Mahogunin Ring Finger-1 Research Articles

MGRN1 depletion promotes intercellular adhesion in melanoma by upregulation of E-cadherin and inhibition of CDC42

Mahogunin Ring Finger 1 is an E3-ubiquitin ligase encoded by the color gene MGRN1. Our previous in vitro and in vivo studies demonstrated that Mgrn1 deletion in mouse melanoma cells induced cell differentiation and adhesion, and decreased cell motility and invasion on collagen I, and lung colonization in an in vivo model. Here, we investigated the role of MGRN1 on human melanoma cell morphology, adhesion and expression of genes/proteins involved in an EMT-like transition. We demonstrated that wild-type BRAF human melanoma cells adopted a clustering-like morphology on collagen I, with permanent MGRN1 abrogation resulting in bigger cell clusters. Enhanced intercellular adhesion was mostly mediated by induction of E-cadherin and higher co-localization with β-catenin. Transcriptional upregulation of E-cadherin likely occurred through downregulation of the ZEB1 repressor. Finally, pulldown assays showed reduced activation of CDC42 in the absence of MGRN1, which was reverted after E-cadherin silencing. Overall, these findings highlight a new MGRN1-dependent pathway regulating melanoma cell shape, motility, and invasion potential.

Distribution and Localization of Mahogunin Ring Finger 1 in the Mouse Central Nervous System.

Mahogunin ring finger 1 (MGRN1), an E3 ubiquitin, is involved in several physiological and neuropathological processes. Although mgrn1 mRNA is widely distributed in the central nervous system (CNS), detailed information on its cellular and subcellular localization is lacking and its physiological role remains unclear. In this study, we aimed to determine the distribution of MGRN1 in the mouse CNS using a newly produced antibody against MGRN1. We found that the MGRN1 protein was expressed in most neuronal cell bodies. An intense MGRN1 expression was also observed in the neuropil of the gray matter in different regions of the CNS, including the main olfactory bulb, cerebral cortex, caudate, putamen, thalamic nuclei, hypothalamic nuclei, medial eminence, superior colliculus, hippocampus, dentate gyrus, and spinal cord. Contrastingly, no MGRN1 expression was observed in glial cells. Double fluorescence and immunoelectron microscopic analyses revealed the intracellular distribution of MGRN1 in pre-synapses and near the outer membrane of the mitochondria in neurons. These findings indicate that MGRN1 is more widely expressed throughout the CNS; additionally, the intracellular expression of MGRN1 suggests that it may play an important role in synaptic and mitochondrial functions.

MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker.

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.

Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells

Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III–IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.

Promoter Methylation of the MGRN1 Gene Predicts Prognosis and Response to Chemotherapy of High-Grade Serous Ovarian Cancer Patients.

Aberrant DNA methylation is considered to play a critical role in the chemoresistance of epithelial ovarian cancer (EOC). In this study, we explored the relationship between hypermethylation of the Mahogunin Ring Finger 1 (MGRN1) gene promoter and primary chemoresistance and clinical outcomes in high-grade serous ovarian cancer (HGSOC) patients. The MALDI-TOF mass spectrometry assays revealed a strong association between hypermethylation of the MGRN1 upstream region and platinum resistance in HGSOC patients. Spearman’s correlation analysis revealed a significantly negative connection between the methylation level of MGRN1 and its expression in HGSOC. In vitro analysis demonstrated that knockdown of MGRN1 reduced the sensitivity of cells to cisplatin and that expression of EGR1 was significantly decreased in SKOV3 cells with low levels of MGRN1 expression. Similarly, EGR1 mRNA expression was lower in platinum-resistant HGSOC patients and was positively correlated with MGRN1 mRNA expression. Kaplan-Meier analyses showed that high methylation of the MGRN1 promoter region and low expression of MGRN1 were associated with worse survival of HGSOC patients. In multivariable models, low MGRN1 expression was an independent factor predicting poor outcome. Furthermore, low expression of EGR1 was also been confirmed to be significantly related to the poor prognosis of HGSOC patients by Kaplan-Meier. The hypermethylation of the MGRN1 promoter region and low expression of MGRN1 were associated with platinum resistance and poor outcomes in HGSOC patients, probably by altering EGR1 expression.

The conserved MASRPF motif in the Attractin homolog, Distracted, is required for association with Drosophila E3-ligase Mgrn1.

In rodents, all three paralogs of the Attractin (Atrn) transmembrane protein family exhibit strong phenotypic overlap and are implicated in the regulation of the same G-protein coupled receptors (GPCR) as E3-ligase Mahogunin ring finger 1 (Mgrn1). Recently it was shown that the highly conserved intracellular MASRPF motif in mammal Multiple epidermal growth factor-like domain 8 protein is required for binding of Mgrn1 to mediate ubiquitination of GPCR Smoothened in vitro. Here, we show that the MASRPF motif of Drosophila Distracted, the ortholog of ATRN and Attractin-like 1, is required for association with Drosophila Mgrn1 (dMgrn1) in vivo.

Mahogunin Ring Finger 1 Is Required for Genomic Stability and Modulates the Malignant Phenotype of Melanoma Cells.

Simple SummaryMelanoma, the most aggressive skin cancer, accounts for the majority of deaths due to this disease. Therefore, identification of genes/proteins involved in melanoma genesis and/or progression is urgent. Mutations abrogating expression of Mahogunin Ring Finger 1 (MGRN1) in mice cause complex phenotypes with hyperpigmentation, and known MGRN1 interactors are important regulators of cell shape and movement. This suggests that MGRN1 may modulate the malignant phenotype of melanoma cells. Analysis of MGRN1-KO mouse melanocytes and melanoma cells showed that lack of MGRN1 leads to cell cycle defects and to a more differentiated, less aggressive phenotype, with increased adhesion to various matrices, decreased motility and high genomic instability. The higher aggressivity of MGRN1-expressing melanoma cells was confirmed in an in vivo mouse melanoma model and is consistent with higher survival of human melanoma patients expressing low levels of MGRN1. Therefore, MGRN1 appears an important determinant of the malignant phenotype of melanoma.The mouse mahoganoid mutation abrogating Mahogunin Ring Finger-1 (MGRN1) E3 ubiquitin ligase expression causes hyperpigmentation, congenital heart defects and neurodegeneration. To study the pathophysiology of MGRN1 loss, we compared Mgrn1-knockout melanocytes with genetically matched controls and melan-md1 (mahoganoid) melanocytes. MGRN1 knockout induced a more differentiated and adherent phenotype, decreased motility, increased the percentage of cells in the S phase of the cell cycle and promoted genomic instability, as shown by stronger γH2AX labelling, increased burden of DNA breaks and higher abundance of aneuploid cells. Lack of MGRN1 expression decreased the ability of melanocytes to cope with DNA breaks generated by oxidizing agents or hydroxyurea-induced replicative stress, suggesting a contribution of genomic instability to the mahoganoid phenotype. MGRN1 knockout in B16-F10 melanoma cells also augmented pigmentation, increased cell adhesion to collagen, impaired 2D and 3D motility and caused genomic instability. Tumors formed by Mgrn1-KO B16-F10 cells had lower mitotic indices, fewer Ki67-positive cells and showed a trend towards smaller size. In short-term lung colonization assays Mgrn1-KO cells showed impaired colonization potential. Moreover, lower expression of MGRN1 is significantly associated with better survival of human melanoma patients. Therefore, MGRN1 might be an important phenotypic determinant of melanoma cells.

The Role of gp91phox and the Effect of Tranexamic Acid Administration on Hair Color in Mice.

We observed that on long-term breeding, gp91phox-knockout (gp91phox−/−) mice developed white hair. Here, we investigate the origin of this hitherto unexplained phenomenon. Moreover, we investigated the effect of tranexamic acid administration on the hair color in gp91phox−/− mice. We administered tranexamic acid (about 12 mg/kg/day) orally to 9-week-old C57BL/6j (control) and gp91phox−/− mice, thrice a week for 12 months. Compared to control mice, gp91phox−/− mice showed more white hair. However, the concentrations of reactive oxygen species and the levels of interleukin (IL)-1β and transforming growth factor (TGF)-β in the skin were lower than those in the control group. Furthermore, increase in white hair was observed in the control mice upon administration of the IL-1β antagonist. On the other hand, administration of tranexamic acid led to brown colored hair on gp91phox−/− mice. Although tranexamic acid treatment did not alter the expression levels of melanocortin receptor 1 and agouti signaling protein on hair follicles, it increased the expression of mahogunin ring finger protein 1 (MGRN1) and collagen XVII. These results suggested that retention of black hair requires the gp91phox/ROS/IL-1β/TGF-β pathway and that elevated levels of MGRN1 and collagen XVII lead to brown hair in gp91phox−/− mice.

Human melanocortin 1 receptor-mediated ubiquitination of nonvisual arrestins. Role of Mahogunin Ring Finger 1 E3 ligase

Signaling from the melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) crucial for melanocyte proliferation and differentiation, is regulated by cytosolic β-arrestins (ARRBs). MC1R signaling is also negatively modulated by the E3-ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1), whose mutation causes hyperpigmentation, congenital heart defects and neurodegeneration in mice. We showed previously that although MC1R interacts stably with human ARRB1 or ARRB2, only ARRB2 mediates receptor desensitization and internalization. We analyzed MC1R-dependent ARRB ubiquitination, and the possible role of MGRN1. ARRB1 expressed in heterologous cells or human melanoma cells migrated in SDS-PAGE as a 55kDa protein whereas ARRB2 migrated as two major bands of apparent molecular weight near 45 and 55kDa, with an intermediate mobility band occasionally detected. These forms were related by post-translational modification rather than by proteolysis. Presence of MC1R favored expression of the 45kDa protein, the form that interacted preferentially with MC1R. MC1R also mediated poly- or multimonoubiquitination of ARRB2. Ubiquitination was agonist-independent, but required a native MC1R conformation and/or normal receptor trafficking to the plasma membrane, as it was not observed for loss-of-function MC1R variants. In a heterologous expression system, MC1R-dependent ARRB ubiquitination was enhanced by overexpression of MGRN1 and was impaired by siRNA-mediated MGRN1 knockdown thus pointing to MGRN1 as the responsible E3-ligase. Co-immunoprecipitation experiments demonstrated interaction of MGRN1 and ARRBs in the presence of MC1R, suggesting a scaffolding role for the GPCR that may determine the selectivity of E3-ubiquitin ligase engagement and the functional outcome of ARRB ubiquitination.

Tumor susceptibility gene 101 regulates predisposition to apoptosis via ESCRT machinery accessory proteins.

ESCRT proteins are implicated in myriad cellular processes, including endosome formation, fusion of autophagosomes/amphisomes with lysosomes, and apoptosis. The role played by these proteins in either facilitating or protecting against apoptosis is unclear. In this study, while trying to understand how deficiency of Mahogunin RING finger 1 (MGRN1) affects cell viability, we uncovered a novel role for its interactor, the ESCRT-I protein TSG101: it directly participates in mitigating ER stress-mediated apoptosis. The association of TSG101 with ALIX prevents predisposition to apoptosis, whereas ALIX-ALG-2 interaction favors a death phenotype. Altered Ca2+ homeostasis in cells and a simultaneous increase in the protein levels of ALIX and ALG-2 are required to elicit apoptosis by activating ER stress-associated caspase 4/12. We further demonstrate that in the presence of membrane-associated, disease-causing prion protein CtmPrP, increased ALIX and ALG-2 levels are detected along with ER stress markers and associated caspases in transgenic brain lysates and cells. These effects were rescued by overexpression of TSG101. This is significant because MGRN1 deficiency is closely associated with neurodegeneration and prenatal and neonatal mortality, which could be due to excess cell death in selected brain regions or myocardial apoptosis during embryonic development.

Chronicle of a Neuronal Death Foretold: Preventing Aging by Keeping MGRN1 at the Nucleus

In this issue of Molecular Cell, Benvegnù etal. (2017) report an unexpected phenomenon by which the E3 ligase mahogunin ring finger-1 (MGRN1) translocates to the nucleus in an age-dependent manner, revealing an intriguing mechanism that allows for an adaptive neuronal response to proteotoxic stress, often seen with aging.

Regulation of Mitofusin1 by Mahogunin Ring Finger-1 and the proteasome modulates mitochondrial fusion

Health and homoeostasis are maintained by a dynamic balance between mitochondrial fission and fusion. Mitochondrial fusion machinery is largely unknown in mammals. Only a few reports have illustrated the role of Fzo1 in mitochondrial fusion known in Saccharomyces cerevisiae. We demonstrate that the ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) interacts with and constitutively ubiquitinates the mammalian homolog, Mitofusin1 (Mfn1) via K63 linkages. In mice models, loss of Mgrn1 function leads to severe developmental defects and adult-onset spongiform neurodegeneration, similar to prion diseases. The tethering of mitochondria to form the ~180kDa Mfn1 complex is independent of MGRN1-mediated ubiquitination. However, successful mitochondrial fusion requires formation of higher oligomers of Mfn1 which in turn needs GTPase activity, intact heptad repeats of Mfn1 and ubiquitination by MGRN1. Following ubiquitination, proteasomal processing of Mfn1 completes the mitochondrial fusion process. This step requires functional p97 activity. These findings suggest a sequence of events where GTPase activity of Mfn1 and tethering of adjacent mitochondria precedes its MGRN1-mediated ubiquitination and proteasomal degradation culminating in mitochondrial fusion.

Oligodendroglial deletion of ESCRT-I component TSG101 causes spongiform encephalopathy.

Vacuolation of the central nervous system (CNS) is observed in patients with transmissible spongiform encephalopathy, HIV-related encephalopathy and some inherited diseases, but the underlying cellular mechanisms remain poorly understood. Mice lacking the mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase develop progressive, widespread spongiform degeneration of the CNS. MGRN1 ubiquitinates and regulates tumour susceptibility gene 101 (TSG101), a central component of the endosomal trafficking machinery. As loss of MGRN1 is predicted to cause partial TSG101 loss-of-function, we hypothesised that CNS vacuolation in Mgrn1 null mice may be caused by the accumulation of multi-cisternal endosome-like 'class E' vacuolar protein sorting (vps) compartments similar to those observed in Tsg101-depleted cells in culture. To test this hypothesis, Tsg101 was deleted from mature oligodendroglia in vivo. This resulted in severe spongiform encephalopathy, histopathologically similar to that observed in Mgrn1 null mutant mice but with a more rapid onset. Vacuoles in the brains of Tsg101-deleted and Mgrn1 mutant mice labelled with endosomal markers, consistent with an endosomal origin. Vacuoles in the brains of mice inoculated with Rocky Mountain Laboratory (RML) prions did not label with these markers, indicating a different origin, consistent with previously published studies that indicate RML prions have a primary effect on neurons and cause vacuolation in an MGRN1-independent manner. Oligodendroglial deletion of Rab7, which mediates late endosome-to-lysosome trafficking and autophagosome-lysosome fusion, did not cause spongiform change. Our data suggest that the formation of multi-cisternal 'class E' vps endosomal structures in oligodendroglia leads to vacuolation. This work provides the first evidence that disrupting multi-vesicular body formation in oligodendroglia can cause white matter vacuolation and demyelination. HIV is known to hijack the endosomal sorting machinery, suggesting that HIV infection of the CNS may also act through this pathway to cause encephalopathy.

ESCRTs and associated proteins in lysosomal fusion with endosomes and autophagosomes.

Endolysosomal and autophagosomal degradation pathways are highly connected at various levels, sharing multiple molecular effectors that modulate them individually or simultaneously. These two lysosomal degradative pathways are primarily involved in the disposal of cargo internalized from the cell surface or long-lived proteins or aggregates and aged organelles present in the cytosol. Both of these pathways involve a number of carefully regulated vesicular fusion events that are dependent on ESCRT proteins. The ESCRT proteins especially ESCRT-I and III participate in the regulation of fusion events between autophagosome/amphisome and lysosome. Along with these, a number of functionally diverse ESCRT associated and regulatory proteins such as, endosomal PtdIns (3) P 5-kinase Fab1, ALIX, mahogunin ring finger 1, atrogin 1, syntaxin 17, ATG12-ATG3 complex, and protein kinase CK2α are involved in fusion events in either or both the lysosomal degradative pathways.

Mahogunin-mediated regulation of Gαi localisation during mitosis and its effect on spindle positioning.

Mahogunin RING Finger 1 (MGRN1) is a ubiquitin E3 ligase known to affect spindle tilt in mitotic cells by regulating α-tubulin ubiquitination and polymerization. In cell culture systems we have found that expressing truncated mutants of MGRN1 leads to various other mitotic anomalies, such as lateral and angular spindle displacements. This seems to be independent of the MGRN1 ligase activity. Our experiments suggest that MGRN1 regulates the balance between the lower molecular weight monomeric Gαi and larger trimeric G-protein complex, along with its abundance in the ternary complex that regulates spindle positioning. The cytosolic isoforms of MGRN1 lead to the enrichment of monomeric Gαi in the cytosol and its subsequent recruitment at the plasma membrane. Excess Gαi at the cell cortex results in an imbalance in the assembly of the ternary complex regulating spindle positioning during mitosis. These observations seem independent of the ligase activity of MGRN1, although we cannot exclude the involvement of an intermediate player that acts as a substrate for MGRN1, and in turn, regulates Gαi.

Mahogunin ring finger 1 confers cytoprotection against mutant SOD1 aggresomes and is defective in an ALS mouse model

Proteotoxicity of misfolded, disease-causing proteins is deeply implicated in the pathomechanisms for neurodegenerative diseases including copper-zinc superoxide dismutase (SOD1)-linked amyotrophic lateral sclerosis (ALS). However, the precise cellular quality control (QC) mechanisms against aggregation of misfolded mutant SOD1 proteins remain elusive. Here, we found that the Mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase, which catalyzes mono-ubiquitination to the substrate, was dysregulated in the cellular and mouse models of ALS and that it preferentially interacted with various mutant forms of SOD1. Intriguingly, the motor neurons of presymptomatic ALS mice have diminished MGRN1 cytoplasmic distribution. MGRN1 was partially recruited to mutant SOD1 inclusions where they were positive for p62 and Lamp2. Moreover, overexpression of MGRN1 reduced mutant SOD1 aggregation and alleviated its proteotoxic effects on cells. Taken together, our findings suggest that MGRN1 contributes to the clearance of toxic mutant SOD1 inclusions likely through autophagic pathway, and, most likely, the sequestration of MGRN1 sensitizes motor neurons to degeneration in the ALS mouse model. Furthermore, the present study identifies the MGRN1-mediated protein QC mechanism as a novel therapeutic target in neurodegenerative diseases.

Mahogunin regulates fusion between amphisomes/MVBs and lysosomes via ubiquitination of TSG101.

Aberrant metabolic forms of the prion protein (PrP), membrane-associated CtmPrP and cytosolic (cyPrP) interact with the cytosolic ubiquitin E3 ligase, Mahogunin Ring Finger-1 (MGRN1) and affect lysosomes. MGRN1 also interacts with and ubiquitinates TSG101, an ESCRT-I protein, involved in endocytosis. We report that MGRN1 modulates macroautophagy. In cultured cells, functional depletion of MGRN1 or overexpression of CtmPrP and cyPrP blocks autophagosome–lysosome fusion, alleviates the autophagic flux and its degradative competence. Concurrently, the degradation of cargo from the endo-lysosomal pathway is also affected. This is significant because catalytic inactivation of MGRN1 alleviates fusion of lysosomes with either autophagosomes (via amphisomes) or late endosomes (either direct or mediated through amphisomes), without drastically perturbing maturation of late endosomes, generation of amphisomes or lysosomal proteolytic activity. The compromised lysosomal fusion events are rescued by overexpression of TSG101 and/or its monoubiquitination in the presence of MGRN1. Thus, for the first time we elucidate that MGRN1 simultaneously modulates both autophagy and heterophagy via ubiquitin-mediated post-translational modification of TSG101.

Mahogunin Ring Finger-1 (MGRN1), a Multifaceted Ubiquitin Ligase: Recent Unraveling of Neurobiological Mechanisms.

In healthy cell, inappropriate accumulation of poor or damaged proteins is prevented by cellular quality control system. Autophagy and ubiquitin proteasome system (UPS) provides regular cytoprotection against proteotoxicity induced by abnormal or disruptive proteins. E3 ubiquitin ligases are crucial components in this defense mechanism. Mahogunin Ring Finger-1 (MGRN1), an E3 ubiquitin ligase of the Really Interesting New Gene (RING) finger family, plays a pivotal role in many biological and cellular mechanisms. Previous findings indicate that lack of functions of MGRN1 can cause spongiform neurodegeneration, congenital heart defects, abnormal left-right patterning, and mitochondrial dysfunctions in mice brains. However, the detailed molecular pathomechanism of MGRN1 in cellular functions and diseases is not well known. This article comprehensively represents the molecular nature, characterization, and functions of MGRN1; we also summarize possible beneficiary aspects of this novel E3 ubiquitin ligase. Here, we review recent literature on the role of MGRN1 in the neuro-pathobiological mechanisms, with precise focus on the processes of neurodegeneration, and thereby propose new lines of potential targets for therapeutic intervention.

Regulation of neuronal survival and morphology by the E3 ubiquitin ligase RNF157.

Neuronal health is essential for the long-term integrity of the brain. In this study, we characterized the novel E3 ubiquitin ligase ring finger protein 157 (RNF157), which displays a brain-dominant expression in mouse. RNF157 is a homolog of the E3 ligase mahogunin ring finger-1, which has been previously implicated in spongiform neurodegeneration. We identified RNF157 as a regulator of survival in cultured neurons and established that the ligase activity of RNF157 is crucial for this process. We also uncovered that independently of its ligase activity, RNF157 regulates dendrite growth and maintenance. We further identified the adaptor protein APBB1 (amyloid beta precursor protein-binding, family B, member 1 or Fe65) as an interactor and proteolytic substrate of RNF157 in the control of neuronal survival. Here, the nuclear localization of Fe65 together with its interaction partner RNA-binding protein SART3 (squamous cell carcinoma antigen recognized by T cells 3 or Tip110) is crucial to trigger apoptosis. In summary, we described that the E3 ligase RNF157 regulates important aspects of neuronal development.

Mahogunin-mediated α-tubulin ubiquitination via noncanonical K6 linkage regulates microtubule stability and mitotic spindle orientation

Mahogunin ring finger-1 (MGRN1) is a cytosolic ubiquitin ligase whose disruption or interaction with some isoforms of cytosolically exposed prion protein leads to spongiform neurodegeneration and also lack of which results in reduced embryonic viability due to mispatterning of the left–right (LR) axis during development. Here we demonstrate an interaction between the cytoskeletal protein α-tubulin and MGRN1. In cultured cell systems, loss of the ubiquitin E3 ligase activity of MGRN1 results in spindle misorientation and decreased α-tubulin polymerization, an effect also seen in primary cells. α-Tubulin was post-translationally modified by MGRN1 via noncanonical K6-linked polyubiquitination. This was significant because expression of catalytically inactive MGRN1 and/or ubiquitin mutant capable of only monoubiquitination resulted in similar mitotic spindle misorientation. The modulatory effect of MGRN1 was specific for α-tubulin and similar changes could not be detected in β- or γ-tubulin. However, catalytic inactivation of MGRN1 did not abrogate monoubiquitination of α-tubulin, thus unraveling a unique dual mode of ubiquitination by an unknown E3 ligase and MGRN1. MGRN1-mediated α-tubulin modification, and hence its stability, may highlight a key event in the LR patterning during embryogenesis.