RING Finger Protein Research Articles

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUNDHepatocellular carcinoma (HCC) exhibits high invasiveness and mortality rates, and the molecular mechanisms of HCC have gained increasing research interest. The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development. Recent studies have shown the potential of the protein RING finger and WD repeat domain 3 (RFWD3) that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers. AIMTo investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways. METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues. Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines. After verifying the silencing efficiency, Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis. Subsequently, cell migration and invasion were assessed by wound healing and transwell assays. In addition, transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis. Next, we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype. Finally, the microarray, ingenuity pathway analysis, and western blot analysis were used to analyze the regulatory network underlying HCC. RESULTSCompared with adjacent tissues, RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage (P < 0.05), which indicated a poor prognosis state. RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis, decreased growth, and inhibited the migration in shRNAi cells compared with those in shCtrl cells (P < 0.05). Furthermore, the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration. Moreover, the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines. Finally, gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSIONWe provide evidence for the expression and function of RFWD3 in HCC. RFWD3 affects the prognosis, proliferation, invasion, and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.

MKRN3 circulating levels in Prader-Willi syndrome: a pilot study.

Hypogonadism in Prader-Willi syndrome (PWS) is generally attributed to hypothalamic dysfunction or to primary gonadal defect. MKRN3, a maternal imprinted gene located on 15q11.2-q13 region, encodes makorin ring finger protein 3, whose deficiency causes precocious puberty, an extremely rare symptom in PWS. This study aimed to evaluate MKRN3 levels in patients with PWS and to analyze its correlation with sexual hormone levels, insulin resistance and Body Mass Index (BMI). We performed an observational cross-sectional study and enrolled 80 patients with genetically confirmed diagnosis of PWS with median age of 9.6years. MKRN3 levels were measurable in 49 PWS patients with a geometric mean of 34.9 ± 22pg/ml (median: 28.4). Unmeasurable levels of MKRN3 were found in 31 patients. No statistically significant differences were found between patients with and without measurable MKRN3 levels for any clinical, biochemical, or genetic characteristics. However, MKRN3 levels were inversely correlated with HOMA-IR index (p: 0.005) and HbA1c (p: 0.046) values. No statistically significant correlations were found between MKRN3 and LH, estradiol and testosterone concentrations, pubertal development and genetic defect, whereas a direct correlation with FSH was found (p: 0.007). The typical genetic defect of PWS should lead to unmeasurable levels of the MKRN3 protein due to the inactivation of the paternal allele. Measurable circulating MKRN3 could suggest the possible involvement of tissue-specific imprinting mechanisms and other regulatory factors in gene expression. Correlations with HOMA-IR index, HbA1c, and FSH suggest peripheral actions of MKRN3, but future studies are warranted to investigate this topic.

Genome-Scale Profiling and High-Throughput Analyses Unravel the Genetic Basis of Arsenic Content Variation in Rice.

Ionomics, the study of the composition of mineral nutrients and trace elements in organisms that represent the inorganic component of cells and tissues, has been widely studied to explore to unravel the molecular mechanism regulating the elemental composition of plants. However, the genetic factors of rice subspecies in the interaction between arsenic and functional ions have not yet been explained. Here, the correlation between As and eight essential ions in a rice core collection was analyzed, taking into account growing condition and genetic factors. The results demonstrated that the correlation between As and essential ions was affected by genetic factors and growing condition, but it was confirmed that the genetic factor was slightly larger with the heritability for arsenic content at 53%. In particular, the cluster coefficient of japonica (0.428) was larger than that of indica (0.414) in the co-expression network analysis for 23 arsenic genes, and it was confirmed that the distance between genes involved in As induction and detoxification of japonica was far than that of indica. These findings provide evidence that japonica populations could accumulate more As than indica populations. In addition, the cis-eQTLs of AIR2 (arsenic-induced RING finger protein) were isolated through transcriptome-wide association studies, and it was confirmed that AIR2 expression levels of indica were lower than those of japonica. This was consistent with the functional haplotype results for the genome sequence of AIR2, and finally, eight rice varieties with low AIR2 expression and arsenic content were selected. In addition, As-related QTLs were identified on chromosomes 5 and 6 under flooded and intermittently flooded conditions through genome-scale profiling. Taken together, these results might assist in developing markers and breeding plans to reduce toxic element content and breeding high-quality rice varieties in future.

RNA Sequencing Reveals Beneficial Effects of Atorvastatin on Endothelial Cells in Acute Kawasaki Disease.

Background Damage to the coronary arteries during the acute phase of Kawasaki disease (KD) is linked to inflammatory cell infiltration, myointimal proliferation, and endothelial cell (EC) dysfunction. To understand the response of ECs to KD treatment, we studied the genome-wide transcriptional changes in cultured ECs incubated with KD sera before and after treatment with or without atorvastatin. Methods and Results RNA sequencing of human umbilical vein ECs incubated with pooled sera from patients with acute KD before or after treatment with intravenous immunoglobulin and infliximab revealed differentially expressed genes in interleukin-1, tumor necrosis factor-α, and inflammatory cell recruitment pathways. Subacute sera pooled from patients treated with intravenous immunoglobulin, infliximab, and atorvastatin uniquely induced expression of NOS3, Kruppel like factor (KLF2, and KLF4 (promotes EC homeostasis and angiogenesis) and ZFP36 ring finger protein (ZFP36) and suppressor of cytokine signaling 3 (SOCS3) (suppresses inflammation), and suppressed expression of TGFB2 and DKK1 (induces endothelial-mesenchymal transition) and sphingosine kinase 1 (SPHK1) and C-X-C motif chemokine ligand 8 (CXCL8) (induces inflammation). Conclusions These results suggest that atorvastatin treatment of patients with acute KD may improve EC health, reduce mediators of inflammation produced by ECs, and block KD-induced myofibroblast proliferation.

The Capsid Protein of Nervous Necrosis Virus Antagonizes Host Type I IFN Production by a Dual Strategy to Negatively Regulate Retinoic Acid-Inducible Gene-I-like Receptor Pathways.

Nervous necrosis virus (NNV), a highly pathogenic RNA virus, is a major pathogen in the global aquaculture industry. To efficiently infect fish, NNV must evade or subvert the host IFN for their replication; however, the precise mechanisms remain to be elucidated. In this study, we reported that capsid protein (CP) of red-spotted grouper NNV (RGNNV) suppressed the IFN antiviral response to promote RGNNV replication in Lateolabrax japonicus brain cells, which depended on the ARM, S, and P domains of CP. CP showed an indirect or direct association with the key components of retinoic acid-inducible gene-I-like receptors signaling, L. japonicus TNFR-associated factor 3 (LjTRAF3) and IFN regulatory factor (LjIRF3), respectively, and degraded LjTRAF3 and LjIRF3 through the ubiquitin-proteasome pathway in HEK293T cells. Furthermore, we found that CP potentiated LjTRAF3 K48 ubiquitination degradation in a L. japonicus ring finger protein 114-dependent manner. LjIRF3 interacted with CP through the S domain of CP and the transcriptional activation domain or regulatory domain of LjIRF3. CP promoted LjIRF3 K48 ubiquitination degradation, leading to the reduced phosphorylation level and nuclear translocation of LjIRF3. Taken together, we demonstrated that CP inhibited type I IFN response by a dual strategy to potentiate the ubiquitination degradation of LjTRAF3 and LjIRF3. This study reveals a novel mechanism of RGNNV evading host immune response via its CP protein that will provide insights into the complex pathogenesis of NNV.

High expression of ZFP36L2 correlates with the prognosis and immune infiltration in lower-grade glioma.

Background: The ZFP36 Ring Finger Protein Like 2 (ZFP36L2) is an RNA-binding protein that regulates gene expression at post-transcriptional level. However, the clinical significance and prognostic value of ZFP36L2 in lower-grade glioma (LGG) remain unclear. Method: ZFP36L2 expression was investigated using public datasets and the prognostic merit of ZFP36L2 with LGG patients was further evaluated. The correlation between the genetic alteration of ZFP36L2 and its mRNA expression was accessed via cBioPortal. Additionally, the prognostic value of the ZFP36L2 methylation levels in LGG was evaluated by MethSurv. The potential biological role of ZFP36L2 in LGG was identified by performing functional analyses. We also examined the correlation between ZFP36L2 expression and the immune infiltration. Finally, the predictive value of ZFP36L2 to immunotherapy was assessed. Result: ZFP36L2 was highly expressed in LGG patients and overexpressed ZFP36L2 predicted poor clinical outcomes. We further identified ZFP36L2 as an independent prognostic factor. The methylation level of ZFP36L2 negatively correlated with the ZFP36L2 expression, and patients with low ZFP36L2 methylation had worse overall survival. The results of functional analysis indicated that ZFP36L2 was involved in multiple immune response-related pathways in LGG. Furthermore, high expression of ZFP36L2 was significantly and positively correlated with immune infiltration. Finally, we found that ZFP36L2 expression was positively correlated with the immune checkpoint PD-L1, and ZFP36L2 low expression cohort gained better benefit from immunotherapy. Conclusion: Our findings demonstrate that ZFP36L2 is a potential biomarker for LGG, highlighting its potential as a therapeutic target in immunotherapy.

The testis-specific E3 ubiquitin ligase RNF133 is required for fecundity in mice

BackgroundUbiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin–proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown.ResultsWe identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 (Rnf133); RING finger protein 148 (Rnf148); RING finger protein 151 (Rnf151); and Zinc finger SWIM-type containing 2 (Zswim2)]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148, Rnf151, and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133.ConclusionsOur studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.

Impact of the RNF213 p.R4810K Variant on Endovascular Therapy for Large‐Vessel Occlusion Stroke

Background We investigated the impact of the ring finger protein 213 p.R4810K variant, a founder variant for moyamoya disease in East Asians, on endovascular therapy outcomes in patients with acute anterior‐circulation large‐vessel occlusion stroke in comparison with noncarriers. Methods Of the consecutive patients with ischemic stroke admitted to our institute from 2011 to 2021, patients who underwent endovascular therapy for acute occlusion of the intracranial internal carotid artery or M1 segment of the middle cerebral artery were included. Outcomes were instant reocclusion, final modified Thrombolysis in Cerebral Infarction reperfusion ≥2b, and early reocclusion. Instant reocclusion was defined as the occurrence of reocclusion during the procedure, and early reocclusion was defined as reocclusion detected on magnetic resonance angiography within 2 weeks after the confirmation of successful reperfusion. Results Of the 277 patients analyzed (128 women; median age, 76 years), 10 patients (3.6%) carried the ring finger protein 213 p.R4810K variant. Variant carriers were younger ( P =0.01) and more frequently had intracranial atherosclerotic disease‐related large‐vessel occlusion as a cause of acute large‐vessel occlusion ( P &lt;0.001) compared with noncarriers. Variant carriers showed a higher rate of instant reocclusion (70.0% versus 5.6%; P &lt;0.001), but there were no significant intergroup differences in the final modified Thrombolysis in Cerebral Infarction ≥2b reperfusion rate between carriers and noncarriers (100.0% versus 81.6%, respectively; P =0.22). Early reocclusion was more frequent in variant carriers compared with noncarriers (60.0% versus 0.4%; P &lt;0.001). Conclusions Instant and early reocclusions were more frequent in variant carriers who underwent endovascular therapy for acute anterior‐circulation large‐vessel occlusion compared with noncarriers.

In silico promoter analysis and expression of the BIG BROTHER gene in different organs of potato

The ubiquitin E3 ligase BIG BROTHER/ENHANCER OF DA1 (BB) gene encoding a RING finger protein was identified as a central growth regulator in Arabidopsis thaliana. It was found that BB restricts cell proliferation and promotes leaf senescence. Besides of Arabidopsis, however, the role and regulation of BB in other plant species is only sparsely known. Supposing that the BB gene, like in Arabidopsis, has an important role in the development of potato we aimed to analyse a 3.0-kb promoter sequence of the potato BB gene, StBB, in silico and study the level of StBB expression by quantitative reverse transcription PCR in different organs. A total of 48 binding sites for 15 transcription factor (TF) families were predicted. Most of them were located in the -1.5-kb promoter region. The dominating family of TFs was DOF. It was found that 20 out of the 24 TFs with known functions are involved in developmental processes such as for example, the flower-, leaf-, stem- and root development or cell cycle regulation. In line with this finding, the StBB mRNA was detected in each organ tested with the largest amounts in petal and stamen. These results suggest a similar function of StBB in potato than that is of BB in Arabidopsis, i.e., restriction of organ overgrowth during development and limitation of the plant growth.

Multifaceted Roles of the E3 Ubiquitin Ligase RING Finger Protein 115 in Immunity and Diseases.

Ubiquitination is a post-translational modification that plays essential roles in various physiological and pathological processes. Protein ubiquitination depends on E3 ubiquitin ligases that catalyze the conjugation of ubiquitin molecules on lysine residues of targeted substrates. RING finger protein 115 (RNF115), also known as breast cancer associated gene 2 (BCA2) and Rab7-interacting RING finger protein (Rabring7), has been identified as a highly expressed protein in breast cancer cells and tissues. Later, it has been demonstrated that RNF115 catalyzes ubiquitination of a series of proteins to modulate a number of signaling pathways, and thereby regulates viral infections, autoimmunity, cell proliferation and death and tumorigenesis. In this review, we introduce the identification, expression and activity regulation of RNF115, summarize the substrates and functions of RNF115 in different pathways, and discuss the roles of RNF115 as a biomarker or therapeutic target in diseases.

Insulin-like growth factor binding protein 7 promotes acute kidney injury by alleviating poly ADP ribose polymerase 1 degradation

The novel biomarker, insulin-like growth factor binding protein 7 (IGFBP7), is used clinically to predict different types of acute kidney injury (AKI) and has drawn significant attention as a urinary biomarker. However, as a secreted protein in the circulation of patients with AKI, it is unclear whether IGFBP7 acts as a key regulator in AKI progression, and if mechanisms underlying its upregulation still need to be determined. Here we found that IGFBP7 is highly expressed in the blood and urine of patients and mice with AKI, possibly via a c-Jun-dependent mechanism, and is positively correlated with kidney dysfunction. Global knockout of IGFBP7 ameliorated kidney dysfunction, inflammatory responses, and programmed cell death in murine models of cisplatin-, kidney ischemia/reperfusion-, and lipopolysaccharide-induced AKI. IGFBP7 mainly originated from kidney tubular epithelial cells. Conditional knockout of IGFBP7 from the kidney protected against AKI. By contrast, rescue of IGFBP7 expression in IGFBP7-knockout mice restored kidney damage and inflammation. IGFBP7 function was determined invitro using recombinant IGFBP7 protein, IGFBP7 knockdown, or overexpression. Additionally, IGFBP7 was found to bind to poly [ADP-ribose] polymerase 1 (PARP1) and inhibit its degradation by antagonizing the E3 ubiquitin ligase ring finger protein 4 (RNF4). Thus, IGFBP7 in circulation acts as a biomarker and key mediator of AKI by inhibiting RNF4/PARP1-mediated tubular injury and inflammation. Hence, over-activation of the IGFBP7/PARP1 axis represents a promising target for AKI treatment.

Glaucocalyxin A Inhibits the Malignancies of Gastric Cancer Cells by Downregulating MDM2 and RNF6 via MiR-3658 and the SMG1-UPF mRNA Decay Pathway.

Gastric cancer (GC) ranks as the most common gastrointestinal cancer and is among the leading causes of cancer death worldwide. Glaucocalyxin A (GLA), an entkauranoid diterpene isolated from Rab-dosia japonica var., possesses various bioactivities. To date, the data on the effect of GLA on GC are still minimal, and the molecular mechanisms remain largely unknown. Herein, we found that GLA could significantly inhibit the proliferation, cell adhesion, and invasion of HGT-1, SNU-1, SNU-6, and NCI-N87 GC cells in a dose-dependent manner. GLA enhanced the apoptosis of the GC cells as evidenced by the increased caspase-3 activity and the elevated levels of cleaved caspase-3 and cleaved PARP in GC cells in the presence of GLA. We then showed that the downregulation of Murine Double Minute Clone 2 (MDM2) and Ring Finger Protein 6 (RNF6) by GLA was implicated in the GLA-induced inhibition of the GC cells. Furthermore, MDM2 and RNF6 were identified as the targets of miR-3658 that was downregulated in the GC cells and upregulated by GLA. Moreover, it was shown that miR-3658 was hypermethylated in the GC cells, and GLA could rescue the expression of miR-3658 via demethylation by abrogating EZH2-mediated epigenetic silencing. In addition to the miR-3658-MDM2/RNF6 regulatory axis, activation of the SMG1-UPF mRNA decay pathway contributed to the downregulation of MDM2 and RNF6 by GLA in the GC cells. The inhibitory effect of GLA on gastric cancer and the expression of MDM2 and RNF6 was also validated in in vivo study. Our findings suggest that has the therapeutic potential for GC by downregulating oncogenes via posttranscriptional regulation.

RING finger protein 128 (RNF128) regulates malignant biological behaviors of colorectal cancer cells via PI3K/AKT signaling pathway.

This study was designed and conducted to clarify the impact of RNF128 expression on malignant biological behaviors of colorectal cancer (CRC) cells and the underlying mechanism. The expression of RNF128 in CRC tissues was analyzed using mRNA sequencing data of TCGA database and was validated by Western blot assay. The experimental studies on biological functions of RNF128 in vitro were conducted to assess its impact on the proliferation, apoptosis, and metastasis of CRC cells. Furthermore, tumor xenograft models in nude mice were established to investigate the relationship between RNF128 expression and tumor growth in vivo. The expression levels of both RNF128 mRNA and protein were significantly increased in CRC tissues (p < .001). The knockdown of RNF128 markedly suppressed the malignant phenotype of HCT116 and SW480 cells in vitro, including cell growth, antiapoptosis, migration, and invasion (p < .001). On the other hand, knockdown of RNF128 exerted a remarkable effect on the growth inhibition of tumor xenografts in vivo (p < .001). Further investigation revealed that RNF128 knockdown lead to a significant decrease in the expression of p-AKT and p-PI3K protein. More importantly, the proliferative, antiapoptotic, metastatic abilities of RNF128-knockdown cells were markedly increased by 740 Y-P treatment (p < .001). These findings further suggested that PI3K/AKT signaling pathway played a key role in RNF128-mediated aggressive phenotype of CRC cells. RNF128 functions as a tumor promoter in the pathogenesis of CRC via regulating PI3K/AKT pathway, and it could be a valuable target for CRC treatment.

Silymarin Prevents High-Fat Diet-Induced Muscle Atrophy by Regulating Protein Degradation and Synthesis in Mice.

Silymarin is found in Silybum marianum. We investigated the effect of silymarin on muscle atrophy in obese mice. The experimental mice were divided into three groups: CON, normal diet; HFD, 60% high-fat diet (HF); and SILY: 50 mg silymarin +60% HF. It was confirmed that increases in body weight and fat mass in the SILY group were significantly inhibited. Moreover, the muscle mass in SILY mice was significantly higher than that in the HFD group. The grip strength in HFD group was significantly reduced, whereas in the SILY group it was higher than that in HFD group. In HFD mice, the mRNA levels of protein degradation factors (muscle ring-finger protein 1 [MuRF-1] and Atrogin-1) were increased and protein synthesis factors (phosphoinositide 3-kinase [PI3K] and Akt) were decreased. However, silymarin was found to elevate the degradation factors as compared with HFD group, whereas it reduced the synthesis factors. The results suggest that silymarin could prevent not only obesity but also muscle atrophy.

Moyamoya Disease Associated with a Deficiency of Complement Component 6

ObjectivesComplement component 6 (C6) deficiency is a very rare genetic defect that leads to significantly diminished synthesis, secretion, or function of C6. In the current report, we demonstrate a previously undescribed, homozygous missense mutation in exon 17 of the C6 gene (c.2545A>G p.Arg849Gly) in a 35-year-old Japanese woman with moyamoya disease and extremely low levels of CH50 (<7.0 U/mL). Materials and MethodsThe complement gene analysis using hybridization capture-based next generation sequencing was performed. CH50 was determined in patient's plasma mixed with plasma from a healthy donor or purified human C6 protein. Western blot was performed on patient's plasma using polyclonal antibodies against C6, with healthy donor's plasma and purified human C6 protein as positive controls while C6-depleted human serum as a negative control. The carriage of ring finger protein 213 variant (c.14576G>A p.Arg4859Lys), a susceptibility gene for moyamoya disease, was examined by direct sequencing. ResultsCH50 mixing test clearly showed a deficiency pattern, being rescued by addition of only 1% healthy donor's plasma or 1 μg/mL purified human C6 protein (1/50-1/100 of physiological concentration). Western blot revealed the absence of C6 protein in the patient's plasma, confirming a quantitative deficiency of C6. The ring finger protein 213 variant was not detected. ConclusionsOur data implies that unrecognized complement deficiencies would be harbored in cerebrovascular diseases with unknown etiologies.

Ubiquitin E3 ligase Atrogin-1 protein is regulated via the rapamycin-sensitive mTOR-S6K1 signaling pathway in C2C12 muscle cells

Atrogin-1 and Muscle-specific RING finger protein 1 (MuRF1) are highly expressed in multiple conditions of skeletal muscle atrophy. The phosphoinositide 3-kinase (PI3K)/Akt/forkhead box (FoxO) signaling pathway is well known to regulate Atrogin-1 and MuRF1 gene expressions. However, Akt activation also activates the mechanistic target of rapamycin complex 1 (mTORC1), which induces skeletal muscle hypertrophy. Whether mTORC1-dependent signaling has a role in regulating Atrogin-1 and/or MuRF1 gene and protein expression is currently unclear. In this study, we showed that activation of insulin-mediated Akt signaling suppresses both Atrogin-1 and MuRF1 protein contents and that inhibition of Akt increases both Atrogin-1 and MuRF1 protein contents in C2C12 myotubes. Interestingly, inhibition of mTORC1 with a specific mTORC1 inhibitor, rapamycin, increased Atrogin-1, but not MuRF1, protein content. Furthermore, activation of AMP-activated protein kinase (AMPK), a negative regulator of the mTORC1 signaling pathway, also showed distinct time-dependent changes between Atrogin-1 and MuRF1 protein contents, suggesting differential regulatory mechanisms between Atrogin-1 and MuRF1 protein content. To further explore the downstream of mTORC1 signaling, we employed a specific S6K1 inhibitor, PF-4708671. We found that Atrogin-1 protein content was dose-dependently increased with PF-4708671 treatment, whereas MuRF1 protein content was decreased at 50 μM of PF-4708671 treatment. However, MuRF1 protein content was unexpectedly increased by PF-4708671 treatment for a longer period. Overall, our results indicate that Atrogin-1 and MuRF1 protein contents are regulated by different mechanisms, the downstream of Akt, and that Atrogin-1 protein content can be regulated by the rapamycin-sensitive mTOR-S6K1-dependent signaling pathway.

Abstract 2101: RNF2 ablation reprograms the tumor-immune microenvironment and stimulates durable NK and CD4+ T cell-dependent anti-tumor immunity

Abstract Expanding the utility of immune-based cancer treatments is a clinical challenge due to tumor-intrinsic factors that suppress the immune response. Here we report the identification of tumoral Ring Finger Protein 2 (RNF2), the core subunit of the Polycomb Repressor Complex 1 (PRC1), as a negative regulator of antitumor immunity in various human cancers, including breast cancer. In syngeneic murine models of triple negative breast cancer, we found that deleting genes encoding PRC1 subunits Rnf2 or BMI1 proto-oncogene, polycomb ring finger (Bmi1), or the downstream effector of Rnf2, Remodeling and Spacing Factor 1 (Rsf1), was sufficient by itself to induce durable tumor rejection and establish immune memory by enhancing infiltration and activation of NK and CD4+ T-cells, but not CD8+ T-cells, into the tumor and enabled their cooperativity. These findings uncover an epigenetic reprogramming of the tumor-immune microenvironment which fosters durable antitumor immunity and memory. Citation Format: Zhuo Zhang, Lin Luo, Chuan Xing, Yu Chen, Peng Xu, Mao Li, Ling Zeng, Chao Li, Sadashib Ghosh, Deborah Della Manna, Tim Townes, William J. Britt, Narendra Wajapeyee, Barry P. Sleckman, Zechen Chong, Jianmei Wu Leavenworth, Eddy Yang. RNF2 ablation reprograms the tumor-immune microenvironment and stimulates durable NK and CD4+ T cell-dependent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2101.

The transcription factor OsMYBc and an E3 ligase regulate expression of a K+ transporter during salt stress.

Sodium (Na+) and potassium (K+) homeostasis is essential for plant survival in saline soils. A member of the High-Affinity K+ Transporter (HKT) family in rice (Oryza sativa), OsHKT1;1, is a vital regulator of Na+ exclusion from shoots and is bound by a MYB transcription factor (OsMYBc). Here, we generated transgenic rice lines in the oshkt1;1 mutant background for genetic complementation using genomic OsHKT1;1 containing a native (Com) or mutated (mCom) promoter that cannot be bound by OsMYBc. In contrast to wild-type (WT) or Com lines, the mCom lines were not able to recover the salt-sensitive phenotype of oshkt1;1. The OsMYBc-overexpressing plants were more tolerant to salt stress than WT plants. A yeast two-hybrid screen using the OsMYBc N-terminus as bait identified a rice MYBc stress-related RING finger protein (OsMSRFP). OsMSRFP is an active E3 ligase that ubiquitinated OsMYBc in vitro and mediated 26S proteasome-mediated degradation of OsMYBc under semi-in vitro and in vivo conditions. OsMSRFP attenuated OsMYBc-mediated OsHKT1;1 expression, and knockout of OsMSRFP led to rice salt tolerance. These findings uncover a regulatory mechanism of salt response that fine-tunes OsHKT1;1 transcription by ubiquitination of OsMYBc.

RNF219 regulates CCR4-NOT function in mRNA translation and deadenylation

Post-transcriptional regulatory mechanisms play a role in many biological contexts through the control of mRNA degradation, translation and localization. Here, we show that the RING finger protein RNF219 co-purifies with the CCR4-NOT complex, the major mRNA deadenylase in eukaryotes, which mediates translational repression in both a deadenylase activity-dependent and -independent manner. Strikingly, RNF219 both inhibits the deadenylase activity of CCR4-NOT and enhances its capacity to repress translation of a target mRNA. We propose that the interaction of RNF219 with the CCR4-NOT complex directs the translational repressive activity of CCR4-NOT to a deadenylation-independent mechanism.

E3 ubiquitin ligase RNF123-deficient mice exhibit reduced parasitemia and mortality in rodent malaria (Plasmodium yoelii 17XL) infection.

Increased levels of several human ubiquitin ligases, including ring finger protein 123 (RNF123), in red blood cells with Plasmodium falciparum infection, have been reported. RNF123 is an E3 ubiquitin ligase that is highly expressed in erythroid cells. However, the function of the RNF123 gene and the relationship between the RNF123 gene and malarial parasite has not been clarified in vivo. In this study, we generated RNF123-deficient mice using the CRISPR/Cas9 system, and analyzed malaria susceptibility and erythrocyte morphology. The levels of parasitemia 5days post-infection and mortality 21days post-infection with the lethal type of rodent malaria (Plasmodium yoelii 17XL) in RNF123-deficient mice was significantly lower than that in wild-type mice. In contrast, red blood cell morphology in RNF123-deficient mice was almost normal. These results suggest that erythrocytic RNF123 plays a role in susceptibility to rodent malaria infection, but does not play a role in erythrocyte morphology.