Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions

Activation of client proteins by the Hsp90 molecular chaperone is dependent on binding and hydrolysis of ATP, which drives a molecular clamp via transient dimerization of the N-terminal domains. The crystal structure of the middle segment of yeast Hsp90 reveals considerable evolutionary divergence from the equivalent regions of other GHKL protein family members such as MutL and GyrB, including an additional domain of new fold. Using the known structure of the N-terminal nucleotide binding domain, a model for the Hsp90 dimer has been constructed. From this structure, residues implicated in the ATPase-coupled conformational cycle and in interactions with client proteins and the activating cochaperone Aha1 have been identified, and their roles functionally characterized in vitro and in vivo

Sensitivity of epidermal growth factor receptor and ErbB2 exon 20 insertion mutants to Hsp90 inhibition

The mature epidermal growth factor receptor (EGFR) neither associates with nor requires the molecular chaperone heat-shock protein 90 (Hsp90). Mutations in EGFR exons 18, 19, and 21 confer Hsp90 chaperone dependence. In non-small cell lung cancer (NSCLC), these mutations are associated with enhanced sensitivity to EGFR inhibitors in vitro and with clinical response in vivo. Although less prevalent, insertions in EGFR exon 20 have also been described in NSCLC. These mutations, however, confer resistance to EGFR inhibitors. In NSCLC, exon 20 insertions have also been identified in the EGFR family member ErbB2. Here, we examined the sensitivity of exon 20 insertion mutants to an Hsp90 inhibitor currently in the clinic. Our data demonstrate that both EGFR and ErbB2 exon 20 insertion mutants retain dependence on Hsp90 for stability and downstream-signalling capability, and remain highly sensitive to Hsp90 inhibition. Use of Hsp90 inhibitors should be considered in NSCLC harbouring exon 20 insertions in either EGFR or ErbB2

Honokiol Induces Calpain-Mediated Glucose-Regulated Protein-94 Cleavage and Apoptosis in Human Gastric Cancer Cells and Reduces Tumor Growth

Background. Honokiol, a small molecular weight natural product, has been shown to possess potent anti-neoplastic and anti-angiogenic properties. Its molecular mechanisms and the ability of anti-gastric cancer remain unknown. It has been shown that the anti-apoptotic function of the glucose-regulated proteins (GRPs) predicts that their induction in neoplastic cells can lead to cancer progression and drug resistance. We explored the effects of honokiol on the regulation of GRPs and apoptosis in human gastric cancer cells and tumor growth. Methodology and Principal Findings. Treatment of various human gastric cancer cells with honokiol led to the induction of GRP94 cleavage, but did not affect GRP78. Silencing of GRP94 by small interfering RNA (siRNA) could induce cell apoptosis. Treatment of cells with honokiol or chemotherapeutics agent etoposide enhanced the increase in apoptosis and GRP94 degradation. The calpain activity and calpain-II (m-calpain) protein (but not calpain-I (mu-calpain)) level could also be increased by honokiol. Honokiol-induced GRP94 down-regulation and apoptosis in gastric cancer cells could be reversed by siRNA targeting calpain-II and calpain inhibitors. Furthermore, the results of immunofluorescence staining and immunoprecipitation revealed a specific interaction of GRP94 with calpain-II in cells following honokiol treatment. We next observed that tumor GRP94 over-expression and tumor growth in BALB/c nude mice, which were inoculated with human gastric cancer cells MKN45, are markedly decreased by honokiol treatment. Conclusions and Significance. These results provide the first evidence that honokiol-induced calpain-II-mediated GRP94 cleavage causes human gastric cancer cell apoptosis. We further suggest that honokiol may be a possible therapeutic agent to improve clinical outcome of gastric cancer

Prise en charge thérapeutique des adolescents atteints d'épilepsie généralisée idiopathique dans une cohorte Marseillaise suivie entre 2001 et 2019

Objectif : du fait des conséquences d’une épilepsie mal contrôlée mais aussi des effets secondaires des antiépileptiques, le choix d’un traitement antiépileptique efficace chez les adolescents atteint d’EGI est primordial mais complexe. En 2015, en raison d’effets tératogènes et cognitifs graves, l’HAS a renforcé les conditions de prescriptions de VPA chez les jeunes filles et adolescentes. L’objectif de notre étude est d’étudier la prise en charge thérapeutique d’une cohorte marseillaise d’adolescents suivi pour une EGI.Méthode : nous avons inclus rétrospectivement 151 adolescents suivis pour EGI entre le 1er janvier 2001 et le 31 décembre 2019. Nous avons évalué la prise en charge mais aussi la prescription de VPA selon le sexe, avant et après les recommandations de 2015.Résultats : le VPA reste le traitement anti épileptique le plus prescrit et le plus efficace dans les EGI, permettant un arrêt des crises dans 75,5% des cas. Il est utilisé en première ligne thérapeutique chez 40,2% des filles et 76,6% des garçons. Depuis juin 2015, on note une diminution de prescription du VPA dans les 2 sexes, plus marquée chez les filles. Le délai pour obtenir un arrêt des crises sous traitement est plus long chez les filles (15,6 mois) que chez les garçons (9,6 mois).Conclusion : un contrôle de la maladie par un arrêt des crises est primordial chez les adolescents épileptiques afin d’éviter les conséquences sociales, psychologiques, et traumatiques. Malgré la volonté de ne pas prescrire le VPA comme thérapie de première intention, il est nécessaire pour contrôler les crises chez la grande majorité des patients atteints d’EGI, y compris les filles. Un suivi médical rapproché à l’adolescence doit permettre d’anticiper un éventuel projet de grossesse pour modifier si besoin le traitement.Abréviations : EGI : épilepsie généralisée idiopathique, EAE : épilepsie absence de l'enfant, EAA : épilepsie absence de l’adolescent, EMJ : épilepsie myoclonique juvénile, CGTC : crise généralisée tonico clonique, VPA : valproate de sodium, LTG : lamotrigine