Paracrine cell-cell communication is central to all developmental processes, ranging from cell diversification to patterning and morphogenesis. Precise calibration of signaling strength is essential for the fidelity of tissue formation during embryogenesis and tissue maintenance in adults. Membrane-tethered ubiquitin ligases can control the sensitivity of target cells to secreted ligands by regulating the abundance of signaling receptors at the cell surface. We discuss two examples of this emerging concept in signaling: (1) the transmembrane ubiquitin ligases ZNRF3 and RNF43 that regulate WNT and bone morphogenetic protein receptor abundance in response to R-spondin ligands and (2) the membrane-recruited ubiquitin ligase MGRN1 that controls Hedgehog and melanocortin receptor abundance. We focus on the mechanistic logic of these systems, illustrated by structural and protein interaction models enabled by AlphaFold. We suggest that membrane-tethered ubiquitin ligases play a widespread role in remodeling the cell surface proteome to control responses to extracellular ligands in diverse biological processes.

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.

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.

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.

DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 x 10-6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.

Cellular quality control provides an efficient surveillance system to regulate mitochondrial turnover. This study elucidates a new interaction between the cytosolic E3 ligase mahogunin RING finger 1 (MGRN1) and the endoplasmic reticulum (ER) ubiquitin E3 ligase GP78 (also known as AMFR). Loss of Mgrn1 function has been implicated in late-onset spongiform neurodegeneration and congenital heart defects, among several developmental defects. Here, we show that MGRN1 ubiquitylates GP78 in trans through non-canonical K11 linkages. This helps maintain constitutively low levels of GP78 in healthy cells, in turn downregulating mitophagy. GP78, however, does not regulate MGRN1. When mitochondria are stressed, cytosolic Ca(2+) increases. This leads to a reduced interaction between MGRN1 and GP78 and its compromised ubiquitylation. Chelating Ca(2+) restores association between the two ligases and the in trans ubiquitylation. Catalytic inactivation of MGRN1 results in elevated levels of GP78 and a consequential increase in the initiation of mitophagy. This is important because functional depletion of MGRN1 by the membrane-associated disease-causing prion protein (Ctm)PrP affects polyubiquitylation and degradation of GP78, also leading to an increase in mitophagy events. This suggests that MGRN1 participates in mitochondrial quality control and could contribute to neurodegeneration in a subset of (Ctm)PrP-mediated prion diseases.

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.

Polyglutamine diseases are a family of inherited neurodegenerative diseases caused by the expansion of CAG repeats within the coding region of target genes. Still the mechanism(s) by which polyglutamine proteins are ubiquitinated and degraded remains obscure. Here, for the first time, we demonstrate that Mahogunin 21 ring finger 1 E3 ubiquitin protein ligase is depleted in cells that express expanded-polyglutamine proteins. MGRN1 co-immunoprecipitates with expanded-polyglutamine huntingtin and ataxin-3 proteins. Furthermore, we show that MGRN1 is predominantly colocalized and recruits with polyglutamine aggregates in both cellular and transgenic mouse models. Finally, we demonstrate that the partial depletion of MGRN1 increases the rate of aggregate formation and cell death, whereas the overexpression of MGRN1 reduces the frequency of aggregate formation and provides cytoprotection against polyglutamine-induced proteotoxicity. These observations suggest that stimulating the activity of MGRN1 ubiquitin ligase might be a potential therapeutic target to eliminate the cytotoxic threat in polyglutamine diseases.

Plant LOSS OF GDU 2 (LOG2) and Mammalian Mahogunin Ring Finger 1 (MGRN1) proteins are RING-type E3 ligases sharing similarity N-terminal to the RING domain. Deletion of this region disrupts the interaction of LOG2 with the plant membrane protein GLUTAMINE DUMPER1 (GDU1). Phylogenetic analysis identified two clades of LOG2/MGRN1-like proteins in vertebrates and plants. The ability of MGRN1 to functionally replace LOG2 was tested. MGRN1 ubiquitylates GDU1 in vitro and can partially substitute for LOG2 in the plant, partially restoring amino acid resistance to a GDU1-myc over-expression, log2-2 background. Altogether, these results suggest a conserved function for the N-terminal domain in evolution.

While the conversion of the normal form of prion protein to a conformationally distinct pathogenic form is recognized to be the primary cause of prion disease, it is not clear how this leads to spongiform change, neuronal dysfunction and death. Mahogunin ring finger-1 (Mgrn1) and Attractin (Atrn) null mutant mice accumulate vacuoles throughout the brain that appear very similar to those associated with prion disease, but they do not accumulate the protease-resistant scrapie form of the prion protein or become sick. A study demonstrating an interaction between cytosolically-exposed prion protein and MGRN1 suggested that disruption of MGRN1 function may contribute to prion disease pathogenesis, but we recently showed that neither loss of MGRN1 nor MGRN1 overexpression influences the onset or progression of prion disease following intracerebral inoculation with Rocky Mountain Laboratory prions. Here, we show that loss of ATRN also has no effect on prion disease onset or progression and discuss possible mechanisms that could cause vacuolation of the central nervous system in Mgrn1 and Atrn null mutant mice and whether the same pathways might contribute to this intriguing phenotype in prion disease.