Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement

PAIP2 et ARNm du VEGF (un modèle original de contrôle de l'expression de gènes angiogéniques)

Le VEGF-A est un des régulateurs majeurs de l'angiogenèse. L'ARNm du VEGF étant très labile, la régulation de sa stabilité est une étape clé dans le contrôle de son expression. Nous avons démontré que PAIP2 est un partenaire protéique de la région 3'UTR de l'ARNm du VEGF, une région impliquée dans les phénomènes de stabilité des ARNm. La surexpression de PAIP2 stabilise l'ARNm endogène du VEGF, conduisant alors à une augmentation de VEGF sécrété. De manière inverse, l'invalidation de PAIP2 par interférence ARN affecte les niveaux basaux d'ARNm du VEGF, diminuant de 50% les quantités de VEGF sécrété. De plus, PAIP2 est capable d'interagir avec HuR, une autre protéine stabilisatrice de l'ARNm du VEGF, suggérant une coopération de ces deux protéines. Des expériences de "Gene Array" ont montré que PAIP2 module également l'expression d'autres gènes angiogéniques (interleukine 8, neuropiline 1, récepteur de l'EGF, intégrines a5 et av et Id3). Ainsi, l'impact de PAIP2 sur la néovascularisation et la croissance tumorales a été étudié par invalidation de son expression in vivo par interférence ARN dans des essais de tumorigenèse. L'administration systémique de siRNA chez la souris s'étant révélée inefficace, nous avons développé des vecteurs lentiviraux permettant d'inhiber l'expression de PAIP2 in vivo. Enfin, une étude rétrospective sur des carcinomes ORL humains a montré une forte corrélation entre l'expression de PAIP2 et celle du VEGF, suggérant que PAIP2 contrôle l'expression du VEGF également au sein de tumeurs humaines. Ainsi, le rôle clé de PAIP2 en tant que modulateur de l'angiogenèse pourrait faire de lui une cible potentielle dans des thérapies anti-angiogéniques.The VEGF-A is one of the most important regulators of angiogenesis. Thus, studying mechanisms governing its expression appears to be crucial to understanding physiological and pathological processes involved in angiogenesis. VEGF expression is tightly regulated, particularly at the level of its mRNA stability, which is essentially mediated through the 3' untranslated region (3'UTR) of the VEGF mRNA. By performing screening experiments, we identified PAIP2 as a protein partner of the VEGF mRNA 3'UTR. PAIP2 overexpression stabilizes endogenous VEGF mRNA, leading to an increase in VEGF secretion. Conversely, PAIP2 silencing by RNA interference impairs the VEGF mRNA basal level, inducing a 50% decrease in the level of secreted VEGF. Moreover, PAIP2 can interact with HuR, another VEGF mRNA-stabilizing protein, suggesting cooperation of both proteins in VEGF mRNA stabilization. "Gene Array" experiments demonstrated that PAIP2 also regulates the expression of other angiogenic genes, such as those encoding interleukin 8, neuropilin 1, EGF receptor, a5 and av integrins and Id3. Hence, we analyzed the role of PAIP2 in neovascularization and tumor growth by silencing PAIP2 expression in vivo using RNA interference. As systemic administration of siRNA to mice proved to be ineffective, we developed lentiviral vectors that allowed PAIP2 silencing in vivo. Finally, a retrospective study into human head and neck carcinomas showed a strong correlation between PAIP2 and VEGF expression, suggesting that PAIP2 also controls VEGF expression in human tumors. Thus, due to its crucial role in regulating angiogenesis, PAIP2 may be used as a potential target in anti-angiogenic therapies.NICE-BU Sciences (060882101) / SudocSudocFranceF

N-terminal alanine-rich (NTAR) sequences drive precise start codon selection resulting in elevated translation of multiple proteins including ERK1/2

Abstract We report the discovery of N-terminal alanine-rich sequences, which we term NTARs, that act in concert with their native 5′-untranslated regions to promote selection of the proper start codon. NTARs also facilitate efficient translation initiation while limiting the production of non-functional polypeptides through leaky scanning. We first identified NTARs in the ERK1/2 kinases, which are among the most important signaling molecules in mammals. Analysis of the human proteome reveals that hundreds of proteins possess NTARs, with housekeeping proteins showing a particularly high prevalence. Our data indicate that several of these NTARs act in a manner similar to those found in the ERKs and suggest a mechanism involving some or all of the following features: alanine richness, codon rarity, a repeated amino acid stretch and a nearby second AUG. These features may help slow down the leading ribosome, causing trailing pre-initiation complexes (PICs) to pause near the native AUG, thereby facilitating accurate translation initiation. Amplification of erk genes is frequently observed in cancer, and we show that NTAR-dependent ERK protein levels are a rate-limiting step for signal output. Thus, NTAR-mediated control of translation may reflect a cellular need to precisely control translation of key transcripts such as potential oncogenes. By preventing translation in alternative reading frames, NTAR sequences may be useful in synthetic biology applications, e.g. translation from RNA vaccines

Fructose modulates GLUT5 mRNA stability in differentiated Caco-2 cells: role of cAMP-signalling pathway and PABP (polyadenylated-binding protein)-interacting protein (Paip) 2.

In intestinal cells, levels of the fructose transporter GLUT5 are increased by glucose and to a greater extent by fructose. We investigated the mechanism by which fructose increases GLUT5 expression. In Caco-2 cells, fructose and glucose increased activity of the -2500/+41 GLUT5 promoter to the same extent. cAMP also activated the GLUT5 promoter. However, if a protein kinase A inhibitor was used to block cAMP signalling, extensive GLUT5 mRNA degradation was observed, with no change in basal transcription levels demonstrating the involvement of cAMP in GLUT5 mRNA stability. Indeed, the half-life of GLUT5 mRNA was correlated ( R2=0.9913) with cellular cAMP levels. Fructose increased cAMP concentration more than glucose, accounting for the stronger effect of fructose when compared with that of glucose on GLUT5 production. We identified several complexes between GLUT5 3'-UTR RNA (where UTR stands for untranslated region) and cytosolic proteins that might participate in mRNA processing. Strong binding of a 140 kDa complex I was observed in sugar-deprived cells, with levels of binding lower in the presence of fructose and glucose by factors of 12 and 6 respectively. This may account for differences in the effects of fructose and glucose. In contrast, the amounts of two complexes of 96 and 48 kDa increased equally after stimulation with either glucose or fructose. Finally, PABP (polyadenylated-binding protein)-interacting protein 2, a destabilizing partner of PABP, was identified as a component of GLUT5 3'-UTR RNA-protein complexes. We conclude that the post-transcriptional regulation of GLUT5 by fructose involves increases in mRNA stability mediated by the cAMP pathway and Paip2 (PABP-interacting protein 2) binding

Tristetraprolin Inhibits Ras-dependent Tumor Vascularization by Inducing Vascular Endothelial Growth Factor mRNA Degradation

Vascular endothelial growth factor (VEGF) is one of the most important regulators of physiological and pathological angiogenesis. Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway and overexpression of VEGF are common denominators of tumors from different origins. We have established a new link between these two fundamental observations converging on VEGF mRNA stability. In this complex phenomenon, tristetraprolin (TTP), an adenylate and uridylate-rich element-associated protein that binds to VEGF mRNA 3′-untranslated region, plays a key role by inducing VEGF mRNA degradation, thus maintaining basal VEGF mRNA amounts in normal cells. ERKs activation results in the accumulation of TTP mRNA. However, ERKs reduce the VEGF mRNA-destabilizing effect of TTP, leading to an increase in VEGF expression that favors the angiogenic switch. Moreover, TTP decreases RasVal12-dependent VEGF expression and development of vascularized tumors in nude mice. As a consequence, TTP might represent a novel antiangiogenic and antitumor agent acting through its destabilizing activity on VEGF mRNA. Determination of TTP and ERKs status would provide useful information for the evaluation of the angiogenic potential in human tumors