Regulation of BAP1 tumor suppressor complex by post-translational modifications

Le régulateur transcriptionnel BAP1 est une déubiquitinase nucléaire (DUB) dont le substrat est l’histone H2A modifiée par monoubiquitination au niveau des residus lysines 118 et 119 (K118/K119). Depuis les dernières années, BAP1 emerge comme un gene suppresseur de tumeur majeur. En effet, BAP1 est inactivé dans un plethore de maladies humaines héréditaires et sporadiques. Cependant, malgré l’accumulation significative des connaissances concernant l’occurrence, la pénétrance et l’impact des défauts de BAP1 sur le développement de cancers, ses mécanismes d’action et de régulation restent très peu compris. Cette étude est dédiée à la caractérisation moléculaire et fonctionnelle du complexe multi-protéique de BAP1 et se présente parmi les premiers travaux décrivant sa régulation par des modifications post-traductionnelles. D’abord, nous avons défini la composition du corps du complexe BAP1 ainsi que ses principaux partenaires d’interaction. Ensuite, nous nous sommes spécifiquement intéressés a investiguer d’avantage deux principaux aspects de la régulation de BAP1. Nous avons d’abord décrit l’inter-régulation entre deux composantes majeures du complexe BAP1, soit HCF-1 et OGT. D’une manière très intéressante, nous avons trouvé que le cofacteur HCF-1 est un important régulateur des niveaux protéiques d’OGT. En retour, OGT est requise pour la maturation protéolytique de HCF-1 en promouvant sa protéolyse par O-GlcNAcylation, un processus de régulation très important pour le bon fonctionnement de HCF-1. D’autre part, nous avons découvert un mécanisme unique de régulation de BAP1 médiée par l’ubiquitine ligase atypique UBE2O. en effet, UBE2O se caractérise par le fait qu’il s’agit aussi bien d’une ubiquitine conjuratrice et d’une ubiquitine ligase. UBE2O, multi-monoubiquitine BAP1 au niveau de son domaine NLS et promeut son exclusion du noyau, le séquestrant ainsi dans le cytoplasme. De façon importante, nos travaux ont permis de mettre de l’emphase sur le rôle de l’activité auto-catalytique de chacune de ces enzymes, soit l’activité d’auto-déubiquitination de BAP1 qui est requise pour la maintenance de sa localisation nucléaire ainsi que l’activité d’auto-ubiquitination d’UBE2O impliquée dans son transport nucléo-cytoplasmique. De manière significative, nous avons trouvé que des défauts au niveau de l’auto-déubiquitination de BAP1 due à des mutations associées à certains cancers indiquent l’importance d’une propre regulation de cette déubiquitinase pour les processus associés à la suppression de tumeurs.BAP1 is a nuclear deubiquitinating enzyme (DUB) that acts as a transcription regulator and a DUB of nucleosomal histone H2AK119. In the recent years, it has become clear that BAP1 is a major tumor suppressor, inactivated in a plethora of hereditary and sporadic human malignancies. Although, we now accumulated a significant body of knowledge in respect to the occurrence, penetrance and impact of BAP1 disruption in cancer, its mechanism of action and regulation remained poorly defined. This work is dedicated to the biochemical and functional characterization of the BAP1 multiprotein complex and presents one of the first cases regarding its regulation by post-translational modifications. First, we defined the initial composition of the BAP1 complex and its main interacting components. Second, we specifically focused on two aspects of BAP1 regulation. We described the cross regulation between the two major components of the complex namely HCF-1 and OGT. We found that HCF-1 is important for the maintenance of the cellular levels of OGT. OGT, in turn, is required for the proper maturation of HCF-1 by promoting O-GlcNAcylation-mediated limited proteolysis of its precursor. Third, we discovered an intricate regulatory mechanism of BAP1 mediated by the atypical ubiquitin ligase UBE2O. UBE2O multi-monoubiquitinates BAP1 on its NLS and promotes its exclusion from the nucleus. Importantly, our work emphasises the role of the autocatalytic activity of both enzymes namely the auto-deubiquitination activity of BAP1, required for the maintenance of nuclear BAP1 and the auto-ubiquitination of UBE2O implicated in its nucleocytoplasmic transport. Significantly, we found that auto-deubiquitination of BAP1 is disrupted by cancer-associated mutations, indicating the involvement of this process in tumor suppression

E4orf1: A Novel Ligand That Improves Glucose Disposal in Cell Culture

Reducing dietary fat intake and excess adiposity, the cornerstones of behavioral treatment of insulin resistance(IR), are marginally successful over the long term. Ad36, a human adenovirus, offers a template to improve IR, independent of dietary fat intake or adiposity. Ad36 increases cellular glucose uptake via a Ras-mediated activation of phosphatidyl inositol 3-kinase(PI3K), and improves hyperglycemia in mice, despite a high-fat diet and without reducing adiposity. Ex-vivo studies suggest that Ad36 improves hyperglycemia in mice by increasing glucose uptake by adipose tissue and skeletal muscle, and by reducing hepatic glucose output. It is impractical to use Ad36 for therapeutic action. Instead, we investigated if the E4orf1 protein of Ad36, mediates its anti-hyperglycemic action. Such a candidate protein may offer an attractive template for therapeutic development. Experiment-1 determined that Ad36 ‘requires’ E4orf1 protein to up-regulate cellular glucose uptake. Ad36 significantly increased glucose uptake in 3T3-L1 preadipocytes, which was abrogated by knocking down E4orf1 with siRNA. Experiment-2 identified E4orf1 as ‘sufficient’ to up-regulate glucose uptake. 3T3-L1 cells that inducibly express E4orf1, increased glucose uptake in an induction-dependent manner, compared to null vector control cells. E4orf1 up-regulated PI3K pathway and increased abundance of Ras–the obligatory molecule in Ad36-induced glucose uptake. Experiment-3: Signaling studies of cells transiently transfected with E4orf1 or a null vector, revealed that E4orf1 may activate Ras/PI3K pathway by binding to Drosophila discs-large(Dlg1) protein. E4orf1 activated total Ras and, particularly the H-Ras isoform. By mutating the PDZ domain binding motif(PBM) of E4orf1, Experiment-4 showed that E4orf1 requires its PBM to increase Ras activation or glucose uptake. Experiment-5: In-vitro, a transient transfection by E4orf1 significantly increased glucose uptake in preadipocytes, adipocytes, or myoblasts, and reduced glucose output by hepatocytes. Thus, the highly attractive anti-hyperglycemic effect of Ad36 is mirrored by E4orf1 protein, which may offer a novel ligand to develop anti-hyperglycemic drugs

Metabolically favorable remodeling of human adipose tissue by human adenovirus type 36

OBJECTIVE: Experimental infection of rats with human adenovirus type 36 (Ad-36) promotes adipogenesis and improves insulin sensitivity in a manner reminiscent of the pharmacologic effect of thiozolinediones. To exploit the potential of the viral proteins as a therapeutic target for treating insulin resistance, this study investigated the ability of Ad-36 to induce metabolically favorable changes in human adipose tissue. RESEARCH DESIGN AND METHODS: We determined whether Ad-36 increases glucose uptake in human adipose tissue explants. Cell-signaling pathways targeted by Ad-36 to increase glucose uptake were determined in the explants and human adipose-derived stem cells. Ad-2, a nonadipogenic human adenovirus, was used as a negative control. As a proof of concept, nondiabetic and diabetic subjects were screened for the presence of Ad-36 antibodies to ascertain if natural Ad-36 infection predicted improved glycemic control. RESULTS: Ad-36 increased glucose uptake by adipose tissue explants obtained from nondiabetic and diabetic subjects. Without insulin stimulation, Ad-36 upregulated expressions of several proadipogenic genes, adiponectin, and fatty acid synthase and reduced the expression of inflammatory cytokine macrophage chemoattractant protein-1 in a phosphotidylinositol 3-kinase (PI3K)-dependent manner. In turn, the activation of PI3K by Ad-36 was independent of insulin receptor signaling but dependent on Ras signaling recruited by Ad-36. Ad-2 was nonadipogenic and did not increase glucose uptake. Natural Ad-36 infection in nondiabetic and diabetic subjects was associated with significantly lower fasting glucose levels and A1C, respectively. CONCLUSIONS: Ad-36 proteins may provide novel therapeutic targets that remodel human adipose tissue to a more metabolically favorable profile

E4orf1 interacts with Dlg1 and activates Ras.

<p>HEK293 were co-transfected with GW1-CMV-HA-Dlg1 and either pcDNA-V5-Ad36 E4orf1 or a control plasmid (pcDNA-V5-DEST), and a pull down for HA was conducted and loaded to SDS-PAGE. WB were probed with V5 antibody to detect Dlg1 and E4orf1 interaction. Also, 3T3-E4 and pTRE Null cell lines were treated with 1,000 ng/ml Doxycycline, and a Ras activation assay was conducted. Following pull down, activated slurry and 6% of the whole cell lysate were loaded to SDS-PAGE and probed with total Ras antibodies, or H-, N-, or K-Ras specific antibodies. Blots were also probed with β-actin, and densitometry was expressed as ratio of activated Ras to normalized total Ras. <b>A</b>) WB show HA-Dlg1 and V5E4orf1 in the same complex after IP, indicating the binding of Ad36 E4orf1 with Dlg1. WB also shows the presence of Dlg1 in the whole cell lysate (WCL). As expected, V5-E4orf1 is absent in pcDNA-V5-DEST group after IP for HA-Dlg1. <b>B</b>. (i) WB for total, H-, and N-Ras activation. T = total (6% WCL) and A = Activated slurry. (ii) Total Ras abundance normalized to β-actin was significantly more in 3T3-E4 compared to pTRE Null vector cell line (p = 0.04). (iii)Total Ras activation densitometry, expressed as ratio of activated to normalized total Ras. Total Ras activation was significantly greater in 3T3-E4 compared to pTRE Null (p = 0.01). (iv) H-ras activation densitometry, expressed as ratio of activated H-Ras to normalized total H-Ras. H-Ras activation was significantly more in 3T3-E4 compared to pTRE Null (p = 0.01).</p

E4orf1 is ‘sufficient’ to up-regulate cellular glucose uptake and to up-regulate the Ras/PI3k pathway.

<p>3T3-E4(pTRE E4orf1) and pTRE Null (pTRE) cell lines were treated with varying doses of Doxycycline for 24 h, and a dose-dependent 2DG uptake was determined. Protein was also harvested from 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours to determine signaling changes with E4orf1 expression. <b>A</b>) E4orf1 is sufficient to induce a dose-response increase in glucose uptake. 3T3-E4 and pTRE Null control cell lines were treated with 500, 750, or 1,000 ng/ml Doxycycline to induce different levels of E4orf1 expression, 2DG uptake was determined. Compared to pTRE, the E4orf1 expressing cell had significantly greater glucose uptake when treated with 750 and 1,000 ng/ml Doxycycline (p * = 0.007 and ** = 2.6×10⁻⁷, respectively). <b>B</b>) Ras WB and densitometry normalized to β-actin for 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. Ras expression was significantly higher in the 3T3-E4 cell line (p = 0.05). <b>C</b>) p-AKT and AKT WB and densitometry expressed as ratio of p-AKT to total AKT in 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. p-AKT is significantly higher in the 3T3-E4 cell line (p = 3.54*10⁻⁵). <b>D</b>) Glut4 WB and densitometry normalized to β-actin for 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. Glut4 expression was significantly higher in the 3T3-E4 cell line (p = 0.05). <b>E</b>) Glut1 WB and densitometry normalized to β-actin for 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. Glut1 expression was significantly higher in the 3T3-E4 cell line (p = 0.01). <b>F</b>) Adiponectin WB and densitometry normalized to β-actin for 3T3-E4 and pTRE Null treated with 1,000 ng/ml Doxycycline for 24 hours. Adiponectin expression is significantly higher in the 3T3-E4 cell line (p = 0.006).</p

Efficacy of low-level laser therapy for body contouring and spot fat reduction

BACKGROUND: Low-level laser therapy (LLLT) is commonly used in medical applications, but scientific studies of its efficacy and the mechanism by which it causes loss of fat from fat cells for body contouring are lacking. This study examined the effectiveness and mechanism by which 635–680 nm LLLT acts as a non-invasive body contouring intervention method. METHODS: Forty healthy men and women ages 18–65 years with a BMI \u3c30 kg/m2 were randomized 1:1 to laser or control treatment. Subject\u27s waistlines were treated 30 min twice a week for 4 weeks. Standardized waist circumference measurements and photographs were taken before and after treatments 1, 3, and 8. Subjects were asked not to change their diet or exercise habits. In vitro assays were conducted to determine cell lysis, glycerol, and triglyceride release. RESULTS: Data were analyzed for those with body weight fluctuations within 1.5 kg during 4 weeks of the study. Each treatment gave a 0.4–0.5 cm loss in waist girth.Cumulative girth loss after 4 weeks was −2.15 cm (−0.78 ± 2.82 vs. 1.35 ± 2.64 cm for the control group,p \u3c 0.05). A blinded evaluation of standardized pictures showed statistically significant cosmetic improvement after 4 weeks of laser treatment. In vitro studies suggested that laser treatment increases fat loss from adipocytes by release of triglycerides, without inducing lipolysis or cell lysis. CONCLUSIONS: LLLT achieved safe and significant girth loss sustained over repeated treatments and cumulative over 4 weeks of eight treatments. The girth loss from the waist gave clinically and statistically significant cosmetic improvement

E4orf1 requires its PBM for glucose uptake and Ras activation.

<p>3T3-L1 cells were transfected with V-5 tagged plasmids expressing E4orf1 (pcDNA-V5-AD36-E4orf1), mutant E4orf1 (pcDNA-V5- AD36-E4ORF1ΔPBM) or a null vector (pcDNA-V5-DEST) and a Ras activation assay was conducted the next day. The activated slurry and 3% of whole cell lystate was loaded to SDS-PAGE and probed for Ras. Also 3T3-L1 cells that constitutively express Ad36 E4orf1, a mutated E4orf1 (deleted PBM; ΔPBM) or Null vector were plated in 12 well plates and a 2DG uptake assay was conducted the following day. <b>A</b>) WB for Ras activation assay: pcDNA-V5-AD36-E4orf1 activates Ras (p = 0.04), whereas the Null vector (pcDNA-V5-DEST) does not. pcDNA-V5- AD36-E4ORF1ΔPBM did not activate Ras, suggesting E4orf1 depends on its PBM region for Ras activation. Densitometry expressed as ratio of activated Ras to normalized total Ras from 3% WCL WB. <b>B</b>) 2DG uptake in constitutive expressing Ad36 E4orf1, a mutated E4orf1 (deleted PBM; ΔPBM) or Null vector normalized to protein: E4orf1 increased glucose uptake compared to Null vector (p = 0.008), and there was no difference in glucose uptake when the PBM region of E4orf1 is mutated.</p