Dynamics of human protein kinase Aurora A linked to drug selectivity

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome

Molecular Mechanism of Pin1–Tau Recognition and Catalysis

Human peptidyl-prolyl isomerase (PPIase) Pin1 plays key roles in developmental processes, cell proliferation, and neuronal function. Extensive phosphorylation of the microtubule binding protein tau has been implicated in neurodegeneration and Alzheimer's disease. For the past 15 years, these two players have been the focus of an enormous research effort to unravel the biological relevance of their interplay in health and disease, resulting in a series of proposed molecular mechanism of how Pin1 catalysis of tau results in biological phenotypes. Our results presented here refute these mechanisms of Pin1 action. Using NMR, isothermal calorimetry (ITC), and small angle x-ray scattering (SAXS), we dissect binding and catalysis on multiple phosphorylated tau with particular emphasis toward the Alzheimer's associated AT180 tau epitope containing phosphorylated THR231 and SER235. We find that phosphorylated (p-) SER235-PRO, but not pTHR231-PRO, is exclusively catalyzed by full-length Pin1 and isolated PPIase domain. Importantly, site-specific measurements of Pin1-catalysis of CDK2/CycA-phosphorylated full-length tau reveal a number of sites that are catalyzed simultaneously with different efficiencies. Furthermore, we show that the turnover efficiency at pSER235 by Pin1 is independent of both the WW domain and phosphorylation on THR231. Our mechanistic results on site-specific binding and catalysis together with the lack of an increase of dephosphorylation rates by PP2A counter a series of previously published models for the role of Pin1 catalysis of tau in Alzheimer's disease. Together, our data reemphasize the complicated scenario between binding and catalysis of multiple phosphorylated tau by Pin1 and the need for directly linking biological phenotypes and residue-specific turnover in Pin1 substrates

Turnover de carbono nos tecidos de frangos de corte em diferentes fases de crescimento

The aim of this study was to evaluate carbon turnover on muscle tissue Pectoralis major, keel, tibia and liver of poultry at different life stages using the carbon stable isotopes technique. 216 male chick lings (Cobb) were used. The trial was conduced in a totally random distribution consisting on seven treatments with distinct days for diet substitution (0, 7, 14, 21, 28, 35, 42 days old). Either Boltzmann sigmoid regression or the exponential time function were used in order to measure the carbon isotopic turnover in a specific period of time. The half-life were: 2.5; 2.8; 2.9; 4.9; 5.1; 5.5 e 6.0 day on muscle tissue; 2.7; 2.9; 3.1; 4.6; 5.3; 4.1 e 8.0 day on keel; 4.4; 3.6; 5.4; 5.8; 4.3; 5.2 e 6.3 day on tibia and 1.3; 1.9; 1.4; 1.5; 1.9; 1.7 and 2.1 day on liver, for to treatments T1, T2, T3, T4, T5, T6 e T7 respectively. According to the results of this study, it is possible to conclude that the tibia has slowest metabolism and may indicate the isotopic signature of initial diets of poultry culture. The fast metabolism tissue, liver, is appropriated to reflect the diets fed at closer period of evaluation.Este trabalho objetivou determinar o turnover do carbono no Pectoralis major, quilha, tíbia e fígado de frangos de corte em seus diferentes períodos de crescimento, utilizando a técnica dos isótopos estáveis. Foram utilizados 216 pintos de corte, machos, Cobb. O delineamento experimental foi inteiramente casualizado e consistiu de sete tratamentos com dias de substituição das dietas distintos: 0; 7; 14; 21; 28; 35; 42 dias de idade. Para mensurar o turnover do carbono nos tecidos em determinado intervalo de tempo, foi utilizado o modelo sigmoidal de regressão de Boltzmann ou função exponencial do tempo. Os valores de meias-vidas foram: 2,5; 2,8; 2,9; 4,9; 5,1; 5,5 e 6,0 dias para músculo peitoral; 2,7; 2,9; 3,1; 4,6; 5,3; 4,1 e 8,0 dias para quilha; 4,4; 3,6; 5,4; 5,8; 4,3; 5,2 e 6,3 dias para tíbia e 1,3; 1,9; 1,4; 1,5; 1,9; 1,7 e 2,1 dias para fígado, nos tratamentos T1, T2, T3, T4, T5, T6 e T7, respectivamente. Pode-se concluir que a tíbia possui metabolismo lento e pode indicar o sinal isotópico das dietas iniciais da criação dos frangos de corte; e o fígado, metabolismo rápido, indicado para refletir a dieta próxima da fase de avaliação.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES