RAD23B mediated proteasomal degradation occurs through p38 MAPK/ATF-2/RAD23B axis under nutrient-deprived conditions in breast cancer.

Metabolic reprogramming in cancer occurs due to interaction of cells with the surrounding tumor microenvironment. In the microenvironment of solid tumors, nutrient deprivation is induced by high consumption of nutrients and insufficient vasculature. Tumor cells alter their metabolic strategies to adapt to the microenvironment. To understand the role of these metabolic changes, in the current study, we have mimicked nutrient deprivation condition in vitro to evaluate the associated signaling pathways in breast cancer cells. In our study, we have shown that nutritional deprivation activated p38 MAPK and activating transcription factor-2 (ATF-2) by increased phosphorylation of Thr180/Tyr182 and Thr71, respectively, in breast cancer cells. Pharmacological inhibition of p38 MAPK showed increased cell viability and reduced expression of ATF-2 and RAD23B under nutrient starvation conditions. Further, silencing of ATF-2 showed increased cell viability and decreased expression of RAD23B under nutrient starvation conditions. This suggests the involvement of p38 MAPK/ATF-2/RAD23B axis as a signaling pathway under nutrition starvation in breast cancer cells. The RAD23B mediated proteasome activity was shown to be much higher under stress conditions indicating a crucial role of RAD23B as a target for breast cancer.

Landscape of protein-protein interactions during hepatitis C virus assembly and release.

Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites.

Knockdown of circ-RAD23B inhibits non-small cell lung cancer progression via the miR-142-3p/MAP4K3 axis.

BACKGROUND: The development of non-small cell lung cancer (NSCLC) is associated with the deregulation of circRNAs. The objective of this study was to investigate the effects of circ-RAD23B in NSCLC. METHODS: Circ-RAD23B expression, miR-142-3p and MAP4K3 was detected by qPCR. Cell proliferation was investigated by CCK-8 assay and colony formation assay. Cell migration and invasion were assessed by transwell assay. Angiogenesis ability was assessed by tube formation assay. Cell cycle distribution and cell apoptosis were monitored by flow cytometry. The predicted binding relationship between miR-142-3p and circ-RAD23B or MAP4K3 was verified by dual-luciferase reporter assay. The protein level of MAP4K3 was detected by western blot. Animal models were established to determine the role of circ-RAD23B in vivo. RESULTS: Circ-RAD23B was shown to be upregulated in NSCLC tissues and cells. Knockdown of circ-RAD23B inhibited proliferation, migration, invasion, angiogenesis and promoted cell cycle arrest and apoptosis in NSCLC cells, and circ-RAD23B knockdown also impeded tumor growth in vivo. Circ-RAD23B acted as miR-142-3p sponge to inhibit miR-142-3p expression and thus enrich the expression of MAP4K3, a target of miR-142-3p. Rescue experiments presented that miR-142-3p inhibition reversed the effects of circ-RAD23B knockdown, and MAP4K3 overexpression abolished the effects of miR-142-3p restoration. In addition, we found that circ-RAD23B knockdown led to decreased phosphorylation expression of ERK1/2, JNK and p38, three key groups of the MAPK signaling pathway. CONCLUSIONS: Circ-RAD23B knockdown inhibited NSCLC development by regulating the miR-142-3p/MAP4K3 axis, which might be associated with the inactivation of the MAPK signaling pathway.

Deubiquitinase PSMD7 promotes the proliferation, invasion, and cisplatin resistance of gastric cancer cells by stabilizing RAD23B.

Ubiquitination, a crucial post-translational modification, controls substrate degradation and can be reversed by deubiquitinases (DUBs). An increasing number of studies are showing that DUBs regulate the malignant behavior and chemotherapy resistance of gastric cancer (GC) by stabilizing various proteins. However, the expression level and biological function of the DUB, proteasome 26S subunit, non-ATPase 7 (PSMD7), in GC remains unknown. Herein, we report for the first time that PSMD7 is frequently overexpressed in GC tissues. Elevated levels of PSMD7 were also detected in GC cell lines. Notably, the upregulation of PSMD7 closely correlated with malignant clinical parameters and reduced the survival of GC patients. Functionally, we found that PSMD7 knockdown consistently suppressed the proliferation, migration, and invasion of AGS and SGC-7901 cells. Ectopic expression of PSMD7 facilitated GC cell proliferation and mobility. Based on protein-protein interaction prediction, RAD23 homolog B (RAD23B) protein was identified as a candidate substrate of PSMD7. PSMD7 positively regulated the abundance of RAD23B and xeroderma pigmentosum, complementation group C (XPC) protein in GC cells. The interaction between PSMD7 and RAD23B was confirmed using protein immunoprecipitation. PSMD7 knockdown enhanced the ubiquitination and degradation of RAD23B protein in GC cells. PSMD7 promoted cell viability, apoptosis resistance, and DNA damage repair in GC cells upon cisplatin (DDP) treatment. Moreover, PSMD7 silencing inhibited tumor growth and enhanced the sensitivity of GC cells to DDP treatment in mice. In summary, PSMD7 was highly expressed in GC and contributed to the malignant behavior and DDP resistance of tumor cells by stabilizing RAD23B.

Molecular and Dynamic Evaluation of Proteins Related to Resistance to Neoadjuvant Treatment with Chemoradiotherapy in Circulating Tumor Cells of Patients with Locally Advanced Rectal Cancer.

The heterogeneity of response to neoadjuvant chemoradiotherapy (NCRT) is still a challenge in locally advanced rectal cancer (LARC). The evaluation of thymidylate synthase (TYMS) and RAD23 homolog B (RAD23B) expression in circulating tumor cells (CTCs) provides complementary clinical information. CTCs were prospectively evaluated in 166 blood samples (63 patients) with LARC undergoing NCRT. The primary objective was to verify if the absence of RAD23B/TYMS in CTCs would correlate with pathological complete response (pCR). Secondary objectives were to correlate CTC kinetics before (C1)/after NCRT (C2), in addition to the expression of transforming growth factor-ß receptor I (TGF-ßRI) with survival rates. CTCs were isolated by ISET and evaluated by immunocytochemistry (protein expression). At C1, RAD23B was detected in 54.1% of patients with no pCR and its absence in 91.7% of patients with pCR (p = 0.014); TYMS- was observed in 90% of patients with pCR and TYMS+ in 51.7% without pCR (p = 0.057). Patients with CTC2 > CTC1 had worse disease-free survival (DFS) (p = 0.00025) and overall survival (OS) (p = 0.0036) compared with those with CTC2 ≤ CTC1. TGF-ßRI expression in any time correlated with worse DFS (p = 0.059). To conclude, RAD23B/TYMS and CTC kinetics may facilitate the personalized treatment of LARC.

Cytoplasmic RAD23B interacts with CORO1C to synergistically promote colorectal cancer progression and metastasis.

Colorectal cancers (CRCs) are characterized by diffuse infiltration of tumor cells into the regional lymph nodes and metastasis to distant organs, and its highly invasive nature contributes to disease recurrence and poor outcomes. The molecular mechanisms underlying CRC cell invasion remain incompletely understood. Here, we identified the upregulation of DNA damage repair-related protein RAD23B in CRC cells and tissues and showed that it associates with coronin 1C or coronin 3 (CORO1C) to facilitate invasion. We found that knockdown of RAD23B expression significantly inhibited the proliferation, invasion, and migration abilities of CRC cells both in vitro and in vivo, and suppressed the talin1/2/integrin/FAK/RhoA/Rac1/CORO1C signaling pathways. Interestingly, RAD23B interacted and co-localized with CORO1C, and CORO1C aggregated toward the margin of cancer cells in both CRC cells and tissues when RAD23B overexpressed. Mechanistically, overexpression of RAD23B and/or CORO1C further increased invadopodia formation and matrix degradation in SW480 and HCT8 CRC cells. Conversely, silencing of RAD23B expression suppressed tumorigenesis and liver metastasis in xenotransplant murine models. Furthermore, we found that RAD23B was significantly overexpressed in tumor tissues (n = 720) compared to adjacent non-tumor tissues (n = 694) of patients with CRC. Finally, we identified a strong correlation between higher levels of cytoplasmic expression of RAD23B, and poor prognosis and liver metastasis in CRC patients. Taken together, our data highlight a novel RAD23B-CORO1C signaling axis in CRC cell invasion and metastasis that may be of clinical significance.

UHRF2 commissions the completion of DNA demethylation through allosteric activation by 5hmC and K33-linked ubiquitination of XRCC1.

The transition of oxidized 5-methylcytosine (5mC) intermediates into the base excision repair (BER) pipeline to complete DNA demethylation remains enigmatic. We report here that UHRF2, the only paralog of UHRF1 in mammals that fails to rescue Uhrf1-/- phenotype, is physically and functionally associated with BER complex. We show that UHRF2 is allosterically activated by 5-hydroxymethylcytosine (5hmC) and acts as a ubiquitin E3 ligase to catalyze K33-linked polyubiquitination of XRCC1. This nonproteolytic action stimulates XRCC1's interaction with the ubiquitin binding domain-bearing RAD23B, leading to the incorporation of TDG into BER complex. Integrative epigenomic analysis in mouse embryonic stem cells reveals that Uhrf2-fostered TDG-RAD23B-BER complex is functionally linked to the completion of DNA demethylation at active promoters and that Uhrf2 ablation impedes DNA demethylation on latent enhancers that undergo poised-to-active transition during neuronal commitment. Together, these observations highlight an essentiality of 5hmC-switched UHRF2 E3 ligase activity in commissioning the accomplishment of active DNA demethylation.

Protein quality control by the proteasome and autophagy: A regulatory role of ubiquitin and liquid-liquid phase separation.

Degradation of dysfunctional, damaged, or misfolded proteins is a crucial component of the protein quality control network to maintain cellular proteostasis. Dysfunction in proteostasis regulation due to imbalances in protein synthesis, folding, and degradation challenges the integrity of the cellular proteome and favors the accumulation of aggregated proteins that can damage cells by a loss of their functions and/or a gain of adverse functions. Ubiquitination is an essential player in proteostasis regulation but also in orchestrating signaling pathways in response to various stress conditions. Both cellular degradation systems, the proteasome and autophagy, employ ubiquitin for selection and targeting of substrates to the degradative machineries. Here we summarize the manifold functions of ubiquitin in protein degradation and discuss its emerging role in the formation of biomolecular condensates through liquid-liquid phase separation, which allows spatiotemporal regulation of protein quality control.

The DNA damage-sensing NER repair factor XPC-RAD23B does not recognize bulky DNA lesions with a missing nucleotide opposite the lesion.

The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were -2.5-3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 ß-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.

Arabidopsis RAD23B regulates pollen development by mediating degradation of KRP1.

The ubiquitin (Ub)/26S proteasome system (UPS) plays a key role in plant growth, development, and survival by directing the turnover of numerous regulatory proteins. In the UPS, the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains function as hubs for ubiquitin-mediated protein degradation. Radiation sensitive 23 (RAD23), which has been identified as a UBL/UBA protein, contributes to the progression of the cell cycle, stress responses, ER proteolysis, and DNA repair. Here, we report that pollen development is arrested at the microspore stage in a rad23b null mutant. We demonstrate that RAD23B can directly interact with KIP-related protein 1 (KRP1) through its UBL-UBA domains. In addition, plants overexpressing KRP1 have defects in pollen development, which is a phenotype similar to the rad23b mutant. RAD23B promotes the degradation of KRP1 in vivo, which is accumulated following treatment with the proteasome inhibitor MG132. Our results indicate that RAD23B plays an important in pollen development by controlling the turnover of the key cell cycle protein, KRP1.

Upregulation of GRIM-19 augments the sensitivity of prostate cancer cells to docetaxel by targeting Rad23b.

The gene associated with retinoid-interferon mortality (GRIM-19) has been reported to be correlated with drug resistance, whereas its functional role in prostate cancer (PC) is not fully understood. This study aims to clarify the potential role and molecular mechanisms of GRIM-19 on the response of PC cells to chemical drug docetaxel. mRNA and protein level of GRIM-19 expression in cells and tissues of PC were measured by quantitative real-time PCR and western blot, respectively. Knock-down of GRIM-19 in PC cells was performed using siRNA. Cell apoptosis was determined by flow cytometric analysis. DNA damage in PC cells was detected by γ-H2AX staining. GRIM-19 was downregulated in PC tissues and cell lines. Knock-down of GRIM-19 increased the resistance of PC cells to docetaxel, and overexpression of GRIM-19 promoted docetaxel-induced apoptotic death in PC cells. Mechanistically, GRIM-19 downregulated the expression of the survival gene Rad23b, which promoted DNA damage repair. Overexpression of Rad23b reversed GRIM-19-mediated response to docetaxel in PC cells. GRIM-19 promoted the sensitivity of PC cells to docetaxel by downregulating Rad23b, which may serve as a promising target to develop a better strategy of chemotherapy for PC.

CircRAD23B facilitates proliferation and invasion of esophageal cancer cells by sponging miR-5095.

Esophageal cancer is one of the most malignant tumors in digestive system, but the pathogenesis of esophageal cancer is still unclear. It has been verified that circular RNAs (circRNAs) play critical roles in development and progression of tumors. However, little research concentrates on the role of circRNAs in the pathogenesis of esophageal cancer. In the present study, we found that circular RNA-RAD23B (circRAD23B) was upregulated in specimens of patients with esophageal cancer. Further investigation revealed that circRAD23B promoted proliferation and invasion of esophageal cancer cells. Next, we identified microRNA-5095 (miR-5095) as a target of circRAD23B, and found that miR-5095 was negatively correlated to the expression of circRAD23B in esophageal cancer. In addition, circRAD23B facilitated expression of PARP2 and AKT2 by sponging miR-5095, which might underlie the growth of esophageal cancer. In summary, these data displayed the crucial role of circRAD23B/miR-5095 regulating PARP2 and AKT2 in esophageal cancer, and provided a novel mechanism in the pathogenesis of esophageal cancer.

Molecular and Kinetic Analyses of Circulating Tumor Cells as Predictive Markers of Treatment Response in Locally Advanced Rectal Cancer Patients.

Neoadjuvant chemoradiation (NCRT) followed by total mesorectal excision is the standard treatment for locally advanced rectal cancer (LARC). To justify a non-surgical approach, identification of pathologic complete response (pCR) is required. Analysis of circulating tumor cells (CTCs) can be used to evaluate pCR. We hypothesize that monitoring of thymidylate synthase (TYMS) and excision repair protein, RAD23 homolog B (RAD23B), can be used to predict resistance to chemotherapy/radiotherapy. Therefore, the aims of this study were to analyze CTCs from patients with LARC who underwent NCRT plus surgery for expression of TYMS/RAD23B and to evaluate their predictive value. Blood samples from 30 patients were collected prior to NCRT (S1) and prior to surgery (S2). CTCs were isolated and quantified by ISET®, proteins were analyzed by immunocytochemistry, and TYMS mRNA was detected by chromogenic in situ hybridization. CTC counts decreased between S1 and S2 in patients exhibiting pCR (p = 0.02) or partial response (p = 0.01). Regarding protein expression, TYMS was absent in 100% of CTCs from patients with pCR (p = 0.001) yet was expressed in 83% of non-responders at S2 (p < 0.001). Meanwhile, RAD23B was expressed in CTCs from 75% of non-responders at S1 (p = 0.01) and in 100% of non-responders at S2 (p = 0.001). Surprisingly, 100% of non-responders expressed TYMS mRNA at both timepoints (p = 0.001). In addition, TYMS/RAD23B was not detected in the CTCs of patients exhibiting pCR (p = 0.001). We found 83.3% of sensitivity for TYMS mRNA at S1 (p = 0.001) and 100% for TYMS (p = 0.064) and RAD23B (p = 0.01) protein expression at S2. Thus, TYMS mRNA and/or TYMS/RAD23B expression in CTCs, as well as CTC kinetics, have the potential to predict non-response to NCRT and avoid unnecessary radical surgery for LARC patients with pCR.

Circular RNA circ-RAD23B promotes cell growth and invasion by miR-593-3p/CCND2 and miR-653-5p/TIAM1 pathways in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is an aggressive malignancy with poor clinical outcomes. Accumulating evidence indicated that dysregulation of circular RNAs (circRNAs) plays a key role in multiple solid tumors. In this study, circ-RAD23B was explored. The expression of circ-RAD23B in NSCLC was detected by RT-qPCR. The clinical value of circ-RAD23B was analyzed by Fisher's exact test and Kaplan-Meier curves. Gain and loss of function experiments were carried out to elucidate the biological functions of circ-RAD23B in NSCLC cell lines. Dual luciferase reporter assay and rescue experiments were used to reveal the mechanism of circ-RAD23B. The findings demonstrated that circ-RAD23B, identified to be amplified and overexpressed in NSCLC, was associated with lymph node invasion, lower differentiation grade and shorter overall survival (OS). Furthermore, circ-RAD23B functions as an oncogene in NSCLC cells. Mechanistically, circ-RAD23B could sponge miR-593-3p and miR-653-5p and thus elevate CCND2 and TIAM1 expression, respectively. Rescue assays proved that circ-RAD23B promotes cell growth via miR-593-3p/CCND2 axis and facilitates cell invasion by miR-653-5p/TIAM1 pathway. Taken together, we propose circ-RAD23B as a promising biomarker and therapeutic target for NSCLC.

Serum starvation raises turnover of phosphorylated p62/SQSTM1 (Serine 349), reveals expression of proteasome and N-glycanase1 interactive protein RAD23B and sensitizes human synovial fibroblasts to BAY 11-7085-induced cell death.

Phosphorylation of p62/SQSTM1 (p62) on Serine 349 (P-Ser349 p62) as well as proteasome dysfunction have been shown to activate the cell protective Keap1/Nrf2 pathway. We showed previously that BAY 11-7085-induced human synovial fibroblast cell death includes autophagy and p62 downregulation. In this work, we have studied expression of P-Ser349 p62 in human synovial fibroblasts. Results showed that P-Ser349 p62 was not detected in synovial cell extracts unless cells were cultured in the presence of proteasome inhibitor (MG132). MG132 revealed P-Ser349 p62 turnover, that was further increased by concomitant autophagy inhibition and markedly enhanced in serum starved cells. Starvation sensitized synovial fibroblasts to BAY 11-7085 while MG132 protected both non-starved and starved cells from BAY 11-7085-induced cell death. Lentivirus mediated overexpression of phosphorylation-mimetic p62 mutant S349E markedly protected synovial fibroblasts from BAY 11-7085. Inhibitor of Keap1-P-S349 p62 interaction, K67, had synergistic effect with MG132. Starvation increased p62 molecular weight, that was reversed by serum and bovine serum albumin re-feeding. Furthermore, starvation markedly induced RAD23B. Increased endo-ß-N-acetylglucosaminidase (ENGase) turnover was detected in starved synovial fibroblasts. PNGase F treatment produced faster migration p62 form in human synovial tissue extracts but starvation-like p62 form of higher molecular weight in synovial cell extracts. Co-transfection of NGLY1, with p62 or p62 mutants S349A and S349E markedly stabilized p62 expressions in HEK293 cells. Tunicamycin upregulated p62 and protected synovial fibroblasts from BAY 11-7085-induced cell death. These results showed that P-Ser349 p62 has pro-survival role in human synovial fibroblasts and that de-glycosylation events are involved in p62 turnover.

Molecular basis for damage recognition and verification by XPC-RAD23B and TFIIH in nucleotide excision repair.

Global genome nucleotide excision repair (GG-NER) is the main pathway for the removal of bulky lesions from DNA and is characterized by an extraordinarily wide substrate specificity. Remarkably, the efficiency of lesion removal varies dramatically and certain lesions escape repair altogether and are therefore associated with high levels of mutagenicity. Central to the multistep mechanism of damage recognition in NER is the sensing of lesion-induced thermodynamic and structural alterations of DNA by the XPC-RAD23B protein and the verification of the damage by the transcription/repair factor TFIIH. Additional factors contribute to the process: UV-DDB, for the recognition of certain UV-induced lesions in particular in the context of chromatin, while the XPA protein is believed to have a role in damage verification and NER complex assembly. Here we consider the molecular mechanisms that determine repair efficiency in GG-NER based on recent structural, computational, biochemical, cellular and single molecule studies of XPC-RAD23B and its yeast ortholog Rad4. We discuss how the actions of XPC-RAD23B are integrated with those of other NER proteins and, based on recent high-resolution structures of TFIIH, present a structural model of how XPC-RAD23B and TFIIH cooperate in damage recognition and verification.

The heat responsive wheat TaRAD23 rescues developmental and thermotolerant defects of the rad23b mutant in Arabidopsis thaliana.

High temperature severely damage the growth and development of crops with climate change. To effectively screen heat responsive proteins in wheat (Triticum aestivum L.), the isobaric tandem mass tag (TMT)-labeled quantitative proteomic analysis and quantitative real-time PCR (qRT-PCR) were performed. Here, we found that a wheat RADIATION SENSITIVE 23 protein, TaRAD23, was up-regulated at both protein and RNA levels by exposing to heat stress. Sequence homology analysis indicated that the TaRAD23 is a conserved protein, which is closely related to the Arabidopsis thaliana proteins AtRAD23B and AtRAD23A. Genetic knockout of AtRAD23B, but not AtRAD23A, shows multiple developmental defects, as well as sensitivity to heat stress. Meanwhile, we observed that constitutive overexpression of TaRAD23 in rad23b fully rescued developmental and thermotolerant defects of the mutant. Furthermore, qRT-PCR analysis of heat responsive genes in rad23b and its complementary lines suggested that suppression of the heat shock transcription factor AtHSFA2 and heat responsive genes (HSP70, HSP90, HSP17.6 and HSA32) may be the cause of the weaker thermotolerance in rad23b. Taken together, the data suggest that the heat responsive TaRAD23 is a functionally highly conserved protein that plays an important role in development, as well as the regulation in heat stress response network.

MicroRNA-196b enhances the radiosensitivity of SNU-638 gastric cancer cells by targeting RAD23B.

Gastric cancer is characterized by resistance to ionizing radiation. The development of resistance to radiotherapy in gastric cancer patients is one of the obstacles to effective radiotherapy. MicroRNAs are small well-conserved non-coding RNA species that regulate post-transcriptional activation. Our study aimed to investigate the role of miR-196b in radiation-induced gastric cancer. In the present study, we found that miR-196b expression was significantly reduced following radiation. The ectopic miR-196b expression sensitized SNU-638 gastric cancer cells and increased γ-H2AX foci upon radiation treatment. Bioinformatics analysis suggested that the DNA repair protein RAD23B was a putative target gene of miR-196b. Overexpression of miR-196b suppressed RAD23B expression in SNU-638 cells. Reporter assays further showed that miR-196b inhibited RAD23B 3'-UTR luciferase activity. Knockdown of RAD23B by small interfering RNA transfection closely mimicked the outcomes of miR-196b transfection, leading to impaired DNA damage repair in gastric cancer cells. Our results show that miR-196b improved radiosensitivity of SNU-638 cells by targeting RAD23B. Our data indicate that miR-196b is a potential target to enhance the effect of radiation treatment on gastric cancer cells. These findings will provide evidence for a new therapeutic target in radiotherapy.

Subcellular distribution of RAD23B controls XPC degradation and DNA damage repair in response to chemotherapy drugs.

The RAD23B-XPC complex in the nucleus plays a key role in the initial damage recognition during global genome nucleotide excision repair (NER). Within the complex, XPC, a product of Xeroderma pigmentosum C, recognizes and interacts with the unpaired bases in the undamaged DNA strand, while RAD23B stabilizes XPC. However, how RAD23B is regulated by other factors is not well known. We report here a mode of spatial regulation of RAD23B that controls XPC stability and DNA damage repair. We first identified that RAD23B was able to directly associate with PAQR3, a newly-discovered tumor suppressor implicated in many types of human cancers. PAQR3 reduced the protein level of XPC, together with accelerated degradation and enhanced polyubiquitination of XPC. Mechanistically, PAQR3 reduces nucleic distribution of RAD23B by tethering it to the Golgi apparatus, thus diminishing the amount of RAD23B proteins available to interact with XPC in the nucleus. The viability of gastric cancer cells upon treatment with chemotherapy drugs including etoposide, cisplatin and doxorubicin was reduced by PAQR3 overexpression, but enhanced by PAQR3 knockdown. The degree of DNA damage induced by these drugs, as measured by immunoblotting with γ-H2AX, was elevated by PAQR3 overexpression and lessened by PAQR3 knockdown. Furthermore, a synthetic peptide comprising the N-terminus of PAQR3 was able to recapitulate the activity of PAQR3 in reducing XPC stability and enhancing chemotherapy drug-induced DNA damage. In conclusion, our study reveals that RAD23B is controlled by subcellular compartmentation, thus affecting XPC-mediated DNA damage repair in cancer cells.

HR23b expression is a potential predictive biomarker for HDAC inhibitor treatment in mesenchymal tumours and is associated with response to vorinostat.

Histone deacetylases (HDAC) are key players in epigenetic regulation of gene expression and HDAC inhibitor (HDACi) treatment seems to be a promising anticancer therapy in many human tumours, including soft tissue sarcomas. HR23b has been shown to be a potential biomarker for sensitivity to HDACi therapy in cutaneous T-cell lymphoma and hepatocellular carcinoma. We aimed to evaluate HR23b as a candidate biomarker for HDACi response in sarcomas and gastrointestinal stromal tumours (GIST). Therefore, HR23b expression was analysed comprehensively by western blot in sarcoma and GIST cell lines covering all major clinically relevant subtypes. MTT assay and ApoTox-Glo(TM) Triplex assay were performed after treatment with vorinostat, belinostat, mocetinostat and entinostat. HR23b protein expression was measured under HDACi treatment. Furthermore, HR23b expression levels were immunohistochemically determined in a large set of 523 clinical samples from sarcoma and GIST patients. Western blot analyses showed that sarcomas differ significantly in their expression of HR23b protein. All HDACi were able to regulate proliferation and apoptosis in vitro. Sensitivity to vorinostat correlated significantly with HR23b protein expression. Immunohistochemical prevalence screening in clinical samples of relevant adult-type tumours revealed that 12.5% of sarcomas (among them malignant peripheral nerve sheath tumours, pleomorphic liposarcomas, leiomyosarcomas, dedifferentiated liposarcomas, synovial sarcomas and angiosarcomas) and 23.2% of GIST show high HR23b expression. Therefore, HDACi have antiproliferative and proapoptotic effects in sarcomas depending on the expression level of HR23b. These findings suggest that HR23b represents a candidate biomarker for HDACi sensitivity in certain sarcoma types and in GIST.