RESUMO
Genetic factors that affect variability in metformin response have been poorly studied in the Latin American population, despite its being the initial drug therapy for type 2 diabetes, one of the most prevalent diseases in that region. Metformin pharmacokinetics is carried out by members of the membrane transporters superfamily (SLCs), being the multidrug and toxin extrusion protein 1 (MATE1), one of the most studied. Some genetic variants in MATE1 have been associated with reduced in vitro metformin transport. They include rs77474263 p.[L125F], a variant present at a frequency of 13.8% in Latin Americans, but rare worldwide (less than 1%). Using exome sequence data and TaqMan genotyping, we revealed that the Mexican population has the highest frequency of this variant: 16% in Mestizos and 27% in Amerindians, suggesting a possible Amerindian origin. To elucidate the metformin pharmacogenetics, a children cohort was genotyped, allowing us to describe, for the first time, a MATE1 rs77474263 TT homozygous individual. An additive effect of the L125F variant was observed on blood metformin accumulation, revealing the highest metformin and lactate serum levels in the TT homozygote, and intermediate metformin values in the heterozygotes. Moreover, a molecular dynamics analysis suggested that the genetic variant effect on metformin efflux could be due to a decreased protein permeability. We conclude that pharmacogenetics could be useful in enhancing metformin pharmacovigilance in populations having a high frequency of the risk genotype, especially considering that these populations also have a higher susceptibility to the diseases for which metformin is the first-choice drug.
Assuntos
Hipoglicemiantes/farmacocinética , Metformina/farmacocinética , Proteínas de Transporte de Cátions Orgânicos/genética , Farmacogenética , Adolescente , Adulto , Criança , Estudos de Coortes , Feminino , Variação Genética , Genótipo , Humanos , Indígenas Norte-Americanos/genética , Ácido Láctico/sangue , Masculino , México , Simulação de Dinâmica MolecularRESUMO
BACKGROUND: We investigated pathogenic DYRK1B variants causative of abdominal obesity-metabolic syndrome 3 (AOMS3) in a group of patients originally diagnosed with type 2 diabetes. All DYRK1B exons were analyzed in a sample of 509 unrelated adults with type 2 diabetes and 459 controls, all belonging to the DMS1 SIGMA-cohort (ExAC). We performed in silico analysis on missense variants using Variant Effect Predictor software. To evaluate co-segregation, predicted pathogenic variants were genotyped in other family members. We performed molecular dynamics analysis for the co-segregating variants. RESULTS: After filtering, Mendelian genotypes were confirmed in two probands bearing two novel variants, p.Arg252His and p.Lys68Gln. Both variants co-segregated with the AOMS3 phenotype in classic dominant autosomal inheritance with full penetrance. In silico analysis revealed impairment of the DYRK1B protein function by both variants. For the first time, we describe age-dependent variable expressivity of this entity, with central obesity and insulin resistance apparent in childhood; morbid obesity, severe hypertriglyceridemia, and labile type 2 diabetes appearing before 40 years of age; and hypertension emerging in the fifth decade of life. We also report the two youngest individuals suffering from AOMS3. CONCLUSIONS: Monogenic forms of metabolic diseases could be misdiagnosed and should be suspected in families with several affected members and early-onset metabolic phenotypes that are difficult to control. Early diagnostic strategies and medical interventions, even before symptoms or complications appear, could be useful.
Assuntos
Diabetes Mellitus Tipo 2 , Adulto , Diabetes Mellitus Tipo 2/genética , Genótipo , Humanos , Mutação , Linhagem , FenótipoRESUMO
Most of the B-cell lymphoma-2 (Bcl-2) protein structure has been elucidated; however, the conformation of its flexible loop domain (FLD) has not yet been experimentally predicted. Its high flexibility under physiological conditions is the reason. FLD behaves as an intrinsically disordered region (IDR) and can adopt regular structures in particular conditions associated with the control of Bcl-2's anti-apoptotic functions. In a previous contribution, we analyzed an engineered Bcl-2 construct (Bcl-2-Δ22Σ3) submitted to 25-ns MD and reported a disordered-to-helix transitions in a region of FLD (rFLD, residues 60-77). However, the conformational preferences in solution of rFLD in the nanosecond to microsecond scale were not analyzed. Herein, an average model was obtained for the native Bcl-2 protein by homology modeling and MD simulation techniques. From this, only the atomic coordinates corresponding to the rFLD were simulated for 1 µs by MD at 310 K. In concordance with previous studies, a disordered-to-helix transitions were exhibited, implying that this "interconversion of folding" in the rFLD suggest a possible set of conformations encoded in its sequence. Principal component analysis (PCA) showed that most of the conformational fluctuation of Bcl-2 is provided by rFLD. Dihedral PCA (dPCA) offered information about all the conformations of rFLD in the µs of the simulation, characterizing a dPCA-based free energy landscape of rFLD, and a conformational ensemble of fast interconverting conformations as other IDRs. Furthermore, despite the conformational heterogeneity of rFLD, the analysis of the dihedral angles (Φ, Ψ) showed that this region does not randomly explore the conformational space in solution.
RESUMO
The anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein interacts with several proteins that regulate the apoptotic properties of cells. In this research, we conduct several all-atom molecular dynamics (MD) simulations under high-temperature unfolding conditions, from 400 to 800 K, for 25 ns. These simulations were performed using a model of an engineered Bcl-2 human protein (Bcl-2-Δ22Σ3), which lacks 22 C-terminal residues of the transmembrane domain. The aim of this study is to gain insight into the structural behavior of Bcl-2-Δ22Σ3 by mapping the conformational movements involved in Bcl-2 stability and its biological function. To build a Bcl-2-Δ22Σ3 three-dimensional model, the protein core was built by homology modeling and the flexible loop domain (FLD, residues 33-91) by ab initio methods. Further, the entire protein model was refined by MD simulations. Afterwards, the production MD simulations showed that the FLD at 400 and 500 K has several conformations reaching into the protein core, whereas at 600 K some of the alpha-helices were lost. At 800 K, the Bcl-2 core is destabilized suggesting a possible mechanism for protein unfolding, where the alpha helices 1 and 6 were the most stable, and a reduction in the number of hydrogen bonds initially occurs. In conclusion, the structural changes and the internal protein interactions suggest that the core and the FLD are crucial components of Bcl-2 in its function of regulate ng access to the recognition sites of kinases and caspases.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/química , Sequência de Aminoácidos , Temperatura Alta , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , Estabilidade ProteicaRESUMO
Epigenetic therapy is an important focus of research for drug development in the treatment of cancer. Valproic acid (VPA) is an HDAC inhibitor that has been evaluated in clinical studies. Despite its success in treating cancer, the mechanism of inhibition of VPA in HDAC is unknown. To this end, we have used docking and molecular dynamic simulations to investigate VPA binding to HDAC, employing both native and rebuilt 3-D structures. The results showed that VPA, via its carboxyl group, coordinates the Zn atom and other local residues (H141-142 and Y360) located at the catalytic site (CS) of HDAC. This causes electrostatic and hydrogen bonding interactions while having little interaction with the hydrophobic side chains, resulting in a low affinity. However, after several docking studies on different native HDAC 3-D structures and after using several snapshots from MD simulations, it became apparent that VPA bound with highest affinity at a site located at the acetyl-releasing channel, termed the hydrophobic active site channel (HASC). The affinity of VPA for HASC was due to its highly hydrophobic properties that allow VPA to take part in van der Waals interactions with Y18, I19, Y20, V25, R37, A38, V41, H42, I135 and W137, while VPA's carboxylate group has several hydrogen bonding interactions with the backbones of S138, I19, N136 and W137. MD simulations showed that the HASC door continuously opened and closed, which affected the affinity of VPA to the HASC, but the affinity toward the HASC was consistently higher than that obtained for the CS, suggesting that the HASC could be involved in the mechanism of inhibition.