubiquitination strengthens association to Rsp5, focusing Rsp5 on cognate substrates while diverting it from non-cognate substrates. The deubiquitinating enzyme, Ubp2, may work to liberate Rsp5 to reprioritize adaptor associations. [ABSTRACT FROM AUTHOR]

Lafora disease (LD, OMIM254780) is a rare and fatal form of progressive myoclonus epilepsy (PME). Among PMEs, LD is unique because of the rapid neurological deterioration of the patients and the appearance in brain and peripheral tissues of insoluble glycogen-like (polyglucosan) inclusions, named Lafora bodies (LBs). LD is caused by mutations in the EPM2A gene, encoding the dual phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Laforin and malin form a functional complex that is involved in the regulation of glycogen synthesis. Thus, in the absence of a functional complex glycogen accumulates in LBs. In addition, it has been suggested that the laforin-malin complex participates in alternative physiological pathways, such as intracellular protein degradation, oxidative stress, and the endoplasmic reticulum unfolded protein response. In this work we review the possible cellular functions of laforin and malin with a special focus on their role in the ubiquitination of specific substrates. We also discuss here the pathological consequences of defects in laforin or malin functions, as well as the therapeutic strategies that are being explored for LD. [ABSTRACT FROM AUTHOR]