FBXO38 is dispensable for PD-1 regulation.

SKP1-CUL1-F-box protein (SCF) ubiquitin ligases are versatile protein complexes that mediate the ubiquitination of protein substrates. The direct substrate recognition relies on a large family of F-box-domain-containing subunits. One of these substrate receptors is FBXO38, which is encoded by a gene found mutated in families with early-onset distal motor neuronopathy. SCFFBXO38 ubiquitin ligase controls the stability of ZXDB, a nuclear factor associated with the centromeric chromatin protein CENP-B. Loss of FBXO38 in mice results in growth retardation and defects in spermatogenesis characterized by deregulation of the Sertoli cell transcription program and compromised centromere integrity. Moreover, it was reported that SCFFBXO38 mediates the degradation of PD-1, a key immune-checkpoint inhibitor in T cells. Here, we have re-addressed the link between SCFFBXO38 and PD-1 proteolysis. Our data do not support the notion that SCFFBXO38 directly or indirectly controls the abundance and stability of PD-1 in T cells.

CRL4-DCAF12 Ubiquitin Ligase Controls MOV10 RNA Helicase during Spermatogenesis and T Cell Activation.

Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an "ancient" RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.

FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas.

BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease. In many DLBCL patients, BCL6 overexpression is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1­CUL1­F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene.

Differential requirements for Smarca5 expression during hematopoietic stem cell commitment.

The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5tg) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.

FBXO38 Ubiquitin Ligase Controls Centromere Integrity via ZXDA/B Stability.

Alterations in the gene encoding the E3 ubiquitin ligase substrate receptor FBXO38 have been associated with several diseases, including early-onset motor neuronopathy. However, the cellular processes affected by the enzymatic action of FBXO38 are not yet known. Here, we identify the zinc finger proteins ZXDA/B as its interaction partners. FBXO38 controls the stability of ZXDA/B proteins via ubiquitination and proteasome-dependent degradation. We show that ZXDA/B proteins associate with the centromeric protein CENP-B and that the interaction between ZXDA/B and FBXO38 or CENP-B is mutually exclusive. Functionally, ZXDA/B factors control the protein level of chromatin-associated CENP-B. Furthermore, their inappropriate stabilization leads to upregulation of CENP-A and CENP-B positive centromeric chromatin. Thus we demonstrate a previously unknown role of cullin-dependent protein degradation in the control of centromeric chromatin integrity.

FBXO38 Ubiquitin Ligase Controls Sertoli Cell Maturation.

The ubiquitin ligase SCFFBXO38 controls centromeric chromatin by promoting the degradation of the ZXDB protein. To determine the importance of this pathway during development, Fbxo38-deficient mice were generated. The loss of FBXO38 resulted in growth retardation affecting several organs, including the male reproductive system. A detailed analysis of the mutant testes revealed pathological changes in the seminiferous tubules, accompanied by a significant decrease in sperm production and reduced fertility. In adult testes, FBXO38 was specifically expressed in Sertoli cells, a somatic population essential for spermatogenesis initiation and progression. Sertoli cells lacking FBXO38 exhibited stabilized ZXDB protein and upregulated centromeric chromatin. Furthermore, the gene expression profile revealed that the absence of FBXO38 led to a defect in Sertoli cell maturation, specifically characterized by dysregulation in genes controlling retinoic acid metabolism and intercellular communication. Consequently, we documented significant changes in their ability to initiate spermatogonial differentiation. In conclusion, we show that FBXO38 acts as a Sertoli cell maturation factor, affecting the Sertoli cell transcription program, centromere integrity, and, subsequently, the ability to control spermatogenesis.

LFA-1-mediated leukocyte adhesion regulated by interaction of CD43 with LFA-1 and CD147.

The activity of the lymphocyte-function associated antigen 1 (LFA-1; CD11a/CD18) must be tightly controlled during the onset of cellular immunity. It is well known that the sialoglycoprotein CD43 can influence LFA-1-mediated cell adhesion in an either anti- or pro-adhesive manner through mechanisms not well understood. By using a yeast-2-hybrid screen and co-immunoprecipitation we identified physical association of CD43 with two novel partners, LFA-1 itself and the Ig-family member CD147 (EMMPRIN, basigin), and characterized how these interactions are involved in LFA-1-mediated cell adhesion. Monoclonal antibodies (mAbs) to both CD43 and CD147 induced similar homotypic cell aggregation and adhesion of Jurkat T cells and U937 myeloid cells. Both CD43 and CD147 mAbs induced dynamic co-capping of LFA-1 together with the CD43 and the CD147 molecule to cell contact zones. However, in contrast to CD43, we were not able to co-immunoprecipitate LFA-1 with CD147, which indicates that CD43 interacts with CD147 and LFA-1 in two distinct but similarly reorganized complexes. Co-transfection of CD43 interfered with the CD147-induced cell adhesion and aggregation, and siRNA-mediated knock down of CD43 in human T cells resulted in enhanced LFA-1 activation induced via CD147 and also the T cell antigen receptor. These results indicate that triggering CD43 and the underlying signaling pathways enhance LFA-1 adhesiveness while CD43 also negatively regulates LFA-1 induction via other receptors by dynamic interaction with either LFA-1 or CD147.

Ubiquitin Ligases Involved in the Regulation of Wnt, TGF-ß, and Notch Signaling Pathways and Their Roles in Mouse Development and Homeostasis.

The Wnt, TGF-ß, and Notch signaling pathways are essential for the regulation of cellular polarity, differentiation, proliferation, and migration. Differential activation and mutual crosstalk of these pathways during animal development are crucial instructive forces in the initiation of the body axis and the development of organs and tissues. Due to the ability to initiate cell proliferation, these pathways are vulnerable to somatic mutations selectively producing cells, which ultimately slip through cellular and organismal checkpoints and develop into cancer. The architecture of the Wnt, TGF-ß, and Notch signaling pathways is simple. The transmembrane receptor, activated by the extracellular stimulus, induces nuclear translocation of the transcription factor, which subsequently changes the expression of target genes. Nevertheless, these pathways are regulated by a myriad of factors involved in various feedback mechanisms or crosstalk. The most prominent group of regulators is the ubiquitin-proteasome system (UPS). To open the door to UPS-based therapeutic manipulations, a thorough understanding of these regulations at a molecular level and rigorous confirmation in vivo are required. In this quest, mouse models are exceptional and, thanks to the progress in genetic engineering, also an accessible tool. Here, we reviewed the current understanding of how the UPS regulates the Wnt, TGF-ß, and Notch pathways and we summarized the knowledge gained from related mouse models.

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane.

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

Wnt-expressing rat embryonic fibroblasts suppress Apo2L/TRAIL-induced apoptosis of human leukemia cells.

Wnt signaling enhances cell proliferation and the maintenance of hematopoietic cells. In contrast, cytotoxic ligand Apo2L/TRAIL induces the apoptosis of various transformed cells. We observed that co-culture of human pre-B leukemia cells KM3 and REH with Wnt1- or Wnt3a-producing rat embryonic fibroblasts efficiently suppressed Apo2L/TRAIL-induced apoptosis of the lymphoid cells. This suppression occurs at the early stages of the Apo2L/TRAIL apoptotic cascade and, interestingly, the activation of the Wnt pathway alone in human leukemia cells is not sufficient for their full anti-apoptotic protection. We hypothesize that a stimulus emanating specifically from Wnt1- or Wnt3a-expressing rat fibroblasts is responsible for the observed resistance to Apo2L/TRAIL. This anti-apoptotic signaling was significantly hampered by the inhibition of the MEK1/ERK1/2 or NFkappaB pathways in KM3 and REH cells. Our results imply that paracrine Wnt-related signals could be important for the survival of pre-B cell-derived malignancies.

Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling.

We have developed animal models of breast cancer that allow the direct examination of the behavior of individual green fluorescent protein-expressing carcinoma cells in live nonmetastatic and metastatic primary tumors in situ. We have combined this model with multiphoton microscopy to image differences in cell behavior within the primary tumor. Differences in cell behavior between nonmetastatic and metastatic cells in culture and within live primary tumors were correlated with results from cDNA microarray analyses to identify potentially important genetic determinants for breast cancer invasion and metastasis. Using multiphoton microscopy, we found five major differences in carcinoma cell behavior between the nonmetastatic and metastatic primary breast tumors involving extracellular matrix, cell motility, and chemotaxis. Behavioral differences were correlated with seven categories of molecules that were differentially expressed and related to these behaviors. We have found that extracellular matrix composition, actin nucleation factors, molecules involved in mechanical stability and survival, and cell polarity and chemotaxis showed large and consistent differences in gene expression. We conclude that aligning cell behavior in vivo with patterns of gene expression can lead to new insights into the microenvironment of carcinoma cells in the primary tumor and the molecular mechanisms behind cell behavior.

Multifunctional adaptor protein Daxx interacts with chromatin-remodelling ATPase Brg1.

Multifunctional adapter and chaperone protein Daxx participates in the regulation of a number of mainly transcription-related processes. Most notably in a complex with chromatin-remodelling ATPase ATRX, Daxx serves as a histone H3.3 chaperone at telomeric regions and certain genes. In this report we document that Daxx interacts with another chromatin-remodelling, ATPase Brg1. We confirm the Daxx-Brg1 association both in vitro and in cells and show that Daxx interacts with Brg1 in high-molecular-weight complexes. Ectopic co-expression of Daxx with Brg1 and PML could shift disperse nuclear localisation of Brg1 into PML bodies. Mapping the Daxx-Brg1 interaction revealed that Daxx preferentially binds the region between Brg1 N-terminal QLQ and HSA domains, but also weakly interacts with its C-terminal part. Brg1 interacted with both the central and N-terminal parts of Daxx. SiRNA-mediated down-regulation of Daxx in SW13 adrenal carcinoma cells markedly enhanced expression of Brg1-activated genes CD44 or SCEL, suggesting that Daxx either directly through Brg1 and/or indirectly via other factors is a negative regulator of their transcription. Our findings point to Brg1 as another chromatin-remodelling protein that might similarly, as ATRX, target Daxx to specific chromatin regions where it can carry out its chromatin- and transcription-regulating functions.

DRE-1/FBXO11-dependent degradation of BLMP-1/BLIMP-1 governs C. elegans developmental timing and maturation.

Developmental timing genes catalyze stem cell progression and animal maturation programs across taxa. Caenorhabditis elegans DRE-1/FBXO11 functions in an SCF E3-ubiquitin ligase complex to regulate the transition to adult programs, but its cognate proteolytic substrates are unknown. Here, we identify the conserved transcription factor BLMP-1 as a substrate of the SCF(DRE-1/FBXO11) complex. blmp-1 deletion suppressed dre-1 mutant phenotypes and exhibited developmental timing defects opposite to dre-1. blmp-1 also opposed dre-1 for other life history traits, including entry into the dauer diapause and longevity. BLMP-1 protein was strikingly elevated upon dre-1 depletion and dysregulated in a stage- and tissue-specific manner. The role of DRE-1 in regulating BLMP-1 stability is evolutionary conserved, as we observed direct protein interaction and degradation function for worm and human counterparts. Taken together, posttranslational regulation of BLMP-1/BLIMP-1 by DRE-1/FBXO11 coordinates C. elegans developmental timing and other life history traits, suggesting that this two-protein module mediates metazoan maturation processes.

FBH1 protects melanocytes from transformation and is deregulated in melanomas.

FBH1 is a member of the UvrD family of DNA helicases and plays a crucial role in the response to DNA replication stress. In particular, upon DNA replication stress, FBH1 promotes double-strand breakage and activation of the DNA-PK and ATM signaling cascades in a helicase-dependent manner. In the present manuscript, we show that FBH1 is often deleted or mutated in melanoma cells, which results in their increased survival in response to replicative stress. Accordingly, FBH1 depletion promotes UV-mediated transformation of human melanocytes. Thus, FBH1 inactivation appears to contribute to oncogenic transformation by allowing survival of cells undergoing replicative stress due to external factors such as UV irradiation.

FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress.

Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)-binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress.

Transformation by oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by up-regulating death receptor 4 and death receptor 5 through a MEK-dependent pathway.

RAS oncogenes play a major role in cancer development by activating an array of signaling pathways, most notably mitogen-activated protein kinases, resulting in aberrant proliferation and inhibition of apoptotic signaling cascades, rendering transformed cells resistant to extrinsic death stimuli. However, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill specific tumor cells through the engagement of its receptors, death receptor 4 (DR4) and death receptor 5 (DR5), and the activation of apoptotic pathways, providing promising targets for anticancer therapies. In this study, we show that TRAIL induces cell death in human colon adenocarcinoma cells in a MEK-dependent manner. We also report a prolonged MEK-dependent activation of ERK1/2 and increased c-FOS expression induced by TRAIL in this system. Our study reveals that transformation of the colon cell line Caco-2 by Ki- and mainly by Ha-ras oncogenes sensitizes these cells to TRAIL-induced apoptosis by causing specific MEK-dependent up-regulation of DR4 and DR5. These observations taken together reveal that RAS-MEK-ERK1/2 signaling pathway can sensitize cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 and overall imply that TRAIL-based therapeutic strategies using TRAIL agonists could be used in cases of human colon cancers bearing RAS mutations.

Molecular mechanisms involved in CD43-mediated apoptosis of TF-1 cells. Roles of transcription Daxx expression, and adhesion molecules.

CD43 (leukosialin, sialophorin), an abundant leukocyte surface sialoglycoprotein, regulates leukocyte adhesion and transmits activating signals in T cells and dendritic cells. Immobilized anti-CD43 monoclonal antibody (mAb) MEM-59 has been previously shown to induce apoptosis of hematopoietic progenitors. In this study we show that it also triggers apoptosis of the myeloid progenitor-derived cell line TF-1. The kinetics of the MEM-59-induced apoptosis were unusually slow, with the first apoptotic cells appearing 36-48 h after their contact with the immobilized antibody; in 5 days, 90% of the cells were dead. CD43-mediated apoptosis was enhanced by coimmobilized anti-CD45 mAb and partly suppressed by coimmobilized anti-CD50 (ICAM-3) or anti-CD99 mAb. The MEM-59-triggered apoptosis of TF-1 cells was also inhibited by the overexpression of an apoptotic regulator, Daxx. CD43-mediated apoptosis was preceded by the repression of the DNA binding activity of the transcription factor AP-1. DNA array screening revealed that the expression of several genes encoding apoptosis-regulating proteins, including 14-3-3 proteins and the granulocyte macrophage colony-stimulating factor (GM-CSF) receptor beta-subunit, was repressed in TF-1 cells bound to immobilized MEM-59. The down-regulation of 14-3-3 proteins and GM-CSF receptor beta was accompanied by translocation of the proapoptotic protein Bad to the mitochondria. These results suggest that engagement of CD43 may, presumably through the repressing transcription, initiate a Bad-dependent apoptotic pathway.

Integration of TGF-beta/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transitions (EMTs) underlie cell plasticity required in embryonic development and frequently observed in advanced carcinogenesis. Transforming growth factor-beta (TGF-beta) induces EMT phenotypes in epithelial cells in vitro and has been associated with EMT in vivo. Here we report that expression of the hairy/enhancer-of-split-related transcriptional repressor Hey1, and the Notch-ligand Jagged1 (Jag1), was induced by TGF-beta at the onset of EMT in epithelial cells from mammary gland, kidney tubules, and epidermis. The HEY1 expression profile was biphasic, consisting of immediate-early Smad3-dependent, Jagged1/Notch-independent activation, followed by delayed, indirect Jagged1/Notch-dependent activation. TGF-beta-induced EMT was blocked by RNA silencing of HEY1 or JAG1, and by chemical inactivation of Notch. The EMT phenotype, biphasic activation of Hey1, and delayed expression of Jag1 were induced by TGF-beta in wild-type, but not in Smad3-deficient, primary mouse kidney tubular epithelial cells. Our findings identify a new mechanism for functional integration of Jagged1/Notch signalling and coordinated activation of the Hey1 transcriptional repressor controlled by TGF-beta/Smad3, and demonstrate functional roles for Smad3, Hey1, and Jagged1/Notch in mediating TGF-beta-induced EMT.

A comparative cDNA microarray analysis reveals a spectrum of genes regulated by Pax6 in mouse lens.

BACKGROUND: Pax6 is a transcription factor that is required for induction, growth, and maintenance of the lens; however, few direct target genes of Pax6 are known. RESULTS: In this report, we describe the results of a cDNA microarray analysis of lens transcripts from transgenic mice over-expressing Pax6 in lens fibre cells in order to narrow the field of potential direct Pax6 target genes. This study revealed that the transcript levels were significantly altered for 508 of the 9700 genes analysed, including five genes encoding the cell adhesion molecules beta1-integrin, JAM1, L1 CAM, NCAM-140 and neogenin. Notably, comparisons between the genes differentially expressed in Pax6 heterozygous and Pax6 over-expressing lenses identified 13 common genes, including paralemmin, GDIbeta, ATF1, Hrp12 and Brg1. Immunohistochemistry and Western blotting demonstrated that Brg1 is expressed in the embryonic and neonatal (2-week-old) but not in 14-week adult lenses, and confirmed altered expression in transgenic lenses over-expressing Pax6. Furthermore, EMSA demonstrated that the BRG1 promoter contains Pax6 binding sites, further supporting the proposition that it is directly regulated by Pax6. CONCLUSIONS: These results provide a list of genes with possible roles in lens biology and cataracts that are directly or indirectly regulated by Pax6.