HERC3 promotes YAP/TAZ stability and tumorigenesis independently of its ubiquitin ligase activity.

YAP/TAZ transcriptional co-activators play pivotal roles in tumorigenesis. In the Hippo pathway, diverse signals activate the MST-LATS kinase cascade that leads to YAP/TAZ phosphorylation, and subsequent ubiquitination and proteasomal degradation by SCFß-TrCP . When the MST-LATS kinase cascade is inactive, unphosphorylated or dephosphorylated YAP/TAZ translocate into the nucleus to mediate TEAD-dependent gene transcription. Hippo signaling-independent YAP/TAZ activation in human malignancies has also been observed, yet the mechanism remains largely elusive. Here, we report that the ubiquitin E3 ligase HERC3 can promote YAP/TAZ activation independently of its enzymatic activity. HERC3 directly binds to ß-TrCP, blocks its interaction with YAP/TAZ, and thus prevents YAP/TAZ ubiquitination and degradation. Expression levels of HERC3 correlate with YAP/TAZ protein levels and expression of YAP/TAZ target genes in breast tumor cells and tissues. Accordingly, knockdown of HERC3 expression ameliorates tumorigenesis of breast cancer cells. Our results establish HERC3 as a critical regulator of the YAP/TAZ stability and a potential therapeutic target for breast cancer.

Tbl1 promotes Wnt-ß-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

Activation of the Wnt-ß-catenin signaling pathway by CHIR99021, a specific inhibitor of GSK3ß, induces Tcf7l1 protein degradation, which facilitates the maintenance of an undifferentiated state in mouse embryonic stem cells (mESCs); however, the precise mechanism is still unclear. Here, we showed that the overexpression of transducin-ß-like protein 1 (Tbl1, also known as Tbl1x) or its family member Tblr1 (also known as Tbl1xr1) can decrease Tcf7l1 protein levels, whereas knockdown of each gene increases Tcf7l1 levels without affecting Tcf7l1 transcription. Interestingly, only Tbl1, and not Tblr1, interacts with Tcf7l1. Mechanistically, Tbl1 translocates from the cytoplasm into the nucleus in association with ß-catenin (CTNNB1) after the addition of CHIR99021 and functions as an adaptor to promote ubiquitylation of the Tcf7l1 protein. Functional assays further revealed that enforced expression of Tbl1 is capable of delaying mESC differentiation. In contrast, knockdown of Tbl1 attenuates the effect of CHIR99021 on Tcf7l1 protein stability and mESC self-renewal. Our results provide insight into the regulatory network of the Wnt-ß-catenin signaling pathway involved in promoting the maintenance of naïve pluripotency.

Human Neuralized is a novel tumour suppressor targeting Wnt/ß-catenin signalling in colon cancer.

While there is growing evidence that many epigenetically silenced genes in cancer are tumour suppressor candidates, their significance in cancer biology remains unclear. Here, we identify human Neuralized (NEURL), which acts as a novel tumour suppressor targeting oncogenic Wnt/ß-catenin signalling in human cancers. The expression of NEURL is epigenetically regulated and markedly suppressed in human colorectal cancer. We, therefore, considered NEURL to be a bona fide tumour suppressor in colorectal cancer and demonstrate that this tumour suppressive function depends on NEURL-mediated oncogenic ß-catenin degradation. We find that NEURL acts as an E3 ubiquitin ligase, interacting directly with oncogenic ß-catenin, and reducing its cytoplasmic levels in a GSK3ß- and ß-TrCP-independent manner, indicating that NEURL-ß-catenin interactions can lead to a disruption of the canonical Wnt/ß-catenin pathway. This study suggests that NEURL is a therapeutic target against human cancers and that it acts by regulating oncogenic Wnt/ß-catenin signalling.

Novel targets of ß-TrCP cooperatively accelerate carbohydrate and fatty acid consumption.

Cellular energy is primarily produced from glucose and fat through glycolysis and fatty acid oxidation (FAO) followed by the tricarboxylic acid cycle in mitochondria; energy homeostasis is carefully maintained via numerous feedback pathways. In this report, we uncovered a new master regulator of carbohydrate and lipid metabolism. When ubiquitin E3 ligase ß-TrCP2 was inducibly knocked out in ß-TrCP1 knockout adult mice, the resulting double knockout mice (DKO) lost fat mass rapidly. Biochemical analyses of the tissues and cells from ß-TrCP2 KO and DKO mice revealed that glycolysis, FAO, and lipolysis were dramatically upregulated. The absence of ß-TrCP2 increased the protein stability of metabolic rate-limiting enzymes including 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), adipose triglyceride lipase (ATGL), carnitine palmitoyltransferase 1A (CPT1A), and carnitine/acylcarnitine translocase (CACT). Our data suggest that ß-TrCP is a potential regulator for total energy homeostasis by simultaneously controlling glucose and fatty acid metabolism and that targeting ß-TrCP could be an effective strategy to treat obesity and other metabolic disorders.

Ubiquitin E3 ligase ß-TrCP negatively regulates surface protein of hepatitis B virus.

Chronic hepatitis B (CHB) still cannot be cured currently, while the pursuit of a functional cure seems to be an accessible goal, in which the condition mainly depends on the serum hepatitis B surface antigen (HBsAg) levels. HBsAg may be downregulated by protein ubiquitination, which may facilitate finding a new potential intervention target for functional cure of CHB. We confirmed that ß-transducin repeat-containing protein (ß-TrCP) was the E3 ubiquitin ligase of HBsAg. ß-TrCP specifically downregulated the expression of Myc-HBsAg. The degradation of Myc-HBsAg occurred via the proteasome pathway. Knockdown of ß-TrCP increased Myc-HBsAg levels in HepG2 cells. The study further indicated that ß-TrCP could affect the K48-linked polyubiquitin chain by acting on Myc-HBsAg. The GS137 G motif of HBsAg protein is required for ß-TrCP-mediated degradation. Furthermore, we found that ß-TrCP could significantly inhibit both intracellular and extracellular HBsAg levels produced by pHBV-1.3. Our study demonstrated that the E3 ubiquitin ligase ß-TrCP induces K48-linked polyubiquitination of HBsAg, promotes the ubiquitination degradation of HBsAg, and downregulates intra- and extracellular HBsAg levels. Therefore, using the ubiquitination degradation pathway of HBsAg, it is possible to reduce HBsAg levels in CHB patients, which may be helpful in obtaining the goal of functional cure in the treatment of CHB patients.

Smurf1: A possible therapeutic target in dry age-related macular degeneration.

Smad ubiquitylation regulatory factor-1 (Smurf1) is one of C2-WW-HECT domain E3 ubiquitin ligases, it can regulate BMP pathway by mediating ubiquitylation degradation of Smad1/Smad5. Many functions about Smurf1 also are still unknown, especially in retina. This research is about to explore the role of Smurf1 in retina degeneration. Tail vein injection of sodium iodate (NaIO3) in C57BL/6J mice was the animal model of retina degeneration. In NaIO3 model, Smurf1 had more expression than normal mice. Specific Smurf1 inhibitor, A01, was injected into vitreous cavity. Results showed that inhibiting Smurf1 could alleviate acute retina injury, such as keeping a better retina structure in living imaging and histologic sections, less cell death and inflammation activation. Tert-butyl hydroperoxide (TBH) was used to establish oxidative stress injury in human retinal pigments epithelial cell line (ARPE-19). Oxidative stress injury gradually caused co-upregulation of Smurf1, TGF-ß1 and phosphorylated NF-κB (pNF-κB). TGF-ß1 could directly induce Smurf1 expression. Inhibiting Smurf1 had an anti-epithelial mesenchymal transition (anti-EMT) function. Similarly, A01 also could inhibit the expression of pNF-κB, NLRP3 and IL-1ß. At last, after searching bioinformatics database, Smurf1 had a possible interaction with beta-transducin repeat containing E3 ubiquitin protein ligase (ß-TrCP), another E3 ubiquitin ligases. ß-TrCP can mediate ubiquitination degradation of p-IκBα. Lentivirus-SMURF1 was used to overexpress Smurf1, and GS143 was used to inhibit ß-TrCP. The results showed Smurf1 could directly induce NF-κB, pNF-κB, and NLRP3 expression, and keep a stable ß-TrCP expression. However, inhibiting ß-TrCP could cause more NF-κB activation and NLRP3 expression. Therefore, ß-TrCP may play a negative role in NF-κB pathway activation. In summary, Smurf1 plays a role in exacerbating oxidative stress injury and inflammation in retina and may become a potential therapeutic target in ROS injury of retina.

The transcript ENST00000444125 of lncRNA LINC01503 promotes cancer stem cell properties of glioblastoma cells via reducing FBXW1 mediated GLI2 degradation.

LINC010503 is a novel oncogenic lncRNA in multiple cancers. In this study, we further explored the expression of LINC010503 transcripts and their regulations on the glioblastoma (GBM) stem cell (GSC) properties. LINC01503 transcription patterns in GBM and normal brain tissues were compared using RNA-seq data from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA)-GBM. GBM cell lines (U251 and U87) were used as in vitro cell models for cellular and molecular studies. The results showed that ENST00000444125 was the dominant transcript of LINC01503 in both normal and tumor tissues. Its expression was significantly elevated in the tumor group and associated with poor survival outcomes. LINC01503 had both cytoplasmic and nuclear distribution. It positively modulated the expression of multiple GSC markers, including CD133, SOX2, NESTIN, ALDH1A1, and MSI1, and tumorsphere formation in U251 and U87 cells. RNA pull-down and RIP-qPCR assay confirmed an interaction between ENST00000444125 and GLI2. ENST00000444125 positively regulated the half-life of the GLI2 protein in GBM cells. ENST00000444125 overexpression reduced GLI2 ubiquitination and partially attenuated FBXW1 overexpression induced GLI2 ubiquitination. ENST00000444125 overexpression could activate Wnt/ß-catenin signaling in GBM cells. However, these activating effects were remarkedly hampered when GLI2 was knocked down. In conclusion, this study revealed that LINC01503 might have isoform-specific dysregulation in GBM. Among the two major transcripts expressed in GBM cells, ENST00000444125 might be the major functional transcript. Its upregulation might enhance the GSC properties of GBM cells via reducing FBXW1-mediated proteasomal degradation of GLI2.

ß-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of ß-TrCP2 in response to genotoxic stress.

F-box proteins ß-TrCP1 and ß-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of ß-TrCP1 and ß-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remain largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that ß-TrCP1 and ß-TrCP2 communicate during genotoxic stress. We found that expression levels of ß-TrCP1 are significantly increased while levels of ß-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of ß-TrCP1 and ß-TrCP2 via the phosphorylation of ß-TrCP1 at Ser158. Phosphorylated ß-TrCP1 potently promotes the proteasomal degradation of ß-TrCP2 and MDM2, resulting in the activation of p53. Additionally, ß-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by ß-TrCP2. Thus, ß-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of ß-TrCP2. Collectively, our findings elucidate an intriguing posttranslational regulatory mechanism of these two paralogs under genotoxic stress and revealed ß-TrCP1 as a key player in maintaining the genome integrity through the attenuation of ß-TrCP2 levels in response to genotoxic stress.

Circular RNA circPVT1 promotes nasopharyngeal carcinoma metastasis via the ß-TrCP/c-Myc/SRSF1 positive feedback loop.

BACKGROUND: Circular RNAs (circRNAs) act as gene expression regulators and are involved in cancer progression. However, their functions have not been sufficiently investigated in nasopharyngeal carcinoma (NPC). METHODS: The expression profiles of circRNAs in NPC cells within different metastatic potential were reanalyzed. Quantitative reverse transcription PCR and in situ hybridization were used to detect the expression level of circPVT1 in NPC cells and tissue samples. The association of expression level of circPVT1 with clinical properties of NPC patients was evaluated. Then, the effects of circPVT1 expression on NPC metastasis were investigated by in vitro and in vivo functional experiments. RNA immunoprecipitation, pull-down assay and western blotting were performed to confirm the interaction between circPVT1 and ß-TrCP in NPC cells. Co-immunoprecipitation and western blotting were performed to confirm the interaction between ß-TrCP and c-Myc in NPC cells. RESULTS: We find that circPVT1, a circular RNA, is significantly upregulated in NPC cells and tissue specimens. In vitro and in vivo experiments showed that circPVT1 promotes the invasion and metastasis of NPC cells. Mechanistically, circPVT1 inhibits proteasomal degradation of c-Myc by binding to ß-TrCP, an E3 ubiquiting ligase. Stablization of c-Myc by circPVT1 alters the cytoskeleton remodeling and cell adhesion in NPC, which ultimately promotes the invasion and metastasis of NPC cells. Furthermore, c-Myc transcriptionally upregulates the expression of SRSF1, an RNA splicing factor, and recruits SRSF1 to enhance the biosynthesis of circPVT1 through coupling transcription with splicing, which forms a positive feedback for circPVT1 production. CONCLUSIONS: Our results revealed the important role of circPVT1 in the progression of NPC through the ß-TrCP/c-Myc/SRSF1 positive feedback loop, and circPVT1 may serve as a prognostic biomarker or therapeutic target in patients with NPC.

De Novo Missense Variants in FBXW11 Cause Diverse Developmental Phenotypes Including Brain, Eye, and Digit Anomalies.

The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include ß-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw.

Combination of artesunate and WNT974 induces KRAS protein degradation by upregulating E3 ligase ANACP2 and ß-TrCP in the ubiquitin-proteasome pathway.

BACKGROUND: KRAS mutation is one of the dominant gene mutations in colorectal cancer (CRC). Up to present, targeting KRAS for CRC treatment remains a clinical challenge. WNT974 (LGK974) is a porcupine inhibitor that interferes Wnt signaling pathway. Artesunate (ART) is a water-soluble semi-synthetic derivative of artemisinin. METHODS: The synergistic effect of ART and WNT974 combination in reducing CRC cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RT-PCR was utilized for the mRNA levels of KRAS, CUL7, ANAPC2, UBE2M, RNF123, SYVN1, or ß-TrCP. Western blot assay was utilized for the protein levels of NRAS, HRAS, KRAS, ANAPC2, ß-TrCP, GSK-3ß, p-Akt (Ser473), t-Akt, p-PI3K (Tyr458), t-PI3K, p-mTOR (Ser2448), t-mTOR. Xenograft mouse model assay was performed for the anti-CRC effect of combination of ART and WNT974 in vivo. IHC assay was utilized for the levels of KRAS, ß-TrCP, GSK-3ß or ANAPC2 in tumor tissues. RESULTS: Our study shows that the combination of WNT974 and ART exhibits synergistic effect in reducing CRC growth. The combination treatment significantly reduces KRAS protein level and activity in CRC cells. Interestingly, the combination treatment increases E3 ligases ANAPC2 expression. Our data show that overexpression of ANAPC2 significantly reduces KRAS protein levels, which is reversed by MG132. Knockdown of ANAPC2 in CRC abolishes the combination treatment-reduce KRAS expression. Besides, the treatment also increases the expressions of GSK-3ß and E3 ligase ß-TrCP that is known to degrade GSK-3ß-phosphorylated KRAS protein. Knockdown of ß-TrCP- and inhibition of GSK-3ß abolish the combination treatment-induce KRAS ubiquitination and reduction in expression. Last but not least, combination treatment suppresses PI3K/Akt/m-TOR signaling pathway. CONCLUSIONS: Our data clearly show that the combination treatment significantly enhances KRAS protein degradation via the ubiquitination ubiquitin-proteasome pathway, which is also demonstrated in xenograft mouse model. The study provides strong scientific evidence for the development of the combination of WNT974 and ART as KRAS-targeting therapeutics for CRC treatment. Video Abstract.

The E3 ubiquitin ligase APC/CCdh1 degrades MCPH1 after MCPH1-ßTrCP2-Cdc25A-mediated mitotic entry to ensure neurogenesis.

Mutations of microcephalin (MCPH1) can cause the neurodevelopmental disorder primary microcephaly type 1. We previously showed that MCPH1 deletion in neural stem cells results in early mitotic entry that distracts cell division mode, leading to exhaustion of the progenitor pool. Here, we show that MCPH1 interacts with and promotes the E3 ligase ßTrCP2 to degrade Cdc25A independent of DNA damage. Overexpression of ßTrCP2 or the knockdown of Cdc25A remedies the high mitotic index and rescues the premature differentiation of Mcph1-deficient neuroprogenitors in vivo MCPH1 itself is degraded by APC/CCdh1, but not APC/CCdc20, in late mitosis and G1 phase. Forced MCPH1 expression causes cell death, underlining the importance of MCPH1 turnover after mitosis. Ectopic expression of Cdh1 leads to premature differentiation of neuroprogenitors, mimicking differentiation defects of Mcph1-knockout neuroprogenitors. The homeostasis of MCPH1 in association with the ubiquitin-proteasome system ensures mitotic entry independent of cell cycle checkpoint. This study provides a mechanistic understanding of how MCPH1 controls neural stem cell fate and brain development.

Reverse Genetics System for a Human Group A Rotavirus.

Group A rotavirus (RV) is a major cause of acute gastroenteritis in infants and young children worldwide. Recently, we established an entirely plasmid-based reverse genetics system for simian RV strain SA11. Although that system was robust enough to generate reassortant RVs, including human RV gene segments, and enabled better understanding of the biological differences between animal and human RV strains, a complete reverse genetics system for human RV strains is desirable. Here, we established a plasmid-based reverse genetics system for G4P[8] human RV strain Odelia. This technology was used to generate a panel of monoreassortant viruses between human and simian RV strains for all of the 11 gene segments demonstrating full compatibility between human and simian RV strains. Furthermore, we generated recombinant viruses lacking the C-terminal region of the viral nonstructural protein NSP1 and used it to define the biological function of the interaction between NSP1 and its target protein ß-transducin repeat-containing protein (ß-TrCP) during viral replication. While the NSP1 truncation mutant lacking the C-terminal 13 amino acids displayed lower ß-TrCP degradation activity, it replicated as efficiently as the wild-type virus. In contrast, the truncation mutant lacking the C-terminal 166 amino acids of NSP1 replicated poorly, suggesting that the C-terminal region of NSP1 plays critical roles in viral replication. The system reported here will allow generation of engineered recombinant virus harboring desired mutations, increase our understanding of the molecular biology of human RV, and facilitate development of novel therapeutics and vaccines.IMPORTANCE Reverse genetics, an approach used to generate viruses from cloned cDNA, has increased our understanding of virus biology. Worldwide research led to the development of an entirely plasmid-based reverse genetics system for the simian RV laboratory strain. Although the technique allows generation of gene-modified recombinant RVs, biological differences between animal and human RVs mean that reverse genetics systems for human RV strains are still needed. Here, we describe a reverse genetics system for the high-yield human RV strain Odelia, which replicates efficiently and is suitable for in vitro molecular studies. Monoreassortant viruses between simian and human RV strains and NSP1 mutant viruses generated by the rescue system enabled study of the biological functions of viral gene segments. This human RV reverse genetics system will facilitate study of RV biology and development of vaccines and vectors.

ß-TrCP suppresses the migration and invasion of trophoblast cells in preeclampsia by down-regulating Snail.

Insufficient trophoblast invasion has been shown to contribute to the occurrence and progression of preeclampsia (PE). Recently, beta-transducin repeat containing E3 ubiquitin protein (ß-TrCP) was shown to function as a ubiquitination regulator in regulating the proliferation and invasion of various cell types. In this study, we employed an in vitro model of trophoblasts to investigate the role played by ß-TrCP in the pathogenesis of PE. The levels of ß-TrCP in newly delivered placentas from 15 pregnant women with PE and 15 healthy pregnant women were detected by quantitative real-time PCR and western blot assays. The effects of ß-TrCP on cell migration, invasion, and epithelial-mesenchymal transition (EMT) in two trophoblast cell lines (HTR-8/SVneo and TEV-1) were examined using wound healing assays, Transwell assays, and western blot assays, respectively. Rescue experiments were performed by treating ß-TrCP knockdown or ß-TrCP expressing trophoblasts with si-Snail transfection or a proteasome inhibitor (MG132). ß-TrCP mRNA and protein expression levels were significantly increased in the PE placentas when compared to the normal control placentas. ß-TrCP overexpression significantly inhibited cell migration and invasion, while silencing of ß-TrCP promoted cell migration and invasion of the two trophoblast cell lines. Furthermore, we demonstrated that ß-TrCP-mediated ubiquitination might inhibit the EMT process of trophoblasts by down-regulating Snail expression. Our results suggest that both ß-TrCP mRNA and protein expression were up-regulated in the PE placentas. ß-TrCP impeded the migration and invasion of trophoblasts by suppressing Snail expression. This implicates the ubiquitin-proteasome pathway in the pathogenesis of PE, and suggests ß-TrCP as a potential target for treating PE.

The ubiquitin ligase subunit ß-TrCP in Sertoli cells is essential for spermatogenesis in mice.

ß-TrCP is the substrate recognition subunit of an SCF-type ubiquitin ligase. We recently showed that deletion of the genes for both ß-TrCP1 and ß-TrCP2 paralogs in germ cells of male mice resulted in accumulation of the transcription factor DMRT1 and spermatogenic failure, whereas systemic ß-TrCP1 knockout combined with ß-TrCP2 knockdown had previously been shown to lead to disruption of testicular organization and accumulation of the transcription factor SNAIL. Here we investigated ß-TrCP function in Sertoli cells by generating mice with targeted deletion of the ß-TrCP2 gene in Sertoli cells on a background of whole-body ß-TrCP1 knockout. Loss of ß-TrCP in Sertoli cells caused infertility due to a reduction in the number of mature sperm. Whereas spermatogonia were not affected, male germ cells entered meiosis prematurely and the number of round spermatids was reduced in the mutant mice. Extracts of Sertoli cells and of the testis from the mutant mice manifested accumulation of SNAIL, and expression of the SNAIL target gene for E-cadherin was down-regulated in Sertoli cells from these animals. Our results indicate that ß-TrCP in Sertoli cells regulates Sertoli cell-germ cell interaction through degradation of SNAIL, with such regulation being critical for sperm development.

Leaky scanning translation generates a second A49 protein that contributes to vaccinia virus virulence.

Vaccinia virus (VACV) strain Western Reserve gene A49L encodes a small intracellular protein with a Bcl-2 fold that is expressed early during infection and has multiple functions. A49 co-precipitates with the E3 ubiquitin ligase ß-TrCP and thereby prevents ubiquitylation and proteasomal degradation of IκBα, and consequently blocks activation of NF-κB. In a similar way, A49 stabilizes ß-catenin, leading to activation of the wnt signalling pathway. However, a VACV strain expressing a mutant A49 that neither co-precipitates with ß-TrCP nor inhibits NF-κB activation, is more virulent than a virus lacking A49, indicating that A49 has another function that also contributes to virulence. Here we demonstrate that gene A49L encodes a second, smaller polypeptide that is expressed via leaky scanning translation from methionine 20 and is unable to block NF-κB activation. Viruses engineered to express either only the large protein or only the small A49 protein both have lower virulence than wild-type virus and greater virulence than an A49L deletion mutant. This demonstrates that the small protein contributes to virulence by an unknown mechanism that is independent of NF-κB inhibition. Despite having a large genome with about 200 genes, this study illustrates how VACV makes efficient use of its coding potential and from gene A49L expresses a protein with multiple functions and multiple proteins with different functions.

Lipin-2 degradation elicits a proinflammatory gene signature in macrophages.

Lipin-2 is a phosphatidate phosphatase with key roles in regulating lipid storage and energy homeostasis. LPIN2-genetic deficiency is associated with an autoinflammatory disorder, underscoring its critical role in innate immune signaling; however, the regulatory mechanisms underlying protein stability remain unknown. Here, we demonstrate that Lipin-2 interacts with ß-TRCP, a substrate receptor subunit of the SCFß-TRCP E3 ligase, and undergoes ubiquitination and proteasomal degradation. ß-TRCP-knockout in RAW264.7 macrophages resulted in Lipin-2 accumulation, leading to the suppression of LPS-induced MAPK activation and subsequent proinflammatory gene expression. Consistent with this, treatment with MLN4924, a Cullin-neddylation inhibitor that suppresses SCF E3 activity, increased Lipin-2 protein and concomitantly decreased Il1b expression. These findings suggested that ß-TRCP-mediated Lipin-2 degradation affects macrophage-elicited proinflammatory responses and could lead to new therapeutic approaches to treat inflammatory diseases.

The nonreceptor tyrosine kinase c-Src attenuates SCF(ß-TrCP) E3-ligase activity abrogating Taz proteasomal degradation.

The polyomavirus middle T antigen (PyMT) oncogene activates the cellular nonreceptor tyrosine kinase c-Src and recruits the Hippo pathway effectors, Yap (yes-associated protein) and Taz (transcriptional coactivator with PDZ-binding motif), as key steps in oncogenesis. Yap and Taz are transcription coactivators shuttling from the cytoplasm to the nucleus. The Hippo pathway kinase Lats1/2 (large tumor suppressor homolog) reduces Yap/Taz nuclear localization and minimizes their cytoplasmic levels by facilitating their ubiquitination by the E3 ligase SCF(ß-TrCP). In contrast, PyMT increases the cytoplasmic Taz level. Here we show that this unique PyMT behavior is mediated by Src. We demonstrate that PyMT-induced Src activation inhibits degradation of both wild-type and tyrosine-less Taz, ruling out Taz modification as a mechanism of escaping degradation. Instead, we found that Src attenuates the SCF(ß-TrCP) E3-ligase activity in blunting Taz proteasomal degradation. The role of Src in rescuing Taz from TrCP-mediated degradation gives rise to higher cell proliferation under dense cell culture. Finally, IkB (NF-kappa-B inhibitor), a known substrate of ß-TrCP, was rescued by Src, suggesting a wider effect of Src on ß-TrCP substrates. These findings introduce the Src tyrosine kinase as a regulator of SCF(ß-TrCP).

ß-TrCP upregulates HIF-1 in prostate cancer cells.

The substantial availability of hypoxia-inducible factor 1 (HIF-1) for pathophysiological states, such as malignancies and ischemia, is primarily regulated post-translationally through the ubiquitin proteolytic system. The balance between degradation and stabilization of HIF-1α protein is determined by specific E3 ligases. In our search for new E3 ligases that might affect HIF-1α protein expression, we studied the effects of beta-transducin repeat-containing protein (ß-TrCP) on the hypoxic pathway in cancer cells. ß-TrCP is overexpressed in many tumors and regulates various cellular processes through mediating the degradation of important targets. Unexpectedly, we found that ß-TrCP overexpression increases HIF-1α protein expression level as well as HIF-1 transcriptional activity by stabilizing HIF-1α protein and preventing its ubiquitination and proteasomal degradation in prostate cancer cells. By using a proteomic approach, we succeeded in demonstrating that ß-TrCP interferes with the association between HIF-1α and HSP70/CHIP, a HIF-1α established E3 ligase complex. Whereas the E3 ligase activity of ß-TrCP is well known, antagonizing another E3 ligase is a new mechanism of action of this important E3. We suggest that destroying or suppressing ß-TrCP and thereby interrupting the HIF-1 pathway, could be valuable antitumor therapy.