Ubiquitin-mediated Proteasome Pathway Research Articles

Swine acute diarrhea syndrome coronavirus Nsp1 suppresses IFN-λ1 production by degrading IRF1 via ubiquitin–proteasome pathway

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus that causes acute watery diarrhea, vomiting, and dehydration in newborn piglets. The type III interferon (IFN-λ) response serves as the primary defense against viruses that replicate in intestinal epithelial cells. However, there is currently no information available on how SADS-CoV modulates the production of IFN-λ. In this study, we utilized IPI-FX cells (a cell line of porcine ileum epithelium) as an in vitro model to investigate the potential immune evasion strategies employed by SADS-CoV against the IFN-λ response. Our results showed that SADS-CoV infection suppressed the production of IFN-λ1 induced by poly(I:C). Through screening SADS-CoV-encoded proteins, nsp1, nsp5, nsp10, nsp12, nsp16, E, S1, and S2 were identified as antagonists of IFN-λ1 production. Specifically, SADS-CoV nsp1 impeded the activation of the IFN-λ1 promoter mediated by MAVS, TBK1, IKKε, and IRF1. Both SADS-CoV and nsp1 obstructed poly(I:C)-induced nuclear translocation of IRF1. Moreover, SADS-CoV nsp1 degraded IRF1 via the ubiquitin-mediated proteasome pathway without interacting with it. Overall, our study provides the first evidence that SADS-CoV inhibits the type III IFN response, shedding light on the molecular mechanisms employed by SADS-CoV to evade the host immune response.

U-box E3 ubiquitin ligase PUB8 attenuates abscisic acid responses during early seedling growth.

ABSCISIC ACID-INSENSITIVE3 (ABI3) and ABI5 are 2 crucial transcription factors in abscisic acid (ABA) signaling, and their homeostasis at the protein level plays a decisive role in seed germination and subsequent seedling growth. Here, we found that PLANT U-BOX 8 (PUB8), a U-box E3 ubiquitin ligase, physically interacts with ABI3 and ABI5 and negatively regulates ABA responses during early Arabidopsis (Arabidopsis thaliana) seedling growth. Loss-of-function pub8 mutants were hypersensitive to ABA-inhibited cotyledon greening, while lines overexpressing PUB8 with low levels of ABI5 protein abundance were insensitive to ABA. Genetic analyses showed that ABI3 and ABI5 were required for the ABA-sensitive phenotype of pub8, indicating that PUB8 functions upstream of ABI3 and ABI5 to regulate ABA responses. Biochemical analyses showed that PUB8 can associate with ABI3 and ABI5 for degradation through the ubiquitin-mediated 26S proteasome pathway. Correspondingly, loss-of-function of PUB8 led to enhanced ABI3 and ABI5 stability, while overexpression of PUB8 impaired accumulation of ABI3 and ABI5 in planta. Further phenotypic analysis indicated that PUB8 compromised the function of ABI5 during early seedling growth. Taken together, our results reveal the regulatory role of PUB8 in modulating the early seedling growth by controlling the homeostasis of ABI3 and ABI5.

CircLRFN5 inhibits the progression of glioblastoma via PRRX2/GCH1 mediated ferroptosis

BackgroundFerroptosis is a novel form of iron-dependent cell death and participates in the malignant progression of glioblastoma (GBM). Although circular RNAs (circRNAs) are found to play key roles in ferroptosis via several mechanisms, including regulating iron metabolism, glutathione metabolism, lipid peroxidation and mitochondrial-related proteins, there are many novel circRNAs regulating ferroptosis need to be found, and they may become a new molecular treatment target in GBM.MethodsThe expression levels of circLRFN5, PRRX2 and GCH1 were detected by qPCR, western blotting, and immunohistochemistry. Lentiviral-based infections were used to overexpress or knockdown these molecules in glioma stem cells (GSCs). The biological functions of these molecules on GSCs were detected by MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay, transwell, neurosphere formation assays, Extreme Limiting Dilution Analysis (ELDA) and xenograft experiments. The content of ferroptosis levels in GSCs was detected by BODIPY 581/591 C11 assay, glutathione (GSH) assay and malondialdehyde (MDA) assay. The regulating mechanisms among these molecules were studied by RNA immunoprecipitation assay, RNA pull-down assay, ubiquitination assay, dual-luciferase reporter assay and chromatin immunoprecipitation assay.ResultsWe found a novel circRNA circLRFN5 is downregulated in GBM and associated with GBM patients’ poor prognosis. CircLRFN5 overexpression inhibits the cell viabilities, proliferation, neurospheres formation, stemness and tumorigenesis of GSCs via inducing ferroptosis. Mechanistically, circLRFN5 binds to PRRX2 protein and promotes its degradation via a ubiquitin-mediated proteasomal pathway. PRRX2 can transcriptionally upregulate GCH1 expression in GSCs, which is a ferroptosis suppressor via generating the antioxidant tetrahydrobiopterin (BH4).ConclusionsOur study found circLRFN5 as a tumor-suppressive circRNA and identified its role in the progression of ferroptosis and GBM. CircLRFN5 can be used as a potential GBM biomarker and become a target for molecular therapies or ferroptosis-dependent therapy in GBM.

The Ash2l SDI Domain Is Required to Maintain the Stability and Binding of DPY30.

ASH2L and DPY30 are important for the assembly and catalytic activity of the complex associated with SET1 (COMPASS), which catalyzes histone methylation and regulates gene expression. However, the regulations among COMPASS components are not fully understood. Here, we leveraged a mouse model and cell lines to observe the outcome of Ash2l depletion and found a significant decrease in DPY30. Analyzing ASH2L ChIP-seq and RNA-seq data excluded transcriptional and translational regulation of ASH2L to DPY30. The decrease in DPY30 was further attributed to the degradation via the ubiquitin-mediated proteasomal pathway. We also verified that three amino acids in the ASH2L Sdc1 DPY30 interaction (SDI) domain are essential for the recognition and binding of DPY30. Lastly, we unexpectedly observed that overexpression of DPY30 in Ash2l-depleted cells rescued the decrease in Ccnd1 and the abnormal cell cycle, which indicates that DPY30 can participate in other complexes to regulate gene expression. Overall, our results, for the first time, reveal that the existence of DPY30 relies on the binding with ASH2L, with degradation of DPY30 via the ubiquitin-proteasome system, and they further indicate that the function of DPY30 can be independent of ASH2L.

TRAF7 contributes to tumor progression by promoting ubiquitin-proteasome mediated degradation of P53 in hepatocellular carcinoma

It has been proved that TRAFs family proteins played malfunctioning roles in the development of human cancers. TRAF7 is the last one of TRAFs family proteins to be found, which was demonstrated to be involved in a serious of cancers development. In this study, we systematically investigated the molecular mechanisms of TRAF7 in facilitating hepatocellular carcinoma (HCC). We discovered that TRAF7 was overexpressed in tumor tissues and the increased TRAF7 expression was closely associated with tumor size, histologic grade, TNM stage and poor prognostication. TRAF7 overexpression repressed cell apoptosis and promoted cell proliferation, invasion and migration, whereas knockdown of TRAF7 in HCC cells had totally opposite effects. Besides, we identified the interaction between TRAF7 and P53 in HCC and demonstrated that TRAF7 promoted ubiquitin-proteasome mediated degradation of P53 at K48 site. The rescue assays further proved that the function of TRAF7 in inhibiting apoptosis and promoting tumor development was depended on P53 in HCC. Overall, this work identified that TARF7 promoted tumorigenesis by targeted degradation P53 for ubiquitin-mediated proteasome pathway. Targeting the TRAF7-P53 axis may provide new insights in the pathogenesis of HCC, and pave the way for developing novel strategies for HCC prevention and treatment.

The E3 Ubiquitin Ligase Ring Finger Protein 5 Ameliorates NASH Through Ubiquitin-Mediated Degradation of 3-Hydroxy-3-Methylglutaryl CoA Reductase Degradation Protein 1.

NAFLD is the most prevalent chronic liver disease worldwide, but no effective pharmacological therapeutics are available for clinical use. NASH is the more severe stage of NAFLD. During this progress, dysregulation of endoplasmic reticulum (ER)-related pathways and proteins is one of the predominant hallmarks. We aimed to reveal the role of ring finger protein 5 (RNF5), an ER-localized E3 ubiquitin-protein ligase, in NASH and to explore its underlying mechanism. We first inspected the expression level of RNF5 and found that it was markedly decreased in livers with NASH in multiple species including humans. We then introduced adenoviruses for Rnf5 overexpression or knockdown into primary mouse hepatocytes and found that palmitic acid/oleic acid (PAOA)-induced lipid accumulation and inflammation in hepatocytes were markedly attenuated by Rnf5 overexpression but exacerbated by Rnf5 gene silencing. Hepatocyte-specific Rnf5 knockout significantly exacerbated hepatic steatosis, inflammatory response, and fibrosis in mice challenged with diet-induced NASH. Mechanistically, we identified 3-hydroxy-3-methylglutaryl CoA reductase degradation protein 1 (HRD1) as a binding partner of RNF5 by systematic interactomics analysis. RNF5 directly bound to HRD1 and promoted its lysine 48 (K48)-linked and K33-linked ubiquitination and subsequent proteasomal degradation. Furthermore, Hrd1 overexpression significantly exacerbated PAOA-induced lipid accumulation and inflammation, and short hairpin RNA-mediated Hrd1 knockdown exerted the opposite effects. Notably, Hrd1 knockdown significantly diminished PAOA-induced lipid deposition, and up-regulation of related genes resulted from Rnf5 ablation in hepatocytes. These data indicate that RNF5 inhibits NASH progression by targeting HRD1 in the ubiquitin-mediated proteasomal pathway. Targeting the RNF5-HRD1 axis may provide insights into the pathogenesis of NASH and pave the way for developing strategies for NASH prevention and treatment.

Stabilization of proto‐oncoprotein ZBTB7A by HSP90 is reversed by p53/KLHL20 proteasomal degradation system upon HSP90 inhibition

ZBTB7A is overexpressed in many human cancers, and is considered a major oncogenic driver. ZBTB7A plays important roles in tumorigenesis by regulating transcription of genes involved in cell survival and proliferation, apoptosis, invasion, and migration/metastasis, key biological capabilities for development of human cancer. One critically unresolved issue is a lack of insight into the mechanisms underlying the aberrant expression and function of ZBTB7A in cancer cells. To that end, we found that HSP90 inhibition decreased ZBTB7A expression in a variety of human cancer cells. ZBTB7A interacts with and is stabilized by HSP90 by inhibition of the ubiquitin-mediated proteasomal degradation pathway. Functional inhibition of HSP90 by 17-AAG, resulted in p53-dependent proteolysis of ZBTB7A, via increased p53 expression and upregulation of the CUL3-dependent E3 ubiquitin ligase, KLHL20. ZBTB7A degradation resulted in derepression of ZBTB7A target genes regulating cell cycle and inhibition of cell proliferation. Our study also revealed a novel function of p53 counteracting proto-oncoprotein ZBTB7A.

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

During influenza A epidemics, bacterial coinfection is a major cause of increased morbidity and mortality. However, the roles of host factors in regulating influenza A virus (IAV)-triggered bacterial coinfection remain elusive. Cyclophilin A (CypA) is an important regulator of infection and immunity. Here, we show that IAV-induced CypA expression facilitates group A Streptococcus (GAS) coinfection both invitro and invivo. Upon IAV infection, CypA interacts with focal adhesion kinase (FAK) and inhibited E3 ligase cCbl-mediated, K48-linked ubiquitination of FAK, which positively regulates integrin α5 expression and actin rearrangement via the FAK/Akt signaling pathway to facilitate GAS colonization and invasion. Notably, CypA deficiency or inhibition by cyclosporine A significantly inhibits IAV-triggered GAS coinfection in mice. Collectively, thesefindings reveal that CypA is critical for GAS infection, and induction of CypA expression is another way for IAV to promote bacterial coinfection, suggesting that CypA is a promising therapeutic target for the secondary bacterial infection.

Mitogen-activated protein kinase 6 negatively regulates secondary wall biosynthesis by modulating MYB46 protein stability in Arabidopsis thaliana.

The R2R3-MYB transcription factor MYB46 functions as a master switch for secondary cell wall biosynthesis, ensuring the exquisite expression of the secondary wall biosynthetic genes in the tissues where secondary walls are critical for growth and development. At the same time, suppression of its function is needed when/where formation of secondary walls is not desirable. Little is known about how this opposing control of secondary cell wall formation is achieved. We used both transient and transgenic expression of MYB46 and mitogen-activated protein kinase 6 (MPK6) to investigate the molecular mechanism of the post-translational regulation of MYB46. We show that MYB46 is phosphorylated by MPK6, leading to site specific phosphorylation-dependent degradation of MYB46 by the ubiquitin-mediated proteasome pathway. In addition, the MPK6-mediated MYB46 phosphorylation was found to regulate in planta secondary wall forming function of MYB46. Furthermore, we provide experimental evidences that MYB83, a paralog of MYB46, is not regulated by MPK6. The coupling of MPK signaling to MYB46 function provides insights into the tissue- and/or condition-specific activity of MYB46 for secondary wall biosynthesis.

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An invitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

AKR1C3 Promotes AR-V7 Protein Stabilization and Confers Resistance to AR-Targeted Therapies in Advanced Prostate Cancer.

The mechanisms resulting in resistance to next-generation antiandrogens in castration-resistant prostate cancer are incompletely understood. Numerous studies have determined that constitutively active androgen receptor (AR) signaling or full-length AR bypass mechanisms may contribute to the resistance. Previous studies established that AKR1C3 and AR-V7 play important roles in enzalutamide and abiraterone resistance. In the present study, we found that AKR1C3 increases AR-V7 expression in resistant prostate cancer cells through enhancing protein stability via activation of the ubiquitin-mediated proteasome pathway. AKR1C3 reprograms AR signaling in enzalutamide-resistant prostate cancer cells. In addition, bioinformatical analysis of indomethacin-treated resistant cells revealed that indomethacin significantly activates the unfolded protein response, p53, and apoptosis pathways, and suppresses cell-cycle, Myc, and AR/ARV7 pathways. Targeting AKR1C3 with indomethacin significantly decreases AR/AR-V7 protein expression in vitro and in vivo through activation of the ubiquitin-mediated proteasome pathway. Our results suggest that the AKR1C3/AR-V7 complex collaboratively confers resistance to AR-targeted therapies in advanced prostate cancer.

Transcriptomic analysis of differentially expressed genes in the oriental armyworm Mythimna separata Walker at different temperatures

The oriental armyworm Mythimna separata Walker is a serious migratory and polyphagous pest that damages major crops and some pastures from the family Gramineae. Temperature is a crucial abiotic factor that affects its survival, development and reproduction, but the thermal responses of this moth at the molecular level are largely unknown. In this research, we sequenced the transcriptomes of oriental armyworms that were reared at three temperatures (20 °C, 25 °C and 30 °C) using an Illumina high-throughput RNA-sequencing (RNA-seq) method. We obtained 54.0 Gb of clean reads and 113,396 transcripts. From a total of 46,681 unigenes identified, 22,911 were annotated to the non-redundant (NR) database. We identified 333 downregulated and 1588 upregulated genes in 20 °C versus 25 °C, and 1096 downregulated and 875 upregulated genes at 30 °C versus 25 °C by differential expression of genes (DEGs). GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses revealed several functional terms related to carbohydrate metabolism, energy metabolism, and xenobiotics metabolism. DEGs involved in glycolysis, the citrate cycle, oxidative phosphorylation, and the composition of myofilaments were significantly downregulated, while most heat shock protein genes (HSPs) and genes in the ubiquitin-mediated proteasome pathway were upregulated at 30 °C. Many cytochrome P450 monooxygenase genes (CYPs) in clan 3 were upregulated at 20 °C, while two genes involved in ecdysteroid biosynthesis, CYP302A1 and CYP315A1, were upregulated at 30 °C. These data may improve the understanding of the complex molecular mechanisms involved in the thermal responses of M. separata.

The tumor suppressor OVCA1 is a short half-life protein degraded by the ubiquitin-proteasome pathway.

Ovarian cancer gene 1 (OVCA1) is a tumor suppressor associated with ovarian cancer, which is involved in cell proliferation regulation, embryonic development and tumorigenesis. Loss of heterozygosity in the OVCA1 gene occurs in 50–86% of cases of ovarian cancer; however, the physiological and biochemical functions of OVCA1 are not yet clear. In the present study, the stability and degradation of OVCA1 were investigated in A2780, Hela and 293 cells. The results revealed that the OVCA1 protein was unstable by MG132 inhibiting proteasome mediated degradation, co-immunoprecipitation and half-life measurement experiments. The cellular protein levels of endogenous OVCA1 were too low to be detected by western blotting. In addition, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal inhibited the degradation of OVCA1 in cells. The co-immunoprecipitation assay revealed that the OVCA1 protein interacted with ubiquitin to form a poly-ubiquitinated complex in cells. The half-life of OVCA1, measured by inhibiting protein synthesis with cycloheximide, was <2 h. The present study demonstrated that OVCA1 may be degraded by the ubiquitin-mediated proteasome pathway and may be considered a short half-life protein. In conclusion, the regulation of OVCA1 protein degradation via the ubiquitin-proteasome pathway may represent a novel direction in the development of ovarian cancer therapy.

The cotton (Gossypium hirsutum) NAC transcription factor (FSN1) as a positive regulator participates in controlling secondary cell wall biosynthesis and modification of fibers.

Cotton (Gossypium hirsutum) fibers are the highly elongated and thickened single-cell trichomes on the seed epidermis. However, little is known about the molecular base of fiber cell wall thickening in detail. In this study, a cotton NAC transcription factor (GhFSN1) that is specifically expressed in secondary cell wall (SCW) thickening fibers was functionally characterized. The GhFSN1 transgenic cotton plants were generated to study how FSN1 regulates fiber SCW formation. Up-regulation of GhFSN1 expression in cotton resulted in an increase in SCW thickness of fibers but a decrease in fiber length. Transcriptomic analysis revealed that GhFSN1 activates or represses numerous downstream genes. GhFSN1 has the ability to form homodimers, binds to its promoter to activate itself, and might be degraded by the ubiquitin-mediated proteasome pathway. The direct targets of GhFSN1 include the fiber SCW-related GhDUF231L1, GhKNL1, GhMYBL1, GhGUT1 and GhIRX12 genes. GhFSN1 binds directly to a consensus sequence (GhNBS), (C/T)(C/G/T)TN(A/T)(G/T)(A/C/G)(A/G)(A/T/G)(A/T/G)AAG, which exists in the promoters of these SCW-related genes. Our data demonstrate that GhFSN1 acts as a positive regulator in controlling SCW formation of cotton fibers by activating its downstream SCW-related genes. Thus, these findings give us novel insights into comprehensive understanding of GhFSN1 function in fiber development.

Kr-POK (ZBTB7c) regulates cancer cell proliferation through glutamine metabolism

Kr-POK (ZBTB7c) is a kidney cancer-related POK transcription factor that not only represses transcription of CDKN1A but also increases expression of FASN. However, precisely how Kr-POK affects cell metabolism by controlling gene expression in response to an energy source in rapidly proliferating cells remains unknown. In this study, we characterized the molecular and functional features of Kr-POK in the context of tumor growth and glutamine metabolism. We found that cells expressing Kr-POK shRNA exhibited more severe cell death than control cells in glucose-deprived medium, and that knockdown of Kr-POK decreased glutamine uptake. Glutamine is critical for tumor cell proliferation. Glutaminase (GLS1), which is activated by p-STAT1, catalyzes the initial reaction in the pathway of glutaminolysis. Kr-POK interacts with PIAS1 to disrupt the interaction between PIAS1 and p-STAT1, and free p-STAT1 can activate GLS1 transcription through an interaction with p300. Kr-POK can be also sumoylated by PIAS1, facilitating Kr-POK degradation by the ubiquitin-mediated proteasomal pathway. Finally, we showed that repression of Kr-POK inhibited tumor growth in vivo in a xenograft model by repressing GLS1 expression. Taken together, our data reveal that Kr-POK activates GLS1 transcription and increases glutamine uptake to support rapid cancer cell proliferation.

Zbtb7c is a molecular ‘off’ and ‘on’ switch of Mmp gene transcription

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases that play roles in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. The expression of MMP gene is tightly regulated and shows cell- and tissue-specific expression patterns. Despite their differential expression, MMP genes have AP-1 (activator protein-1) binding elements within their promoters. Interestingly, c-JUN phosphorylation by cytokine signaling decreased its interaction with NCoR, but increased its interaction with p300, resulting in activation of MMP gene transcription. Here, we found that Zbtb7c (Kr-pok) is a critical component of a transcriptional repressor complex containing c-Jun and NCoR. c-Jun, bound at AP-1, interacts with Zbtb7c, which in turn recruits an NCoR/Hdac3 complex to repress several Mmp (-8, -10, -13, and -16) genes. The molecular interaction between c-Jun and Zbtb7c also prevents phosphorylation of c-Jun by p-Jnk, However, Zbtb7c phosphorylation by p-Jnk (induced by TNFα), and its (Zbtb7c) subsequent degradation by the ubiquitin-mediated proteasomal pathway, leads to c-Jun phosphorylation by p-Jnk. Promoter-bound p-c-Jun then recruits the coactivator p300 to upregulate Mmp gene. Overall, these findings show that Zbtb7c is a key molecule that recruits an NCoR/Hdac3 complex to inhibit phosphorylation of c-Jun, and thereby repress Mmp gene expression.

Camptothecin targets WRN protein: mechanism and relevance in clinical breast cancer.

Werner syndrome protein (WRN) is a RecQ helicase that participates in DNA repair, genome stability and cellular senescence. The five human RecQ helicases, RECQL1, Bloom, WRN, RECQL4 and RECQL5 play critical roles in DNA repair and cell survival after treatment with the anticancer drug camptothecin (CPT). CPT derivatives are widely used in cancer chemotherapy to inhibit topoisomerase I and generate DNA double-strand breaks during replication. Here we studied the effects of CPT on the stability and expression dynamics of human RecQ helicases. In the cells treated with CPT, we observed distinct effects on WRN compared to other human RecQ helicases. CPT altered the cellular localization of WRN and induced its degradation by a ubiquitin-mediated proteasome pathway. WRN knockdown cells as well as CPT treated cells became senescent and stained positive for senescence-associated β-galactosidase at a higher frequency compared to control cells. However, the senescent phenotype was attenuated by ectopic expression of WRN suggesting functional implication of WRN degradation in CPT treated cells. Approximately 5-23% of breast cancer tumors are known to respond to CPT-based chemotherapy. Interestingly, we found that the extent of CPT-induced WRN degradation correlates with increasing sensitivity of breast cancer cells to CPT. The abundance of WRN decreased in CPT-treated sensitive cells; however, WRN remained relatively stable in CPT-resistant breast cancer cells. In a large clinical cohort of breast cancer patients, we find that WRN and topoisomerase I expression correlate with an aggressive tumor phenotype and poor prognosis. Our novel observations suggest that WRN abundance along with CPT-induced degradation could be a promising strategy for personalizing CPT-based cancer chemotherapeutic regimens.

RPN1a, a subunit of the 26S proteasome, controls trichome development in Arabidopsis

The ubiquitin-mediated 26S proteasome pathway (UPS) is of great importance to plant growth and development. Previously research showed that a subunit of the 26S proteasome, named RPN1a, was involved in trichome's branching in Arabidopsis. Mutation in RPN1a give rise to more trichome branches on leaves. Here, we found that T-DNA insertion mutation in RPN1a resulted in increased trichome branches on main stem, and trichome number on rosette leaves and the main stem compared with the wild type plant. Expression analysis results showed that the transcription levels of ZFP6, ZFP5, GIS, GL1, GL2, GL3, TTG1 and MYB23, which promote trichome initiation, were up-regulated in the rpn1a mutant, and expression of FRC4, which is responsible for increased trichome branching, was also increased in the rpn1a mutant. Moreover, the mRNA expression level of RPN1a was significantly repressed by GA (gibberellin) and CK (cytokinin) treatment, which are two important phytohormones that play essential roles in trichome development. These results demonstrate that RPN1a may be involved in trichome development through the GA and CK signaling pathways.

C-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine

Tetrandrine, a constituent of Chinese herb Stephania tetrandra, causes cell death in prostate cancer, but the molecular mechanisms leading to apoptosis is not known. Here we demonstrated that tetrandrine selectively inhibits the growth of prostate cancer PC3 and DU145 cells compared to normal prostate epithelial PWR-1E cells. Tetrandrine-induced cell death in prostate cancer cells is caused by reactive oxygen species (ROS)-mediated activation of c-Jun NH2-terminal kinase (JNK1/2). JNK1/2-mediated proteasomal degradation of c-FLIPL/S and Bcl2 proteins are key events in the sensitization of prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine. Tetrandrine-induced JNK1/2 activation caused the translocation of Bax to mitochondria by disrupting its association with Bcl2 which was accompanied by collapse of mitochondrial membrane potential (MMP), cytosolic release of cytochrome c and Smac, and apoptotic cell death. Additionally, tetrandrine-induced JNK1/2 activation increased the phosphorylation of Bcl2 at Ser70 and facilitated its degradation via the ubiquitin-mediated proteasomal pathway. In parallel, tetrandrine-mediated ROS generation also caused the induction of ligand-independent Fas-mediated apoptosis by activating procaspase-8 and Bid cleavage. Inhibition of procaspase-8 activation attenuated the cleavage of Bid, loss of MMP and caspase-3 activation suggest that tetrandrine-induced Fas-mediated apoptosis is associated with the mitochondrial pathway. Furthermore, most of the signaling effects of tetrandrine on apoptosis were significantly attenuated in the presence of antioxidant N-acetyl-l-cysteine, thereby confirming the involvement of ROS in these events. In conclusion, the results of the present study indicate that tetrandrine-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic pathway contributes to cell death.

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G2/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.