Jaewhan Song

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.

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.

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.

Magnetic iron oxide nanoparticles induce autophagy preceding apoptosis through mitochondrial damage and ER stress in RAW264.7 cells.

Magnetic nanoparticles have been widely used in a broad range of disciplines owing to their unique properties. However, many unexpected risks have been reported in their use. In this study, we investigated the uptake process and toxic mechanism of magnetic iron oxide nanoparticles (M-FeNPs) using RAW264.7 cells, a murine peritoneal macrophage cell line. M-FeNPs markedly enhanced the mobility of cells. At 24h after exposure, M-FeNPs were located freely in the cytosol or within autolysosomes containing various organelles, especially the endoplasmic reticulum (ER). Cell viability decreased in a dose-dependent manner in conjunction with the arrest in S phase. ATP production also rapidly decreased together with mitochondrial damage, the number of cells that generate ROS increased, and the secretions of pro-inflammatory cytokines enhanced. The levels of oxidative stress- and ER stress-related genes were up-regulated, whereas the levels of transcription-related genes were down-regulated. Additionally, the levels of autophagy- and ER stress-related proteins increased, and the number of apoptotic cells increased with time. We also investigated the function of the autolysosome in the cellular response after exposure of M-FeNPs. When cells were exposed to M-FeNPs for 24h with BaFA1 pretreatment, the plasma membrane disintegrated, cytosolic components disappeared, and the number of apoptotic cells significantly increased. Taken together, these results show that M-FeNPs induce autophagy preceding apoptosis through mitochondrial dysfunction and ER stress in RAW264.7 cells. Furthermore, blocking of autolysosome formation may accelerate apoptotic cell death and ER stress.

PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis

The activity of the phosphatase and tensin homologue (PTEN) is known to be suppressed via post-translational modification. However, the mechanism and physiological significance by which post-translational modifications lead to PTEN suppression remain unclear. Here we demonstrate that PTEN destabilization is induced by EGFR- or oncogenic PI3K mutation-mediated AKT activation in cervical cancer. EGFR/PI3K/AKT-mediated ubiquitination and degradation of PTEN are dependent on the MKRN1 E3 ligase. These processes require the stabilization of MKRN1 via AKT-mediated phosphorylation. In cervical cancer patients with high levels of pAKT and MKRN1 expression, PTEN protein levels are low and correlate with a low 5-year survival rate. Taken together, our results demonstrate that PI3K/AKT signals enforce positive-feedback regulation by suppressing PTEN function.

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

Background We investigated whether Makorin ring finger protein 1 (MKRN1), an E3 ligase, affects p14ARF-associated cellular senescence and tumorigenesis by posttranslational modification in gastric tumorigenesis. Methods A 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. Results MKRN1 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). Conclusions We demonstrated that MKRN1 functions as a novel E3 ligase of p14ARF and that it potentially regulates cellular senescence and tumorigenesis in gastric cancer.

CHIP controls necroptosis through ubiquitylation- and lysosome-dependent degradation of RIPK3

Receptor-interacting protein kinase 3 (RIPK3) functions as a key regulator of necroptosis. Here, we report that the RIPK3 expression level is negatively regulated by CHIP (carboxyl terminus of Hsp70-interacting protein; also known as STUB1) E3 ligase-mediated ubiquitylation. Chip−/− mouse embryonic fibroblasts and CHIP-depleted L929 and HT-29 cells exhibited higher levels of RIPK3 expression, resulting in increased sensitivity to necroptosis induced by TNF (also known as TNFα). These phenomena are due to the CHIP-mediated ubiquitylation of RIPK3, which leads to its lysosomal degradation. Interestingly, RIPK1 expression is also negatively regulated by CHIP-mediated ubiquitylation, validating the major role of CHIP in necrosome formation and sensitivity to TNF-mediated necroptosis. Chip−/− mice (C57BL/6) exhibit inflammation in the thymus and massive cell death and disintegration in the small intestinal tract, and die within a few weeks after birth. These phenotypes are rescued by crossing with Ripk3−/− mice. These results imply that CHIP is a bona fide negative regulator of the RIPK1–RIPK3 necrosome formation leading to desensitization of TNF-mediated necroptosis.

Activation of nuclear PTEN by inhibition of Notch signaling induces G2/M cell cycle arrest in gastric cancer

Mutation in PTEN has not yet been detected, but its function as a tumor suppressor is inactivated in many cancers. In this study we determined that, activated Notch signaling disables PTEN by phosphorylation and thereby contributes to gastric tumorigenesis. Notch inhibition by small interfering RNA or γ-secretase inhibitor (GSI) induced mitotic arrest and apoptosis in gastric cancer cells. Notch inhibition induced dephosphorylation in the C-terminal domain of PTEN, which led to PTEN nuclear localization. Overexpression of activated Notch1-induced phosphorylation of PTEN and reversed GSI-induced mitotic arrest. Dephosphorylated nuclear PTEN caused prometaphase arrest by interaction with the cyclin B1-CDK1 complex, resulting in their accumulation in the nucleus and subsequent apoptosis. We found a correlation between high expression levels of Notch1 and low survival rates and, similarly, between reduced nuclear PTEN expression and increasing the TNM classification of malignant tumours stages in malignant tissues from gastric cancer patients. The growth of Notch1-depleted gastric tumors was significantly retarded in xenografted mice, and in addition, PTEN deletion restored growth similar to control tumors. We also demonstrated that combination treatment with GSI and chemotherapeutic agents significantly reduced the orthotopically transplanted gastric tumors in mice without noticeable toxicity. Overall, our findings suggest that inhibition of Notch signaling can be employed as a PTEN activator, making it a potential target for gastric cancer therapy.

Hdm2 negatively regulates telomerase activity by functioning as an E3 ligase of hTERT.

In this study, we identified posttranslational regulation of human telomerase reverse-transcriptase (hTERT) by the E3 ligase Hdm2. The telomerase activity generated by exogenous hTERT in U2OS cells was reduced on adriamycin treatment. The overexpressed levels of hTERT were also decreased under the same conditions. These processes were reversed by treatment with a proteasome inhibitor or depletion of Hdm2. Furthermore, intrinsic telomerase activity was increased in HCT116 cells with ablation of Hdm2. Immunoprecipitation analyses showed that hTERT and Hdm2 bound to each other in multiple domains. Ubiquitination analyses showed that Hdm2 could polyubiquitinate hTERT principally at the N-terminus, which was further degraded in a proteasome-dependent manner. An hTERT mutant with all five lysine residues at the N-terminus of hTERT that mutated to arginine became resistant to Hdm2-mediated ubiquitination and degradation. In U2OS cells, depletion of Hdm2 or addition of the Hdm2-resistant hTERT mutant strengthened the cellular protective effects against apoptosis. Similar results were obtained with the Hdm2-stable H1299 cell line. These observations indicate that Hdm2 is an E3 ligase of hTERT.

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

ABSTRACT 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.