RESUMO

The Rho GTPase Rac1 is involved in the control of cytoskeleton reorganization and other fundamental cellular functions. Aberrant activity of Rac1 and its regulators is common in human cancer. In particular, deregulated expression/activity of Rac GEFs, responsible for Rac1 activation, has been associated to a metastatic phenotype and drug resistance. Thus, the development of novel Rac1-GEF interaction inhibitors is a promising strategy for finding new preclinical candidates. Here, we studied structure-activity relationships within a new family of N,N'-disubstituted guanidine as Rac1 inhibitors. We found that compound 1D-142, presents superior antiproliferative activity in human cancer cell lines and higher potency as Rac1-GEF interaction inhibitor in vitro than parental compounds. In addition, 1D-142 reduces Rac1-mediated TNFα-induced NF-κB nuclear translocation during cell proliferation and migration in NSCLC. Notably, 1D-142 allowed us to show for the first time the application of a Rac1 inhibitor in a lung cancer animal model.

Assuntos

Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Desenvolvimento de Medicamentos , Guanidina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Guanidina/síntese química , Guanidina/química , Humanos , Hidroxilação , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Proteínas rac1 de Ligação ao GTP/metabolismo

RESUMO

Plants in arid zones are constantly exposed to drought stress. The ASR protein family (Abscisic, Stress, Ripening) -a subgroup of the late embryogenesis abundant superfamily- is involved in the water stress response and adaptation to dry environments. Tomato ASR1, as well as other members of this family, is an intrinsically disordered protein (IDP) that functions as a transcription factor and a chaperone. Here we employed different biophysical techniques to perform a deep in vitro characterization of ASR1 as an IDP and showed how both environmental factors and in vivo targets modulate its folding. We report that ASR1 adopts different conformations such as α-helix or polyproline type II in response to environmental changes. Low temperatures and low pH promote the polyproline type II conformation (PII). While NaCl increases PII content and slightly destabilizes α-helix conformation, PEG and glycerol have an important stabilizing effect of α-helix conformation. The binding of Zn2+in the low micromolar range promotes α-helix folding, while extra Zn2+ results in homo-dimerization. The ASR1-DNA binding is sequence specific and dependent on Zn2+. ASR1 chaperone activity does not change upon the structure induction triggered by the addition of Zn2+. Furthermore, trehalose, which has no effect on the ASR1 structure by itself, showed a synergistic effect on the ASR1-driven heat shock protection towards the reporter enzyme citrate synthase (CS). These observations prompted the development of a FRET reporter to sense ASR1 folding in vivo. Its performance was confirmed in Escherichia coli under saline and osmotic stress conditions, representing a promising probe to be used in plant cells. Overall, this work supports the notion that ASR1 plasticity is a key feature that facilitates its response to drought stress and its interaction with specific targets.

Assuntos

Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Estresse Fisiológico , Temperatura Baixa , Secas , Glicerol/metabolismo , Concentração de Íons de Hidrogênio , Solanum lycopersicum/metabolismo , Polietilenoglicóis/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Desdobramento de Proteína , Trealose/metabolismo , Zinco/metabolismo

RESUMO

Phasins are a group of proteins associated to granules of polyhydroxyalkanoates (PHAs). Apart from their structural role as part of the PHA granule cover, different structural and regulatory functions have been found associated to many of them, and several biotechnological applications have been developed using phasin protein fusions. Despite their remarkable functional diversity, the structure of these proteins has not been analyzed except in very few studies. PhaP from Azotobacter sp. FA8 (PhaPAz) is a representative of the prevailing type in the multifunctional phasin protein family. Previous work performed in our laboratory using this protein have demonstrated that it has some very peculiar characteristics, such as its stress protecting effects in recombinant Escherichia coli, both in the presence and absence of PHA. The aim of the present work was to perform a structural characterization of this protein, to shed light on its properties. Its aminoacid composition revealed that it lacks clear hydrophobic domains, a characteristic that appears to be common to most phasins, despite their lipid granule binding capacity. The secondary structure of this protein, consisting of α-helices and disordered regions, has a remarkable capacity to change according to its environment. Several experimental data support that it is a tetramer, probably due to interactions between coiled-coil regions. These structural features have also been detected in other phasins, and may be related to their functional diversity.

Assuntos

Azotobacter/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Cromatografia em Gel , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Estrutura Secundária de Proteína

RESUMO

It is now clear that proteins are flexible entities that in solution switch between conformations to achieve their function. Hydrogen/Deuterium Exchange Mass Spectrometry (HX/MS) is an invaluable tool to understand dynamic changes in proteins modulated by cofactor binding, post-transductional modifications, or protein-protein interactions. ERK2MAPK, a protein involved in highly conserved signal transduction pathways of paramount importance for normal cellular function, has been extensively studied by HX/MS. Experiments of the ERK2MAPK in the inactive and active states (in the presence or absence of bound ATP) have provided valuable information on the plasticity of the MAPK domain. However, interpretation of the HX/MS data is difficult, and changes are mostly explained in relation to available X-ray structures, precluding a complete atomic picture of protein dynamics. In the present work, we have used all atom Molecular Dynamics simulations (MD) to provide a theoretical framework for the interpretation of HX/MS data. Our results show that detailed analysis of protein-solvent interaction along the MD simulations allows (i) prediction of the number of protons exchanged for each peptide in the HX/MS experiments, (ii) rationalization of the experimentally observed changes in exchange rates in different protein conditions at the residue level, and (iii) that at least for ERK2MAPK, most of the functionally observed differences in protein dynamics are related to what can be considered the native state conformational ensemble. In summary, the combination of HX/MS experiments with all atom MD simulations emerges as a powerful approach to study protein native state dynamics with atomic resolution.