ß-TrCP1-variant 4, a novel splice variant of ß-TrCP1, is a negative regulator of ß-TrCP1-variant 1 in ß-catenin degradation.

ß-transducin repeats-containing protein-1 (ß-TrCP1) serves as the substrate recognition subunit for SCFß-TrCP E3 ubiquitin ligases, which specifically ubiquitinate phosphorylated substrates. Three variants of ß-TrCP1 are known and act as homodimer or heterodimer complexes. Here, we identified a novel full-sequenced variant, ß-TrCP1-variant 4, which harbours exon II instead of exon III of variant 1, with no change in the open reading frame. The expression of ß-TrCP1-variant 4 is lower than that of variant 1 or 2 in ovarian cancer cell lines, whereas it is abundantly expressed in normal and cancerous ovarian tissues. Moreover, ß-TrCP1-variant 2 was aberrantly expressed more than variant 1 in ovarian cancer tissues whereas variant 1 was expressed more in normal tissues. Similar to variants 1 and 2, ß-TrCP1-variant 4 directly interacts with ß-catenin, one of the substrates of SCFß-TrCP E3 ubiquitin ligase and down-regulates the transcriptional activity and protein expression of ß-catenin with a significantly weaker effect than that by variants 1 and 2. However, the co-expression of ß-TrCP1-variant 4 with variant 1 in same proportion has no effect, whereas other combinations effectively down-regulate the activity of ß-catenin, indicating that the heterodimer of variants 1 and 4 has no function. Thus, ß-TrCP1-variant 4 could play a critical role in SCFß-TrCP E3 ligase-mediated ubiquitination by acting as a negative regulator of ß-TrCP1-variant 1.

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.

Regulation of mitosis-meiosis transition by the ubiquitin ligase ß-TrCP in male germ cells.

The mitosis-meiosis transition is essential for spermatogenesis. Specific and timely downregulation of the transcription factor DMRT1, and consequent induction of Stra8 expression, is required for this process in mammals, but the molecular mechanism has remained unclear. Here, we show that ß-TrCP, the substrate recognition component of an E3 ubiquitin ligase complex, targets DMRT1 for degradation and thereby controls the mitosis-meiosis transition in mouse male germ cells. Conditional inactivation of ß-TrCP2 in male germ cells of ß-TrCP1 knockout mice resulted in sterility due to a lack of mature sperm. The ß-TrCP-deficient male germ cells did not enter meiosis, but instead underwent apoptosis. The induction of Stra8 expression was also attenuated in association with the accumulation of DMRT1 at the Stra8 promoter in ß-TrCP-deficient testes. DMRT1 contains a consensus ß-TrCP degron sequence that was found to bind ß-TrCP. Overexpression of ß-TrCP induced the ubiquitylation and degradation of DMRT1. Heterozygous deletion of Dmrt1 in ß-TrCP-deficient spermatogonia increased meiotic cells with a concomitant reduction of apoptosis. Collectively, our data indicate that ß-TrCP regulates the transition from mitosis to meiosis in male germ cells by targeting DMRT1 for degradation.

CENP-W inhibits CDC25A degradation by destabilizing the SCFß-TrCP-1 complex at G2/M.

Skp, Cullin, F-box (SCF)ß-TrCP-1 ubiquitin ligases play a central role in cell cycle regulation and tumorigenesis via proteolytic cleavage of many essential cell cycle regulators. In this study, we propose that centromere protein (CENP)-W, a newly identified kinetochore component, is a novel negative regulator of the SCFß-TrCP-1 complex. CENP-W interacts with Cullin (CUL)-1 and ß-Transducin repeat-containing protein (ß-TrCP)-1 through highly overlapped binding sites with S-phase kinase-associated protein (SKP)-1. CENP-W is incorporated into the SCFß-TrCP-1 complex to promote complex disassembly. Unlike other known regulators that increase SCFß-TrCP-1 ubiquitin ligase activity by promoting complex reassociation, CENP-W-mediated complex disorganization induced ß-TrCP1 degradation and consequently decreased its activity. The association between CENP-W and the SCFß-TrCP-1 complex was prominent during the G2/M transition in the nucleus. Especially, CENP-W knockdown decreased the cell division cycle-25A protein level, leading to a delay in mitotic progression. We propose that CENP-W participates in cell cycle regulation by modulating SCFß-TrCP-1 ubiquitin ligase activity.-Cheon, Y., Lee, S. CENP-W inhibits CDC25A degradation by destabilizing the SCFß-TrCP-1 complex at G2/M.

ATG5 nonautophagically regulates inflammation and differentiation in mouse embryonic stem cells.

Embryonic stem cells (ESCs), with abilities of infinite proliferation (self-renewal) and to differentiate into distinct cell types (pluripotency), show attenuated inflammatory response against cytokines or pathogens, which is recognized as a unique characteristic of ESCs compared with somatic cells. However, the underlying molecular mechanisms remain unclear, and whether the attenuated inflammatory state is involved in ESC differentiation is completely unknown. Our recent study demonstrated that macroautophagy/autophagy-related protein ATG5 inhibits the inflammatory response of mouse ESCs (MmESCs) by promoting the degradation of BTRC/ß-TrCP1 and further the downregulation of NFKB/NF-κB signaling. In addition, maintenance of an attenuated inflammation status in MmESCs is required for their differentiation. In conclusion, ATG5 is a key regulator for the regulation of inflammatory response and differentiation of MmESCs.

Long Non-Coding RNA B3GALT5-AS1 Suppresses Keloid Progression by Regulating the ß-Trcp1-Mediated Ubiquitination of HuR.

Background: lncRNA ß­1,3­galactosyltransferase 5­AS1 (B3GALT5-AS1) plays a vital regulatory role in colon and gastric cancers. However, the biological functions and regulatory mechanisms of B3GALT5-AS1 in keloid progression remain unknown. This study aims to investigate the molecular mechanisms in the B3GALT5-AS1-regulated keloid proliferation and invasion. Methods: Secondary mining of the lncRNA sequencing data from GSE158395 was conducted to screen differentially expressed lncRNAs between keloid and normal tissues. MTT, cell migration and invasion assays were performed to detect the effects of B3GALT5-AS1 on keloid fibroblasts (KFs) proliferation and metastasis. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were also determined to evaluate glycolysis in KFs. RNA pull-down and RNA-protein immunoprecipitation assays were used to confirm the interaction between B3GALT5-AS1 and Hu-Antigen R (HuR). Further ubiquitination and rescue experiments were performed to elucidate the regulatory relationship between B3GALT5-AS1 and HuR. Results: B3GALT5-AS1 was significantly down-regulated in keloid tissues and fibroblasts. B3GALT5-AS1 overexpression significantly inhibited KFs proliferation, glycolysis, invasion, and migration and promoted cell apoptosis, whereas silencing B3GALT5-AS1 inhibited these effects. Moreover, B3GALT5-AS1 binds to HuRand reduces its stability through ß-Transducin repeats-containing protein 1 (ß-Trcp1)-mediated ubiquitination. Overexpression of HuR reversed the inhibition of B3GALT5-AS1 on cell proliferation, migration, and invasion in KFs, where glycolysis pathway was involved. Conclusion: Our findings illustrate that B3GALT5-AS1 has great effect on inhibition of keloid formation, which provides a potential target for keloid therapy.

SIRT1 Stabilizes ß-TrCP1 to Inhibit Snail1 Expression in Maintaining Intestinal Epithelial Integrity to Alleviate Colitis.

BACKGROUND & AIMS: Dysfunction of the intestinal epithelial barrier comprising the junctional complex of tight junctions and adherent junctions leads to increased intestinal permeability, which is a major cause of uncontrolled inflammation related to inflammatory bowel disease (IBD). The NAD+-dependent deacetylase SIRT1 is implicated in inflammation and the pathologic process of IBD. We aimed to elucidate the protective role and underlying mechanism of SIRT1 in cell-cell junction and intestinal epithelial integrity. METHODS: The correlation of SIRT1 expression and human IBD was analyzed by GEO or immunohistochemical analyses. BK5.mSIRT1 transgenic mice and wild-type mice were given dextran sodium sulfate (DSS) and the manifestation of colitis-related phenotypes was analyzed. Intestinal permeability was measured by FITC-dextran and cytokines expression was analyzed by quantitative polymerase chain reaction. The expression of the cell junction-related proteins in DSS-treated or SIRT1-knockdown Caco2 or HCT116 cells was analyzed by Western blotting. The effects of nicotinamide mononucleotide in DSS-induced mice colitis were investigated. Correlations of the SIRT1-ß-TrCP1-Snail1-Occludin/Claudin-1/E-cadherin pathway with human IBD samples were analyzed. RESULTS: Reduced SIRT1 expression is associated with human IBD specimens. SIRT1 transgenic mice exhibit much-reduced manifestations of DSS-induced colitis. The activation of SIRT1 by nicotinamide mononucleotide bolsters intestinal epithelial barrier function and ameliorates DSS-induced colitis in mice. Mechanistically, DSS downregulates SiRT1 expression, leading to destabilization of ß-TrCP1 and upregulation of Snail1, accompanied by reduced expression of E-cadherin, Occludin, and Claudin-1, consequently resulting in increased epithelial permeability and inflammation. The deregulated SIRT1-ß-TrCP1-Snail1-Occludin/Claudin-1/E-cadherin pathway correlates with human IBD. CONCLUSIONS: SIRT1 is pivotal in maintaining the intestinal epithelial barrier integrity via modulation of the ß-TrCP1-Snail1-E-cadhein/Occludin/Claudin-1 pathway.

ATG5 attenuates inflammatory signaling in mouse embryonic stem cells to control differentiation.

Attenuated inflammatory response is a property of embryonic stem cells (ESCs). However, the underlying mechanisms are unclear. Moreover, whether the attenuated inflammatory status is involved in ESC differentiation is also unknown. Here, we found that autophagy-related protein ATG5 is essential for both attenuated inflammatory response and differentiation of mouse ESCs and that attenuation of inflammatory signaling is required for mouse ESC differentiation. Mechanistically, ATG5 recruits FBXW7 to promote ubiquitination and proteasome-mediated degradation of ß-TrCP1, resulting in the inhibition of nuclear factor κB (NF-κB) signaling and inflammatory response. Moreover, differentiation defects observed in ATG5-depleted mouse ESCs are due to ß-TrCP1 accumulation and hyperactivation of NF-κB signaling, as loss of ß-TrCP1 and inhibition of NF-κB signaling rescued the differentiation defects. Therefore, this study reveals a previously uncharacterized mechanism maintaining the attenuated inflammatory response in mouse ESCs and further expands the understanding of the biological roles of ATG5.

Insights into the role of HIV-1 Vpu in modulation of NF-ĸB signaling pathways.

HIV-1 inhibits the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) to prevent the induction of a proinflammatory state but also activates the NF-κB pathway to promote viral transcription. Thus, optimal regulation of this pathway is important for the viral life cycle. In recent work, Pickering et al. (3) demonstrate that HIV-1 viral protein U has contrasting effects on the two distinct paralogs of ß-transducin repeat-containing protein (ß-TrCP1 and ß-TrCP2) and that this interaction has important implications for the regulation of both the canonical and non-canonical NF-κB pathways. Additionally, the authors identified the viral requirements for the dysregulation of ß-TrCP. In this commentary, we discuss how these findings further our understanding of how the NF-κB pathway functions during viral infection.

MK2 promotes Tfcp2l1 degradation via ß-TrCP ubiquitin ligase to regulate mouse embryonic stem cell self-renewal.

Tfcp2l1 can maintain mouse embryonic stem cell (mESC) self-renewal. However, it remains unknown how Tfcp2l1 protein stability is regulated. Here, we demonstrate that ß-transducin repeat-containing protein (ß-TrCP) targets Tfcp2l1 for ubiquitination and degradation in a mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2)-dependent manner. Specifically, ß-TrCP1 and ß-TrCP2 recognize and ubiquitylate Tfcp2l1 through the canonical ß-TrCP-binding motif DSGDNS, in which the serine residues have been phosphorylated by MK2. Point mutation of serine-to-alanine residues reduces ß-TrCP-mediated ubiquitylation and enhances the ability of Tfcp2l1 to promote mESC self-renewal while repressing the speciation of the endoderm, mesoderm, and trophectoderm. Similarly, inhibition of MK2 reduces the association of Tfcp2l1 with ß-TrCP1 and increases the self-renewal-promoting effects of Tfcp2l1, whereas overexpression of MK2 or ß-TrCP genes decreases Tfcp2l1 protein levels and induces mESC differentiation. Collectively, our study reveals a posttranslational modification of Tfcp2l1 that will expand our understanding of the regulatory network of stem cell pluripotency.

lncRNA SLC7A11-AS1 Promotes Chemoresistance by Blocking SCFß-TRCP-Mediated Degradation of NRF2 in Pancreatic Cancer.

Drug resistance is the major obstacle of gemcitabine-based chemotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC). Many long non-coding RNAs (lncRNAs) are reported to play vital roles in cancer initiation and progression. Here, we report that lncRNA SLC7A11-AS1 is involved in gemcitabine resistance of PDAC. SLC7A11-AS1 is overexpressed in PDAC tissues and gemcitabine-resistant cell lines. Knockdown of SLC7A11-AS1 weakens the PDAC stemness and potentiates the sensitivity of resistant PDAC cells toward gemcitabine in vitro and in vivo. SLC7A11-AS1 promotes chemoresistance through reducing intracellular reactive oxygen species (ROS) by stabilizing nuclear factor erythroid-2-related factor 2 (NRF2), the key regulator in antioxidant defense. Mechanically, SLC7A11-AS1 is co-localized with ß-TRCP1 in the nucleus. The exon 3 of SLC7A11-AS1 interacts with the F-box motif of ß-TRCP1, the critical domain that recruits ß-TRCP1 to the SCFß-TRCP E3 complex. This interaction prevents the consequent ubiquitination and proteasomal degradation of NRF2 in the nucleus. Our results demonstrate that the overexpression of SLC7A11-AS1 in gemcitabine-resistant PDAC cells can scavenge ROS by blocking SCFß-TRCP-mediated ubiquitination and degradation of NRF2, leading to a low level of intracellular ROS, which is required for the maintenance of cancer stemness. These findings suggest SLC7A11-AS1 as a therapeutic target to overcome gemcitabine resistance for PDAC treatment.

ß-TrCP1 promotes cell proliferation via TNF-dependent NF-κB activation in diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL), a heterogeneous group of invasive disease, is the most common type of B-cell non-Hodgkin's lymphomas. The mechanism of its development is closely related to the constitutive activation of NF-κB. In this study, we investigated the function and the mechanism of ß-TRCP1 in DLBCL. CCK8 and EdU assays showed that ß-TRCP1 could promote the growth of DLBCL cells under the stimulation of TNFα. Furthermore, overexpression of ß-TRCP1 enhanced NF-κB activation in the presence of TNFα. Moreover, ectopic expression of ß-TRCP1 decreased IκB-α expression but increased phospho-p65 expression. In addition, ß-TRCP1 promoted cell cycle progression by accelerating G1-S phase transition. We also found that silencing of ß-TrCP1 increased mitoxantrone-induced cell growth arrest and apoptosis. Based on these, we proposed that the expression of ß-TRCP1 promoted cell proliferation via TNF-dependent NF-κB activation in DLBCL cells.

Two major alternative splice variants of beta-TrCP1 interact with CENP-W with different binding preferences.

Beta-transducin repeat containing protein 1 (ß-TrCP1) is a versatile F-box protein that is responsible for substrate recognition of SCFß-TrCP1 ubiquitin ligase. In human cells, two major alternatively spliced isoforms (b and f) of ß-TrCP1 were found. Recently, we identified that CENP-W interacts with the ß-TrCP1 and regulates the cellular distribution of ß-TrCP1. In this study, we examined whether CENP-W, a new kinetochore component, may differentially regulate the two major isoforms of human ß-TrCP1 (b and f), especially in the cytoplasmic-nuclear shuttling of ß-TrCP1. An in vivo binding assay was performed to examine whether CENP-W binds differently to the two isoforms of ß-TrCP1. EGFP-conjugated ß-TrCP1 isoforms were co-transfected with NLS-defective mutant CENP-W and their cellular distribution were observed using a fluorescence microscopy. Although CENP-W interacts with both b and f isoforms, it has a greater affinity for the b isoform rather than f isoform. Moreover, CENP-W effectively regulates the nuclear-cytoplasmic shuttling of these two ß-TrCP1 isoforms, but with a slight preference towards the b isoform. The Elongin C-binding motif existing in the b isoform may be involved in their specific association. CENP-W showed a higher affinity toward the ß-TrCP1 b isoform, and translocated isoform b more efficiently than isoform f, which may allow a fine regulation of of ß-TrCP1 in the cells.

Involvement of p38-ßTrCP-Tristetraprolin-TNFα axis in radiation pneumonitis.

Early release of tumor necrosis factor-alpha (TNF-α) during radiotherapy of thoracic cancers plays an important role in radiation pneumonitis, whose inhibition may provide lung radioprotection. We previously reported radiation inactivates Tristetraprolin (TTP), a negative regulator of TNF-α synthesis, which correlated with increased TNF-α release. However, the molecular events involved in radiation-induced TTP inactivation remain unclear. To determine if eliminating Ttp in mice resulted in a phenotypic response to radiation, Ttp-null mice lungs were exposed to a single dose of 15 Gy, and TNF-α release and lung inflammation were analyzed at different time points post-irradiation. Ttp-/- mice with elevated (9.5±0.6 fold) basal TNF-α showed further increase (12.2±0.9 fold, p<0.02) in TNF-α release and acute lung inflammation within a week post-irradiation. Further studies using mouse lung macrophage (MH-S), human lung fibroblast (MRC-5), and exogenous human TTP overexpressing U2OS and HEK293 cells upon irradiation (a single dose of 4 Gy) promoted p38-mediated TTP phosphorylation at the serine 186 position, which primed it to be recognized by an ubiquitin ligase (E3), beta transducing repeat containing protein (ß-TrCP), to promote polyubiquitination-mediated proteasomal degradation. Consequently, a serine 186 to alanine (SA) mutant of TTP was resistant to radiation-induced degradation. Similarly, either a p38 kinase inhibitor (SB203580), or siRNA-mediated ß-TrCP knockdown, or overexpression of dominant negative Cullin1 mutants protected TTP from radiation-induced degradation. Consequently, SB203580 pretreatment blocked radiation-induced TNF-α release and radioprotected macrophages. Together, these data establish the involvement of the p38-ßTrCP-TTP-TNFα signaling axis in radiation-induced lung inflammation and identified p38 inhibition as a possible lung radioprotection strategy.

Centromere protein W interacts with beta-transducin repeat-containing protein 1 and modulates its subcellular localization.

Beta-transducin repeat-containing protein 1 (ß-TrCP1) is a substrate-recognition module of SCFß-TrCP1 ubiquitin ligases and its subcellular distribution is known to be critical for target specificity. Heterogeneous nuclear ribonucleoprotein (hnRNP) U, an abundant nuclear protein, is known to be a unique regulator of ß-TrCP1 shuttling between the cytoplasm and the nucleus. In this study, we report that centromere protein W (CENP-W), which is frequently overexpressed in a variety of human cancers, may also contribute to ß-TrCP1 shuttling. Although hnRNP U and CENP-W can interact with ß-TrCP1 and transport it independently, these proteins do not compete for ß-TrCP1 binding, but rather cooperate to form a stable shuttling complex. Intriguingly, we found that overexpression of CENP-W leads to accumulation of ß-TrCP1 in the nucleus. Thus, we propose that CENP-W may function as a booster of ß-TrCP1 nuclear import to increase the oncogenicity of ß-TrCP1.

Transcriptional Regulation of CRD-BP by c-myc: Implications for c-myc Functions.

The coding region determinant binding protein, CRD-BP, is a multifunctional RNA binding protein involved in different processes such as mRNA turnover, translation control, and localization. It is mostly expressed in fetal and neonatal tissues, where it regulates many transcripts essential for normal embryonic development. CRD-BP is scarce or absent in normal adult tissues but reactivated and/or overexpressed in various neoplastic and preneoplastic tumors and in most cell lines. Its expression has been associated with the most aggressive form of some cancers. CRD-BP is an important regulator of different genes including a variety of oncogenes or proto-oncogenes (c-myc, ß-TrCP1, GLI1, etc.). Regulation of CRD-BP expression is critical for proper control of its targets as its overexpression may play an important role in abnormal cell proliferation, suppression of apoptosis, invasion, and metastasis. Molecular bases of the regulatory mechanisms governing CRD-BP expression are still not completely elucidated. In this article, we have identified c-myc as a novel transcriptional regulator of CRD-BP. We show that c-myc binds to CRD-BP promoter and induces its transcription. This induction of CRD-BP expression contributes to the role of c-myc in the regulation of translation, increase in cell size, and acceleration of cell cycle progression via a mechanism involving upregulation of ß-TrCP1 levels and activities and accelerated degradation of PDCD4.