Makorin Ring Finger Protein 1 Research Articles

SF3A2 promotes progression and cisplatin resistance in triple-negative breast cancer via alternative splicing of MKRN1.

Triple-negative breast cancer (TNBC) is the deadliest subtype of breast cancer owing to the lack of effective therapeutic targets. Splicing factor 3a subunit 2 (SF3A2), a poorly defined splicing factor, was notably elevated in TNBC tissues and promoted TNBC progression, as confirmed by cell proliferation, colony formation, transwell migration, and invasion assays. Mechanistic investigations revealed that E3 ubiquitin-protein ligase UBR5 promoted the ubiquitination-dependent degradation of SF3A2, which in turn regulated UBR5, thus forming a feedback loop to balance these two oncoproteins. Moreover, SF3A2 accelerated TNBC progression by, at least in part, specifically regulating the alternative splicing of makorin ring finger protein 1 (MKRN1) and promoting the expression of the dominant and oncogenic isoform, MKRN1-T1. Furthermore, SF3A2 participated in the regulation of both extrinsic and intrinsic apoptosis, leading to cisplatin resistance in TNBC cells. Collectively, these findings reveal a previously unknown role of SF3A2 in TNBC progression and cisplatin resistance, highlighting SF3A2 as a potential therapeutic target for patients with TNBC.

MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation

BackgroundThe Makorin ring finger protein 1 (MKRN1) gene, also called RNF61, is located on the long arm of chromosome 7 and is a member of the RING finger protein family. The E3 ubiquitin ligase MKRN1 is closely linked to tumour development, but the exact mechanism needs to be elucidated. In this study, we aimed to investigate the specific mechanism and role of MKRN1 in colorectal cancer (CRC) development.MethodsMKRN1 expression in CRC was analysed using the Cancer Cell Line Encyclopaedia and the Cancer Genome Atlas (TCGA) databases. Rectal tumour tissues were frozen to explore the MKRN1 expression in CRC and its clinical significance. The impact of MKRN1 on CRC cell proliferation and migration was observed using CCK8, colony formation, wound healing, and transwell assays. A combination of MKRN1 quantitative proteomics, ubiquitination modification omics analysis, and a string of in vitro and in vivo experiments revealed the potential mechanisms by which MKRN1 regulates CRC metastasis.ResultsMKRN1 expression was significantly elevated in CRC tissues compared to paracancerous tissues and was positively linked with prognosis (P < 0.01). MKRN1 downregulation inhibits CRC cell proliferation, migration, and invasion. Conversely, MKRN1 overexpression promotes the proliferation, migration, and invasion of CRC cells. Mechanistically, MKRN1 induces epithelial-mesenchymal transition (EMT) in CRC cells via ubiquitination and degradation of Smad nuclear-interacting protein 1 (SNIP1). Furthermore, SNIP1 inhibits transforming growth factor-β (TGF-β) signalling, and MKRN1 promotes TGF-β signalling by degrading SNIP1 to induce EMT in CRC cells. Finally, using conditional knockout mice, intestinal lesions and metastatic liver microlesions were greatly reduced in the intestinal knockout MKRN1 group compared to that in the control group.ConclusionsHigh MKRN1 levels promote TGF-β signalling through ubiquitination and degradation of SNIP1, thereby facilitating CRC metastasis, and supporting MKRN1 as a CRC pro-cancer factor. The MKRN1/SNIP1/TGF-β axis may be a potential therapeutic target in CRC.

Host MKRN1-Mediated Mycobacterial PPE Protein Ubiquitination Suppresses Innate Immune Response.

Mycobacterium tuberculosis (Mtb), as an important intracellular pathogen, can invade and survive in macrophages and is capable of escaping the clearance of immune system. Despite decades of research efforts, the precise mechanism of immune escape and the virulence factors encoded by Mtb involved remain to be explored. Mtb-specific genomic regions of deletion (RD)-encoded proteins and PE/PPE family proteins have been implicated in immune evasion. Here, we screened more than forty RD-encoded proteins which might be involved in facilitating bacterial survival in macrophages, and found that a Mtb PPE68/Rv3873 protein, encoded by Mtb-RD1, is essential for efficient Mtb intracellular survival in macrophages. In terms of mechanism, we found that the ubiquitin ligase (E3) Makorin Ring Finger Protein 1 (MKRN1) of macrophage interacted with PPE68 and promoted the attachment of lysine (K)-63-linked ubiquitin chains to the K166 site of PPE68. K63-ubiquitination of PPE68 further bound src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) to suppress K63-linked polyubiquitin chains of tumor necrosis factor receptor-associated factor 6 (TRAF6), and then remarkably suppressed TRAF6-driven NF-κB and AP-1 signaling and TNF-α, IL-6 and NO production. We demonstrate that the K63-linked ubiquitination of PPE68 by MKRN1 contributed to the PPE68-mediated mycobacterial immune escape. Our finding identifies a previously unrecognized mechanism by which host MKRN1-mediated-ubiquitination of mycobacterial PPE protein suppresses innate immune responses. Disturbing the interaction between host MKRN1 ubiquitin system and mycobacterial PPE protein might be a potential therapeutic target for tuberculosis.

Hyperoside Ameliorates DSS-Induced Colitis through MKRN1-Mediated Regulation of PPARγ Signaling and Th17/Treg Balance.

Hyperoside (HYP), a naturally occurring flavonoid compound, exerts multiple biological functions including myocardial protection, antiredox, and anti-inflammatory activities. However, the role of HYP on inflammatory bowel disease (IBD) and the underlying mechanism need to be further established. Here, we show that HYP treatment profoundly alleviated dextran sulfate sodium-induced ulcerative colitis in mice, characterized by reduced pathological scores, preserved tissue integrity, suppressed colonic inflammation, and balanced Th17/Treg response. Mechanistically, HYP was shown to restrain the expression of the E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), which in turn promoted the ubiquitination and proteasomal degradation of peroxisome proliferator-activated receptor gamma (PPARγ), an essential regulator of Th17 and Treg differentiation. Consequently, HYP treatment enhanced PPARγ signaling and hence promoted Treg differentiation while suppressing Th17 cell development during colitis. Thus, our data indicate that HYP acts through the MKRN1/PPARγ axis to modulate the Th17/Treg axis and thereby confers protection against experimental colitis. The findings extend our understanding about HYP action and may provide a potential therapeutic target for IBD.

MKRN1 Ubiquitylates p21 to Protect against Intermittent Hypoxia-Induced Myocardial Apoptosis.

Although chronic intermittent hypoxia- (IH-) induced myocardial apoptosis is an established pathophysiological process resulting in a poor prognosis for patients with obstructive sleep apnea syndrome, its underlying mechanism remains unclear. This study is aimed at exploring the role of makorin ring finger protein 1 (MKRN1) in IH-induced myocardial apoptosis and elucidating its molecular activity. First, the GSE2271 dataset was downloaded from the Gene Expression Omnibus database to identify the differentially expressed genes. Then, an SD rat model of IH, together with rat cardiomyocyte H9C2 and human cardiomyocyte AC16 IH models, was constructed. TUNEL, Western blot, and immunohistochemistry assays were used to detect cell apoptosis. Dihydroethidium staining was conducted to analyze the concentration of reactive oxygen species. In addition, RT-qPCR, Western blot, and immunohistochemistry were performed to measure the expression levels of MKRN1 and p21. The direct interaction between MKRN1 and p21 was determined using coimmunoprecipitation and ubiquitination analysis. MKRN1 expression was found to be downregulated in IH rat myocardial tissues as well as in H9C2 and AC16 cells. Upregulated expression of MKRN1 in H9C2 and AC16 cells alleviated the IH-induced reactive oxygen species production and cell apoptosis. Mechanistically, MKRN1 promoted p21 protein ubiquitination and the proteasome pathway degradation to negatively regulate p21 expression. Thus, MKRN1 regulates p21 ubiquitination to prevent IH-induced myocardial apoptosis.

Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K+ channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K+ channels, however, remain unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent nonglycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, nonglycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.

In vitro ubiquitination of Mycobacterium tuberculosis by E3 ubiquitin ligase, MKRN1.

Ubiquitination has a role as a host defense mechanism against pathogens. To channelize autophagic mycobacteria to destruction, ubiquitin ligase like, Makorin Ring Finger Protein 1 (MKRN1) was speculated to play a role in ubiquitinating M. tuberculosis. We have developed a flow cytometry based in vitro ubiquitin ligase assay to understand the role of MKRN1 in ubiquitinating mycobacteria and confirmed the results by western blotting. MKRN1 was cloned and expressed in E. coli BL21 (DE3) strain. The recombinant MKRN1 protein was solubilised, purified and refolded to restore the activity. In addition, through autoubiquitination assay, the activity of protein was confirmed. The corresponding E1 and E2 enzymes for MKRN1, UBE1 and UBE2D3 respectively, were selected using BioGrid tool. Surprisingly, flow cytometric assay revealed that at a concentration of 300nM of MKRN1, 38% of M. tuberculosis was found to be ubiquitinated in vitro with 3.5% of the cells havingbound MKRN1. Immunoblot results also substantiates the ubiquitination of M. tuberculosis. MKRN1 did not ubiquitinate B. Subtilis and therefore, we speculate that the E3 Ub ligase activity might be specific to M. tuberculosis. This clearly demonstrates that recombinant MKRN1 ubiquitinates M. tuberculosis which opens up a novel, potential role of MKRN1 against mycobacteria which has to be unfolded.

The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

BackgroundCells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via ribosome-associated quality control.ResultsHere, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during ribosome-associated quality control. We show that MKRN1 directly binds to the cytoplasmic poly(A)-binding protein (PABPC1) and associates with polysomes. MKRN1 is positioned upstream of poly(A) tails in mRNAs in a PABPC1-dependent manner. Ubiquitin remnant profiling and in vitro ubiquitylation assays uncover PABPC1 and ribosomal protein RPS10 as direct ubiquitylation substrates of MKRN1.ConclusionsWe propose that MKRN1 mediates the recognition of poly(A) tails to prevent the production of erroneous proteins from prematurely polyadenylated transcripts, thereby maintaining proteome integrity.

Attenuating MKRN1 E3 ligase-mediated AMPKα suppression increases tolerance against metabolic stresses in mice.

The 5' adenosine monophosphate-activated protein kinase (AMPK) is an essential energy sensor in the cell, which, at low energy levels, instigates the cellular energy-generating systems along with suppression of the anabolic signaling pathways. The activation of AMPK through phosphorylation is a well-known process; however, activation alone is not sufficient, and knowledge about the other regulatory networks of post-translational modifications connecting the activities of AMPK to systemic metabolic syndromes is important, which is still lacking. The recent studies on Makorin Ring Finger Protein 1 (MKRN1) mediating the ubiquitination and proteasome-dependent degradation of AMPK( implicate that the post-translational modification of AMPK, regulating its protein homeostasis, could impose significant systemic metabolic effects (Lee et al. Nat Commun 9:3404). In this study, MKRN1 was identified as a novel E3 ligase for both AMPKα1 and α2. Mouse embryonic fibroblasts, genetically deleted for Mkrmn1, and Ampkα1 and α2, became stabilized with the suppression of lipogenesis pathways and an increase in nutrient consumption and mitochondria regeneration. Of note, the Mkrn1 knockout mice fed normal chow displayed no obvious phenotypic defects or abnormality, whereas the Mkrn1-null mice exhibited strong tolerance to metabolic stresses induced by high-fat diet (HFD). Thus, these mice, when compared with the HFD-induced wild type, were resistant to obesity, diabetes, and non-alcoholic fatty liver disease. Interestingly, in whole-body Mkrn1 knockout mouse, only the liver and white and brown adipose tissues displayed anincrease in the active phosphorylated AMPK levels, but no other organs, such as the hypothalamus, skeletal muscles, or pancreas, displayed such increases. Specific ablation of MKRN1 in the mouse liver using adenovirus prevented HFD-induced lipid accumulation in the liver and blood, implicating MKRN1 as a possible therapeutic target for metabolic syndromes, such as obesity, type II diabetes, and fat liver diseases. This study would provide a crucial perspective on the importance of post-translational regulation of AMPK in metabolic pathways and will help researchers develop novel therapeutic strategies that target not only AMPK but also its regulators.

Loss of the E3 ubiquitin ligase MKRN1 represses diet-induced metabolic syndrome through AMPK activation

AMP-activated protein kinase (AMPK) plays a key role in controlling energy metabolism in response to physiological and nutritional status. Although AMPK activation has been proposed as a promising molecular target for treating obesity and its related comorbidities, the use of pharmacological AMPK activators has been met with contradictory therapeutic challenges. Here we show a regulatory mechanism for AMPK through its ubiquitination and degradation by the E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1). MKRN1 depletion promotes glucose consumption and suppresses lipid accumulation due to AMPK stabilisation and activation. Accordingly, MKRN1-null mice show chronic AMPK activation in both liver and adipose tissue, resulting in significant suppression of diet-induced metabolic syndrome. We demonstrate also its therapeutic effect by administering shRNA targeting MKRN1 into obese mice that reverses non-alcoholic fatty liver disease. We suggest that ubiquitin-dependent AMPK degradation represents a target therapeutic strategy for metabolic disorders.

SF3B1 deficiency impairs human erythropoiesis via activation of p53 pathway: implications for understanding of ineffective erythropoiesis in MDS

BackgroundSF3B1 is a core component of splicing machinery. Mutations in SF3B1 are frequently found in myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ringed sideroblasts (RARS), characterized by isolated anemia. SF3B1 mutations have been implicated in the pathophysiology of RARS; however, the physiological function of SF3B1 in erythropoiesis remains unknown.MethodsshRNA-mediated approach was used to knockdown SF3B1 in human CD34+ cells. The effects of SF3B1 knockdown on human erythroid cell differentiation, cell cycle, and apoptosis were assessed by flow cytometry. RNA-seq, qRT-PCR, and western blot analyses were used to define the mechanisms of phenotypes following knockdown of SF3B1.ResultsWe document that SF3B1 knockdown in human CD34+ cells leads to increased apoptosis and cell cycle arrest of early-stage erythroid cells and generation of abnormally nucleated late-stage erythroblasts. RNA-seq analysis of SF3B1-knockdown erythroid progenitor CFU-E cells revealed altered splicing of an E3 ligase Makorin Ring Finger Protein 1 (MKRN1) and subsequent activation of p53 pathway. Importantly, ectopic expression of MKRN1 rescued SF3B1-knockdown-induced alterations. Decreased expression of genes involved in mitosis/cytokinesis pathway including polo-like kinase 1 (PLK1) was noted in SF3B1-knockdown polychromatic and orthochromatic erythroblasts comparing to control cells. Pharmacologic inhibition of PLK1 also led to generation of abnormally nucleated erythroblasts.ConclusionsThese findings enabled us to identify novel roles for SF3B1 in human erythropoiesis and provided new insights into its role in regulating normal erythropoiesis. Furthermore, these findings have implications for improved understanding of ineffective erythropoiesis in MDS patients with SF3B1 mutations.

Makorin Ring Finger Protein 1 as Adjunctive Marker in Liquid-based Cervical Cytology.

To assess the utility of makorin ring finger protein 1 (MKRN1) as a marker of cervical pathology.A PROspective specimen collection and retrospective Blinded Evaluation study was conducted. Liquid-based cytology samples were collected from 187 women, embedding all residuals as cell blocks for immunohistochemical staining of MKRN1 and P16 INK4a. Results of liquid-based cervical cytology, immunostained cell block sections, and human papillomavirus (HPV) hybrid capture (with real-time polymerase chain reaction) were analyzed. Clinical outcomes were analyzed overall and in subsets of specimens yielding atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesions.Makorin ring finger protein 1 positivity and grades (1–3) of cervical intraepithelial neoplasia (CIN) increased in tandem (CIN1, 32.4%; CIN2, 60.0%; and CIN3, 80.0%), reaching 92.3% in invasive cancer. Sensitivity, specificity, positive predictive value, and negative predictive value in detecting CIN2+ via MKRN1 were 73.8%, 76.8%, 75.6%, and 75.0%, respectively. The performance of liquid-based cytology was poorer by comparison (61.3%, 69.5%, 66.2%, and 64.8%, respectively), and HPV assay (versus MKRN1 immunohistochemical staining) displayed lower specificity (67.7%). Combined HPV + MKRN1 testing proved highest in sensitivity, specificity, positive predictive value, and negative predictive value (71.8%, 85.5%, 82.3%, and 76.5%, respectively), whereas corresponding values for cytology + HPV (60.6%, 81.8%, 75.4%, and 69.2%) and cytology + MKRN1 (58.8%, 84.1%, 78.3%, and 67.7%) were all similar. In instances of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesions, the HPV + MKRN1 combination performed best by above measures (100%, 72.7%, 73.9%, and 100%), followed by cytology + MKRN1 (100%, 50.0%, 60.7%, and 100%).Makorin ring finger protein 1 displayed greater sensitivity and specificity than liquid-based cytology and proved more specific than HPV assay. In combination testing, MKRN1 + HPV showed the highest sensitivity and specificity levels. The MKRN1 biomarker may be a useful adjunct in primary cervical cytology screening.

Makorin ring finger protein 1 (MKRN1) as an adjunct marker in liquid-based cervical cytology

Makorin ring finger protein 1 (MKRN1) as an adjunct marker in liquid-based cervical cytology

The expression of makorin ring finger protein 1 (MKRN1), pAKT, pmTOR, and PTEN in cervical neoplasia

Objective: The protein kinase B (AKT) pathway plays a key role in the regulation of cell death and survival, and has been proposed as a central signaling event in carcinogenesis. We investigated the expression of MKRN1, which is a transcriptional coregulator and an E3 ligase, and sought to define the role of the AKT pathway in cervical neoplasia.

Acceleration of Gastric Tumorigenesis Through MKRN1-Mediated Posttranslational Regulation of p14ARF

BackgroundWe investigated whether Makorin ring finger protein 1 (MKRN1), an E3 ligase, affects p14ARF-associated cellular senescence and tumorigenesis by posttranslational modification in gastric tumorigenesis.MethodsA link between MKRN1 and ARF was examined in MKRN1 null mouse embryonic fibroblasts (MEFs) and in human fibroblasts and gastric cancer cells by silencing MKRN1 using small interfering RNA (siRNA) and short hairpin RNA (shRNA). Ubiquitination and proteasomal degradation assays were used to assess p14ARF degradation associated with MKRN1. MKRN1 and p14ARF expression levels were analyzed with immunohistochemistry in malignant and normal tissues from gastric cancer patients and with χ2 tests. The tumor growth of gastric cancer cells stably expressing MKRN1 shRNA, p14ARF shRNA, or both was examined in mouse xenograft models (n = 4–6) and analyzed with unpaired t tests. All statistical tests were two-sided.ResultsMKRN1 knockout MEFs exhibited premature senescence and growth retardation with increased p19ARF protein expression. Similar results were obtained for human fibroblasts or gastric cancer cell lines by MKRN1 knockdown. Biochemical analyses confirmed that MKRN1 targets p14ARF for ubiquitination and subsequent proteasome-dependent degradation. A statistically significant association was shown between MKRN1 overexpression and p14ARF underexpression (P = .016). Xenograft analyses using p53-functional AGS or -dysfunctional SNU601 cells displayed statistically significant tumor growth retardation by silencing MKRN1, which was reversed under depletion of p14ARF (AGS cells, MKRN1 knockdown tumors vs MKRN1 and p14ARF knockdown tumors: 164.6 vs 464.8mm3, difference = 300.2mm3, 95% CI = 189.1 to 411.3mm3, P < .001).ConclusionsWe demonstrated that MKRN1 functions as a novel E3 ligase of p14ARF and that it potentially regulates cellular senescence and tumorigenesis in gastric cancer.

Ubiquitination and degradation of the FADD adaptor protein regulate death receptor-mediated apoptosis and necroptosis

Fas-associated protein with death domain (FADD) is a pivotal component of death receptor-mediated extrinsic apoptosis and necroptosis. Here we show that FADD is regulated by Makorin Ring Finger Protein 1 (MKRN1) E3 ligase-mediated ubiquitination and proteasomal degradation. MKRN1 knockdown results in FADD protein stabilization and formation of the rapid death-inducing signalling complex, which causes hypersensitivity to extrinsic apoptosis by facilitating caspase-8 and caspase-3 cleavage in response to death signals. We also show that MKRN1 and FADD are involved in the regulation of necrosome formation and necroptosis upon caspase inhibition. Downregulation of MKRN1 results in severe defects of tumour growth upon tumour necrosis factor-related apoptosis-inducing ligand treatment in a xenograft model using MDA-MB-231 breast cancer cells. Suppression of tumour growth by MKRN1 depletion is relieved by simultaneous FADD knockdown. Our data reveal a novel mechanism by which fas-associated protein with death domain is regulated via an ubiquitination-induced degradation pathway.

MKRN1 Induces Degradation of West Nile Virus Capsid Protein by Functioning as an E3 Ligase

West Nile virus capsid protein (WNVCp) displays pathogenic toxicity via the apoptotic pathway. However, a cellular mechanism protective against this toxic effect has not been observed so far. Here, we identified Makorin ring finger protein 1 (MKRN1) as a novel E3 ubiquitin ligase for WNVCp. The cytotoxic effects of WNVCp as well as its expression levels were inhibited in U2OS cells that stably expressed MKRN1. Immunoprecipitation analyses revealed an interaction between MKRN1 and WNVCp. Domain analysis indicated that the C terminus of MKRN1 and the N terminus of WNVCp were required for the interaction. MKRN1 could induce WNVCp ubiquitination and degradation in a proteasome-dependent manner. Interestingly, the WNVCp mutant with amino acids 1 to 105 deleted WNVCp was degraded by MKRN1, whereas the mutant with amino acids 1 to 90 deleted was not. When three lysine sites at positions 101, 103, and 104 of WNVCp were replaced with alanine, MKRN1-mediated ubiquitination and degradation of the mutant were significantly inhibited, suggesting that these sites are required for the ubiquitination. Finally, U2OS cell lines stably expressing MKRN1 were resistant to cytotoxic effects of WNV. In contrast, cells depleted of MKRN1 were more susceptible to WNVCp cytotoxicity. Confirming this, overexpression of MKRN1 significantly reduced, but depletion of MKRN1 increased, WNV proliferation in 293T cells. Taken together, our results suggest that MKRN1 can protect cells from WNV by inducing WNVCp degradation.

Differential regulation of p53 and p21 by MKRN1 E3 ligase controls cell cycle arrest and apoptosis

Makorin Ring Finger Protein 1 (MKRN1) is a transcriptional co-regulator and an E3 ligase. Here, we show that MKRN1 simultaneously functions as a differentially negative regulator of p53 and p21. In normal conditions, MKRN1 could destabilize both p53 and p21 through ubiquitination and proteasome-dependent degradation. As a result, depletion of MKRN1 induced growth arrest through activation of p53 and p21. Interestingly, MKRN1 used earlier unknown sites, K291 and K292, for p53 ubiquitination and subsequent degradation. Under severe stress conditions, however, MKRN1 primarily induced the efficient degradation of p21. This regulatory process contributed to the acceleration of DNA damage-induced apoptosis by eliminating p21. MKRN1 depletion diminished adriamycin or ultraviolet-induced cell death, whereas ectopic expression of MKRN1 facilitated apoptosis. Furthermore, MKRN1 stable cell lines that constantly produced low levels of p53 and p21 exhibited stabilization of p53, but not p21, with increased cell death on DNA damage. Our results indicate that MKRN1 exhibits dual functions of keeping cells alive by suppressing p53 under normal conditions and stimulating cell death by repressing p21 under stress conditions.