Leu promotes C2C12 cell differentiation by regulating the GSK3ß/ß-catenin signaling pathway through facilitating the interaction between SESN2 and RPN2.

BACKGROUND: Leucine (Leu) is an essential amino acid that facilitates skeletal muscle satellite cell differentiation, yet its mechanism remains underexplored. Sestrin2 (SESN2) serves as a Leu sensor, binding directly to Leu, while ribophorin II (RPN2) acts as a signaling factor in multiple pathways. This study aimed to elucidate Leu's impact on mouse C2C12 cell differentiation and skeletal muscle injury repair by modulating RPN2 expression through SESN2, offering a theoretical foundation for clinical skeletal muscle injury prevention and treatment. RESULTS: Leu addition promoted C2C12 cell differentiation compared to the control, enhancing early differentiation via myogenic determinant (MYOD) up-regulation. Sequencing revealed SESN2 binding to and interacting with RPN2. RPN2 overexpression up-regulated MYOD, myogenin and myosin heavy chain 2, concurrently decreased p-GSK3ß and increased nuclear ß-catenin. Conversely, RPN2 knockdown yielded opposite results. Combining RPN2 knockdown with Leu rescued increased p-GSK3ß and decreased nuclear ß-catenin compared to Leu absence. Hematoxylin and eosin staining results showed that Leu addition accelerated mouse muscle damage repair, up-regulating Pax7, MYOD and RPN2 in the cytoplasm, and nuclear ß-catenin, confirming that the role of Leu in muscle injury repair was consistent with the results for C2C12 cells. CONCLUSION: Leu, bound with SESN2, up-regulated RPN2 expression, activated the GSK3ß/ß-catenin pathway, enhanced C2C12 differentiation and expedited skeletal muscle damage repair. © 2024 Society of Chemical Industry.

RPN2 in cancer: An overview.

Oncogenes together with tumor suppresser genes are confirmed to regulate tumor phenotype in human cancers. RPN2, widely verified as an oncogene, encodes a protein that is part of an N-oligosaccharyl transferase, and is observed to be aberrantly expressed in human malignancies. Accumulating evidence unveils the vital functions of RPN2, contributing to tumorigenicity, metastasis, progression, and multi-drug resistance. Furthermore, previous studies partly indicated that RPN2 was involved in tumor progression via contributing to N-glycosylation and regulating multiple signaling pathways. In addition, RPN2 was also confirmed as a downstream target involved in tumor progression. Moreover, with demonstrated prognosis value and therapeutic target, RPN2 was also determined as a promising biomarker for forecasting patients' prognostic and therapy efficacy. In the present review, we aimed to summarize the present studies of RPN2 in cancer, and enhance the understanding of RPN2's extensive functions and clinical significances.

circTOP2A functions as a ceRNA to promote glioma progression by upregulating RPN2.

Competing endogenous RNA (ceRNA)-mediated signaling pathway dysregulation provides great insight into comprehensively understanding the molecular mechanism and combined targeted therapy for glioblastoma. circRNA is characterized by high stability, tissue/developmental stage-specific expression and abundance in brain and plays significant roles in the initiation and progression of cancer. Our previous published data have demonstrated that RPN2 was significantly upregulated in glioma and promoted tumor progression via the activation of the Wnt/ß-catenin pathway. Furthermore, we proved that miR-422a regulated the Wnt/ß-catenin signaling pathway by directly targeting RPN2. In this study, based on the glioblastoma microarray profiles, we identified the upstream circTOP2A, which completely bound to miR-422a and was co-expressed with the RPN2. circTOP2A was significantly overexpressed in glioma and conferred a poor prognosis. circTOP2A could regulate RPN2 expression by sponging miR-422a, verified by western blot, dual-luciferase reporter gene assay, and RNA pull-down assay. Functional assays including CCK8, transwell and FITC-annexin V were performed to explore the RPN2-mediated role of the circTOP2A effect on the glioma malignant phenotype. Additionally, TOP/FOP and immunofluorescence analysis were used to confirm that sh-circTOP2A could suppress the Wnt/ß-catenin pathway partly through RPN2. Finally, a tumor xenograft model was applied to validate the biological function of circTOP2A in vivo. Taken together, our findings reveal the critical role of circTOP2A in promoting glioma proliferation and invasion via a ceRNA mechanism and provide an exploitable biomarker and therapeutic target for glioma patients.

The N6-methyladenosine-mediated lncRNA WEE2-AS1 promotes glioblastoma progression by stabilizing RPN2.

Background: Glioblastoma (GBM) is the most common primary brain malignancy and has high aggressiveness and a poor prognosis. N6-methyladenosine (m6A) represents the most prevalent methylation modification of lncRNAs and has been shown to play important roles in the pathophysiological processes of tumors. However, the distribution and function of m6A modifications in lncRNAs in GBM tissues have not been fully revealed. Methods: The global depiction of m6A-modified lncRNA expression patterns in GBM tumor tissues was screened via m6A high-throughput sequencing. Gain- and loss-of-function assays were performed to investigate the role of WEE2-AS1 in GBM. Mass spectrometry and RNA-pulldown, RNA immunoprecipitation (RIP), luciferase reporter and coimmunoprecipitation assays were performed to explore the mechanism of m6A-mediated upregulation of WEE2-AS1 expression and the downstream mechanism promoting the malignant progression of GBM. Results: Herein, we report the differential expression profile of m6A-modified lncRNAs in human GBM tissues for the first time. WEE2-AS1 was identified as a novel m6A-modified lncRNA that promotes GBM progression and was post-transcriptionally stabilized by IGF2BP3, an m6A reader. Moreover, we confirmed that WEE2-AS1 promoted RPN2 protein stabilization by preventing CUL2-mediated RPN2 K322 ubiquitination, thereby contributing to GBM malignant progression by activating the PI3K-Akt signaling pathway. In translational medicine, we found that blocking WEE2-AS1 expression improved the therapeutic sensitivity of dasatinib, a central nervous system penetrant that is FDA-approved in GBM. Conclusions: Overall, this work highlights that WEE2-AS1 may serve as a potential prognostic biomarker and therapeutic target in GBM, the knockdown of which significantly improves the efficacy of dasatinib, providing a promising strategy for improving targeted combination therapy for GBM patients.

Ribophorin II promotes the epithelial-mesenchymal transition and aerobic glycolysis of laryngeal squamous cell carcinoma via regulating reactive oxygen species-mediated Phosphatidylinositol-3-Kinase/Protein Kinase B activation.

Ribophorin II (RPN2), a part of an N-oligosaccharyl transferase complex, plays vital roles in the development of multiple cancers. Nevertheless, its biological role in laryngeal squamous cell carcinoma (LSCC) remains unclear. The RPN2 expression levels in LSCC tissues and cell lines (AMC-HN-8 and TU212) were measured using real-time PCR, immunohistochemistry, or Western blot. The influences of RPN2 on the proliferation, migration, epithelial-mesenchymal transition, and aerobic glycolysis of LSCC cells were investigated after upregulation or downregulation of RPN2 in vitro and in vivo. Mechanically, we assessed the impact of RPN2 on the reactive oxygen species (ROS)/Phosphatidylinositol-3-Kinase (PI3K)/Protein Kinase B (Akt) signaling pathway. We found that compared with the control, RPN2 was highly expressed in LSCC tissues and cells. Overexpression of RPN2 elevated the proliferation, migration, glucose uptake, lactate production release, and levels of Vimentin, hexokinase-2 (HK-2), pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase A (LDHA), and ROS, but inhibited E-cadherin expression in AMC-HN-8 cells. Knockdown of RPN2 in TU212 cells showed opposite effects on the above indexes. Meanwhile, RPN2 upregulation increased the levels of p-PI3K/PI3K and p-Akt/Akt, which were attenuated by N-acetyl-L-cysteine (NAC), an ROS inhibitor. Both NAC and PI3K inhibitor LY294002 could reverse the effects of RPN2 overexpression on the malignant phenotypes of LSCC cells. In xenografted mice, silencing RPN2 expression reduced tumor growth, ROS production, and levels of Ki-67, Vimentin, LDHA, and p-Akt/Akt, but enhanced E-cadherin expression. In conclusion, our data suggested that RPN2 promoted the proliferation, migration, EMT, and glycolysis of LSCC via modulating ROS-mediated PI3K/Akt activation.

Sevoflurane represses the progression of glioma by the regulation of circ_0037655/miR-130a-5p/RPN2 axis.

Sevoflurane (SEV) is a common anesthetic to inhibit glioma progression. The previous studies have indicated the molecular mechanisms of SEV function in glioma. The objective of this study was to explore the association of circ_00037655 with SEV in glioma. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was analyzed using Edu assay and colony formation assay. Flow cytometry was applied to determine cell apoptosis. Protein analysis was performed via western blot. Cell migration and invasion were assessed by transwell assay. Circ_0037655, microRNA-130a-5p (miR-130a-5p) and ribophorin II (RPN2) levels were detected using the quantitative real-time polymerase chain reaction (qRT-PCR). Dual-luciferase reporter, RNA immunoprecipitation (RIP) and pull-down assays were used to analyze target interaction. The effect of circ_0037655 on SEV in vivo was researched by xenograft models. SEV reduced cell viability, proliferation, migration and invasion but induced apoptosis of glioma cells. Circ_0037655 expression was inhibited after SEV treatment in glioma cells. The effects of SEV on glioma cell behaviors were attenuated by upregulation of circ_0037655. Circ_0037655 interacted with miR-130a-5p and miR-130a-5p targeted RPN2. Circ_0037655 or miR-130a-5p regulated the anti-tumor function of SEV in glioma by targeting miR-130a-5p or RPN2. Circ_0037655 affected the expression of RPN2 via targeting miR-130a-5p. Circ_0037655 relieved SEV-induced glioma growth inhibition in vivo by mediating miR-130a-5p and RPN2 levels. SEV inhibited the malignant progression of glioma cells partly by regulating the circ_0037655/miR-130a-5p/RPN2 axis.

Epigenetically-regulated RPN2 gene influences lymphocyte activation and is involved in pathogenesis of rheumatoid arthritis.

BACKGROUND: To identify RA-associated genes and to ascertain epigenetic factors and functional mechanisms underlying RA pathogenesis. METHODS: Peripheral blood mononuclear cells (PBMC) transcriptome- and proteome- wide gene expressions were profiled in a case-control study sample. Differentially expressed genes (DEGs) were discovered and validated independently. In-house PBMC genome-wide SNP genotyping data, miRNA expression data and DNA methylation data in the same sample were utilized to identify SNPs [expression quantitative trait locus (eQTLs) and protein quantitative trait locus (pQTLs)], miRNAs, and DNA methylation positions (DMPs) regulating key DEG of interest. Lentivirus transfection was conducted to study the effects of RPN2 on T lymphocyte activation, proliferation, apoptosis, and inflammatory cytokine expression. Rpn2 protein level in plasma was quantitated by ELISA to assess its performance in discriminating RA cases and controls. RESULTS: Twenty-two DEGs were discovered in PBMCs. The most significant DEG, i.e., RPN2, was validated to be up-regulated with RA in PBMCs. A complex regulatory network for RPN2 gene expression in PBMCs was constructed, which consists of 38 eQTL and 53 pQTL SNPs, 3 miRNAs and 2 DMPs. Besides, RPN2 expression was significantly up-regulated with RA in primary T lymphocytes, as well as in PHA-activated T lymphocytes. RPN2 over-expression in T lymphocytes significantly inhibited apoptosis and IL-4 expression and promoted proliferation and activation. PBMCs-expressed RPN2 mRNA and plasma Rpn2 protein demonstrated superior and modest performances in discriminating RA cases and controls, respectively. CONCLUSIONS: RPN2 gene influences T lymphocyte growth and activation and is involved in the pathogenesis of RA. Rpn2 may serve as a novel protein biomarker for RA diagnosis.

Ribophorin II Overexpression Is Associated with Poor Response to Induction Chemotherapy with Docetaxel, Cisplatin, and Fluorouracil in P16-Negative Locally Advanced Head and Neck Squamous Cell Carcinoma.

This study aims to evaluate the relationship between human ribophorin II (RPN2) and the effect of treatment using induction therapy with docetaxel, cisplatin, and fluorouracil (TPF) for p-16 negative locally advanced head and neck squamous cell carcinoma (HNSCC). A total of 203 patients with locally advanced p-16 negative HNSCC who received induction chemotherapy with TPF at the Kaohsiung Chang Gung Memorial Hospital between 2009 and 2014 were enrolled. Immunohistochemistry (IHC) for RPN2 was examined and correlated with treatment outcome. Our study showed that RPN2 overexpression was significantly correlated with a poor response to induction chemotherapy with TPF. Both RPN2 overexpression and clinical N1 to N3 stages represented adverse prognostic factors for progression-free survival (PFS) and overall survival (OS). RPN2 might be a predictive marker for treatment response to induction chemotherapy. Further clinical trials are needed to determine the therapeutic significance of RPN2 in patients with HNSCC.

Overexpression of ribophorin II is required for viability of nasopharyngeal cancer cells by regulating JAK1/STAT3 activation.

OBJECTIVE: This study aimed to elucidate the role of ribophorin II (RPN2) in nasopharyngeal cancer (NPC) cell survival and death. RPN2 expression was upregulated in 22 human NPC specimens and 5-8F and CNE1 cells compared with that in adjacent normal tissues and normal nasopharyngeal NP69 cells. MATERIALS AND METHODS: CCK-8 and colony formation assays indicated that the silencing of RPN2 hindered the proliferation and growth of 5-8F and CNE1 cells. RESULTS: RPN2 expression was upregulated in 22 human NPC specimens as well as in 5-8F and CNE1 cells compared with that in adjacent normal tissues and NP69 cells. CCK-8 and colony formation assays indicated that the silencing of RPN2 reduced the proliferation and growth of 5-8F and CNE1 cells. Annexin V/PI flow cytometry and Bcl-2/Bax analysis showed that RPN2 silencing led to increased apoptosis. Moreover, JAK1 was found to interact with RPN2, and total JAK1, STAT3, and phosphorylated STAT3 levels were dramatically decreased in cells with RPN2 silencing. Furthermore, the nuclear localization of STAT3 was blocked by the silencing of RPN2. The administration of the STAT3 activator colivelin could offset the inhibitory effect of RPN2 silencing on the survival and apoptosis of NPC cells. CONCLUSION: RPN2 is upregulated in NPC tissues or cells, and RPN2 silencing repressed NPC cell proliferation and elicited apoptosis. RPN2 overexpression is possibly associated with JAK1/STAT3 silencing and activation. Finally, RPN2 represents a promising target for NPC treatment.

RPN2 Predicts Poor Prognosis and Promotes Bladder Cancer Growth and Metastasis via the PI3K-Akt Pathway.

BACKGROUND: Ribophorin II (RPN2) is a highly conserved glycoprotein involved in the N-linked glycosylation of multiple proteins. RPN2 was reported to be associated with malignant phenotype in several tumors. However, the function of RPN2 in bladder cancer (BCa) remains unclear. METHODS: Expression of RPN2 in BCa and adjacent tissues was compared by bioinformatics analysis, immunohistochemistry, and Western blotting. qRT-PCR was performed to explore the correlation between RPN2 expression and various clinical features in 38 patients. We assessed the effects of RPN2 on the biological activity of BCa both in vitro and in vivo, and explored its potential mechanisms based on gene set enrichment analysis (GSEA). RESULTS: We found that RPN2 was highly expressed in human BCa compared with normal adjacent tissues. There was a significant positive correlation between higher RPN2 mRNA levels and tumor T stage, lymph node (LN) metastasis and the degree of pathological differentiation in 38 patients with BCa. We further demonstrated that RPN2 silencing inhibited the growth and metastasis of BCa both in vitro and in vivo. Western blotting revealed that RPN2 knockdown suppressed epithelial-mesenchymal transition (EMT) and inhibited the PI3K-Akt pathway. CONCLUSION: These data suggest that RPN2 functions as an oncogene to promote tumor development and is a promising prognostic factor and therapeutic target in BCa.

circ_SMAD2 regulate colorectal cancer cells proliferation through targeting miR-1258/RPN2 signaling pathway.

Circular RNAs (circRNAs) are associated with various diseases, including cancers. However, their roles in colorectal cancer (CRC) have not been established. Hsa_circ_0000847 (circ_SMAD2) is a novel circRNA that was found to be elevated in CRC cell lines and tissues. High circ_SMAD2 levels were positively correlated with CRC clinicopathological features. Functional assays revealed that circ_SMAD2 enhanced CRC cell invasion, proliferation, and tumor growth. Mechanistically, circ_SMAD2 elevated Ribophorin II (RPN2) levels by inhibiting miR-1258. Therefore, circ_SMAD2 is a potential indicator for CRC progression.

Circ_0046599 Promotes the Development of Hepatocellular Carcinoma by Regulating the miR-1258/RPN2 Network.

BACKGROUND: Many studies have confirmed that circular RNAs (circRNAs) play a key role in the biological progression of cancers. However, the function of a novel circRNA, circ_0046599, in hepatocellular carcinoma (HCC) progression has not been explored. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression of circ_0046599, microRNA (miR)-1258 and Ribophorin II (RPN2). Subcellular fractionation location assay was used to localize circ_0046599 in HCC cells. The circular characteristic of circ_0046599 was verified using Ribonuclease R (RNase R) digestion assay. Besides, cell counting kit 8 (CCK8) assay, colony formation assay, wound healing assay and transwell assay were used to detect cell proliferation, migration and invasion, respectively. The lactate production and glucose level were determined by Lactate and Glucose Assay Kits. Furthermore, the protein levels of glycolysis, metastasis and proliferation-related marker proteins, as well as RPN2 were tested by Western blot (WB) analysis. Moreover, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to confirm the interactions among circ_0046599, miR-1258 and RPN2. In addition, mice xenograft models were applied to observe the effect of circ_0046599 silencing on HCC tumor growth in vivo. RESULTS: Circ_0046599 was highly expressed in HCC tissues and cells, and its knockdown could suppress HCC cell proliferation, migration, invasion and glycolysis process. MiR-1258 could be targeted by circ_0046599, and its inhibitor could invert the suppressing effect of circ_0046599 knockdown on HCC progression. Further, RPN2 was a target of miR-1258. Overexpressed RPN2 could reverse the inhibiting effect of miR-1258 overexpression on HCC progression. Also, knockdown of circ_0046599 could restrain HCC tumor growth in vivo. CONCLUSION: Our results provided new evidence that circ_0046599 could promote the progression of HCC by increasing RPN2 expression via sponging miR-1258.

Overexpression of RPN2 suppresses radiosensitivity of glioma cells by activating STAT3 signal transduction.

BACKGROUND: Radiation therapy is the primary method of treatment for glioblastoma (GBM). Therefore, the suppression of radioresistance in GBM cells is of enormous significance. Ribophorin II (RPN2), a protein component of an N-oligosaccharyl transferase complex, has been associated with chemotherapy drug resistance in multiple cancers, including GBM. However, it remains unclear whether this also plays a role in radiation therapy resistance in GBM. METHODS: We conducted a bioinformatic analysis of RPN2 expression using the UCSC Cancer Genomics Browser and GEPIA database and performed an immunohistochemical assessment of RPN2 expression in biopsy specimens from 34 GBM patients who had received radiation-based therapy. We also studied the expression and function of RPN2 in radiation-resistant GBM cells. RESULTS: We found that RPN2 expression was upregulated in GBM tumors and correlated with poor survival. The expression of RPN2 was also higher in GBM patients with tumor recurrence, who were classified to be resistant to radiation therapy. In the radiation-resistant GBM cells, the expression of RPN2 was also higher than in the parental cells. Depletion of RPN2 in resistant cells can sensitize these cells to radiation-induced apoptosis, and overexpression of RPN2 had the reverse effect. Myeloid cell leukemia 1 (MCL1) was found to be the downstream target of RPN2, and contributed to radiation resistance in GBM cells. Furthermore, STAT3 was found to be the regulator of MCL1, which can be activated by RPN2 dysregulation. CONCLUSION: Our study has revealed a novel function of RPN2 in radiation-resistant GBM, and has shown that MCL1 depletion or suppression could be a promising method of therapy to overcome the resistance promoted by RPN2 dysregulation.

An Extended Conformation for K48 Ubiquitin Chains Revealed by the hRpn2:Rpn13:K48-Diubiquitin Structure.

Rpn13/Adrm1 is recruited to the proteasome by PSMD1/Rpn2, where it serves as a substrate receptor that binds preferentially to K48-linked ubiquitin chains, an established signal for protein proteolysis. Here, we use NMR to solve the structure of hRpn13 Pru:hRpn2 (940-953):K48-diubiquitin. Surprisingly, hRpn2-bound hRpn13 selects a dynamic, extended conformation of K48-diubiquitin that is unique from previously determined structures. NMR experiments on free K48-diubiquitin demonstrate the presence of the reported "closed" conformation observed by crystallography, but also this more extended state, in which the hRpn13-binding surface is exposed. This extended K48-diubiquitin conformation is defined by interactions between L73 from G76-linked (distal) ubiquitin and a Y59-centered surface of K48-linked (proximal) ubiquitin. Furthermore, hRpn13 exchanges between the two ubiquitins within 100 ms, although prefers the proximal ubiquitin due to interactions with the K48 linker region. Altogether, these data lead to a revised model of how ubiquitinated substrates interact with the proteasome.

Overexpression of RPN2 promotes osteogenic differentiation of hBMSCs through the JAK/STAT3 pathway.

Osteoporosis is characterized by decreased bone mass and degenerating bone structure, which cause severe bone fragility and increase the risk for fractures. Human bone mesenchymal stem cells (hBMSCs) differentiate into osteoblasts through osteogenesis, and disturbances in the balance between bone generation and degeneration underlie the pathogenesis of senile osteoporosis. The highly conserved glycoprotein Ribophorin II (RPN2) is involved in multiple biological reactions, but the role of RPN2 in the osteogenic differentiation of hBMSCs and their molecular etiology is incompletely understood. Here, we show that RPN2 expression is up-regulated in hBMSCs during osteogenic differentiation. In vitro assays revealed that silencing of RPN2 inhibited hBMSC differentiation into osteoblasts. Moreover, RPN2 overexpression enhanced the expression of linked genes and resulted in high alkaline phosphatase activity. Our results suggest that RPN2 targets Janus kinase 1 (JAK1), and RPN2 overexpression was observed to induce JAK1 ubiquitination. Depletion of JAK1 facilitated osteogenic differentiation of RPN2-silenced hBMSCs. Moreover, western blot analysis revealed that RPN2 silencing suppressed the stimulation and nuclear translocation of the downstream signal transducer and activator of transcription 3 sensor; this could be reversed via RPN2 overexpression. This research sheds light on an innovative molecular mechanism that is associated with hBMSC differentiation into osteoblasts and may facilitate bone anabolism through RPN2.

RPN2 promotes metastasis of hepatocellular carcinoma cell and inhibits autophagy via STAT3 and NF-κB pathways.

This study aimed to investigate the function and the molecular mechanism of Ribophorin II (RPN2) in regulating Hepatocellular carcinoma (HCC) cell growth, metastasis, and autophagy. Quantitative real-time PCR (qPCR), western blotting analysis, and immunofluorescence assay were utilized to detect the RPN2 expression in HCC cell lines and specimens of HCC patients. We discovered that RPN2 expression was upregulated in HCC cell lines and tissues of HCC patients, which correlated with the low histological grade and low survival rate. Enhanced RPN2 expression stimulated cell proliferation, metastasis, invasion, and epithelial-mesenchymal transition (EMT), and decreased Microtubule-associated protein light chain 3B (LC3B) synthesis and reduced the expression of p62 protein. Further studies suggested that matrix metalloproteinase 9 (MMP-9) was partially upregulated by RPN2 via Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65. Interestingly, we found that phosphorylated RPN2 activated the signal transducer and activator of transcription 3 (STAT3) in HCC cells. It was also accountable for RPN2-stimulated elevated expression of MMP-9 and for invading HCC cells. It can be concluded that over-expression of RPN2 in HCC aggravated the malignant progression into cancerous cells. This research provided new evidences that RPN2 could facilitate tumor invasion by increasing the expression of MMP-9 in HCC cells.

Phosphorylation of Tyr-950 in the proteasome scaffolding protein RPN2 modulates its interaction with the ubiquitin receptor RPN13.

Protein substrates are targeted to the 26S proteasome through several ubiquitin receptors. One of these receptors, RPN13, is recruited to the proteasome by binding of its N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain to C-terminal residues of the scaffolding protein RPN2. The RPN13 PRU domain is followed by a flexible linker and a C-terminal deubiquitylase adaptor (DEUBAD) domain, which recruits and activates the deubiquitylase UCH37. Both RPN13 and UCH37 have been implicated in human cancers, and inhibitors of the RPN2-RPN13 interaction are being developed as potential therapeutic anticancer agents. Our current study builds on the recognition that a residue central to the RPN2-RPN13 interaction, RPN2 Tyr-950, is phosphorylated in Jurkat cells. We found that the Tyr-950 phosphorylation enhances binding to RPN13. The crystal structure of the RPN2-RPN13 pTyr-950-ubiquitin complex was determined at 1.76-Å resolution and reveals specific interactions with positively charged side chains in RPN13 that explain how phosphorylation increases binding affinity without inducing conformational change. Mutagenesis and quantitative binding assays were then used to validate the crystallographic interface. Our findings support a model in which RPN13 recruitment to the proteasome is enhanced by phosphorylation of RPN2 Tyr-950, have important implications for efforts to develop specific inhibitors of the RPN2-RPN13 interaction, and suggest the existence of a previously unknown stress-response pathway.

Ribophorin II promotes cell proliferation, migration, and invasion in esophageal cancer cells in vitro and in vivo.

Esophageal cancer is a common digestive tract cancer, which is a serious threat to human health. Ribophorin II (RPN2) is a part of an N-oligosaccharyltransferase complex, which is excessively expressed in many kinds of cancers. In the present study, we explore the biological role of RNP2 in esophageal cancer. First, we found that the expression of RPN2 was higher in esophageal cancer tissues than in adjacent non-tumor tissues, and negatively correlated with E-cadherin expression. RPN2 expression levels in esophageal cancer tissues were positively associated with differentiation and tumor node metastasis (TNM) stage. Furthermore, the expression of RPN2 was increased significantly in esophageal cancer cell lines compared with normal cells. The effect of RPN2 down-regulation on cell proliferation, cell migration, and cell invasion was examined by cell counting kit-8 (CCK8), wound healing assay, and Transwell assay, respectively. Silencing RPN2 effectively inhibited cell proliferation of esophageal cancer cells in vitro and in vivo Cell migration and invasion were also weakened dramatically by siRPN2 treatment of esophageal cancer cells. In addition, protein expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP-2), and E-cadherin in esophageal cancer cells was determined by Western blot analysis. PCNA, MMP-2, E-cadherin, Snail and phosphorylation-Smad2/3 expression was also regulated notably by siRPN2 treatment. These findings indicate that RPN2 exhibits oncogenetic capabilities in esophageal cancer, which could provide novel insights into esophageal cancer prevention and treatment.

Canthin-6-One Accelerates Alpha-Synuclein Degradation by Enhancing UPS Activity: Drug Target Identification by CRISPR-Cas9 Whole Genome-Wide Screening Technology.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of protein aggregates (namely Lewy bodies) in dopaminergic neurons in the substantia nigra region of the brain. Alpha-synuclein (α-syn) is the major component of Lewy bodies in PD patients, and impairment of the ubiquitin-proteasome system has been linked to its accumulation. In this work, we developed a tetracycline-inducible expression system, with simultaneous induced expression of α-syn-EGFP and a bright red fluorescent protein marker (mCherry) to screen for potential compounds for degrading α-syn. We identified canthin-6-one as an α-syn lowering compound which promoted both wild type and mutants α-syn degradation in an ubiquitin-proteasome-system (UPS) dependent manner. By CRISPR/Cas9 genome-wide screening technology, we identified RPN2/PSMD1, the 26S proteasome non-ATPase regulatory subunit 1, as the targeting gene for pharmacological activity of canthin-6-one. Finally, we showed that canthin-6-one up-regulates PSMD1 and enhances UPS function by activating PKA.