Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 219
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 10(1): 3192, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324803

RESUMO

Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 Å away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients.


Assuntos
Proteínas Reguladoras de Apoptose/química , Vírus da Hepatite B/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-bcl-2/química , Transativadores/química , Transativadores/fisiologia , Proteína bcl-X/química , Animais , Cristalografia por Raios X , Modelos Animais de Doenças , Células Hep G2 , Vírus da Hepatite B/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutação , Ligação Proteica , Transativadores/genética , Replicação Viral/fisiologia
2.
Food Chem Toxicol ; 132: 110644, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31252023

RESUMO

Supercritical fluid technologies offer an innovative method for food industry and drug discovery from natural sources. The aim of the study is to investigate the anti-tumor activity of piperine rich extract by supercritical fluid (SFE) from black pepper (Piper nigrum). In silico docking simulations predicted anti-tumor molecular mechanism and protein-piperine hydrophobic interactions, showing hydrogen bonds between piperine and residue Ser5 inside the ATP binding site in CDK2. Moreover, piperine interacts with peptide substrate residue Lys8 inside its binding site in Cyclin A molecule. Other predicted interaction showed piperine inside the hydrophobic groove of Bcl-xL. Confirming the docking simulation, in vitro assays with SFE (40 °C/30 MPa) showed cytotoxicity to MCF-7 cells (IC50 = 27.8 ±â€¯6.8 µg/ml) correlated to increased apoptosis. Balb/c mice-bearing Ehrlich Ascites Carcinoma (EAC) group that received the SFE (100 mg/kg/day) showed tumor growth inhibition (60%) and increased mice survival (50%), probably related to cell cycle arrest (G2/M) and increased apoptosis. In vivo treatments with SFE increased the expression of pro-apoptotic proteins (p53 and Bax), inhibited cell cycle proteins (CDK2, Cyclin A) and anti-apoptotic protein (Bcl-xL). Thus, confirming in silico predicted inhibitory interactions. These results clearly showed promising performance of the piperine-rich fraction recovered from black pepper, drawing attention to its use as complementary therapy for cancer.


Assuntos
Alcaloides/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Benzodioxóis/uso terapêutico , Piperidinas/uso terapêutico , Alcamidas Poli-Insaturadas/uso terapêutico , Alcaloides/química , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Benzodioxóis/química , Benzodioxóis/isolamento & purificação , Benzodioxóis/farmacologia , Dióxido de Carbono/química , Quinase 2 Dependente de Ciclina/química , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Células MCF-7 , Masculino , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Piper nigrum/química , Piperidinas/química , Piperidinas/isolamento & purificação , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/química , Alcamidas Poli-Insaturadas/isolamento & purificação , Alcamidas Poli-Insaturadas/farmacologia , Extração em Fase Sólida/métodos , Proteína bcl-X/química
3.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067648

RESUMO

Interactions between the pro-survival and pro-apoptotic members of the Bcl-2 family of proteins dictate whether a cell lives or dies. Much of our knowledge of the molecular details of these interactions has come from biochemical and structural studies on the pro-survival protein Bcl-xL. The first high-resolution structure of any Bcl-2 family member was of Bcl-xL, which revealed the conserved topology amongst all family members. Subsequent structures of Bcl-xL complexes with pro-apoptotic ligands demonstrated the general features of all pro-survival:pro-apoptotic complexes. Structural studies involving Bcl-xL were also the basis for the discovery of the first small-molecule pro-survival protein inhibitors, leading ultimately to the development of a new class of drugs now successfully used for cancer treatment in the clinic. This article will review our current knowledge of the structural biology of Bcl-xL and how this has impacted our understanding of the molecular details of the intrinsic apoptotic pathway.


Assuntos
Proteína bcl-X/química , Animais , Sítios de Ligação , Humanos , Ligação Proteica , Multimerização Proteica , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/metabolismo
4.
Biochim Biophys Acta Proteins Proteom ; 1867(7-8): 691-700, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31004798

RESUMO

Bcl-xL is a member of the Bcl-2 family of apoptotic regulators, responsible for inhibiting the permeabilization of the mitochondrial outer membrane, and a promising anti-cancer target. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (a) a soluble folded conformation, (b) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (c) refolded membrane-inserted conformations, for which no structural data are available. Previous studies established that in the cell Bcl-xL exists in a dynamic equilibrium between soluble and membranous states, however, no direct evidence exists in support of either anchored or inserted conformation of the membranous state in vivo. In this in vitro study, we employed a combination of fluorescence and EPR spectroscopy to characterize structural features of the bilayer-inserted conformation of Bcl-xL and the lipid modulation of its membrane insertion transition. Our results indicate that the core hydrophobic helix α6 inserts into the bilayer without adopting a transmembrane orientation. This insertion disrupts the packing of Bcl-xL and releases the regulatory N-terminal BH4 domain (α1) from the rest of the protein structure. Our data demonstrate that both insertion and refolding of Bcl-xL are modulated by lipid composition, which brings the apparent pKa of insertion to the threshold of physiological pH. We hypothesize that conformational rearrangements associated with the bilayer insertion of Bcl-xL result in its switching to a so-called non-canonical mode of apoptotic inhibition. Presented results suggest that the alteration in lipid composition before and during apoptosis can serve as an additional factor regulating the permeabilization of the mitochondrial outer membrane.


Assuntos
Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Proteína bcl-X/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Domínios Proteicos , Proteína bcl-X/metabolismo
5.
ChemMedChem ; 14(1): 100-106, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30380184

RESUMO

Progress in drug delivery is hampered by a lack of efficient strategies to target drugs with high specificity and precise spatiotemporal regulation. The remote control of nanoparticles and drugs with light allows regulation of their action site and dosage. Peptide-based drugs are highly specific, non-immunogenic, and can be designed to cross the plasma membrane. In order to combine target specificity and remote control of drug action, here we describe a versatile strategy based on a generalized template to design nanoswitchable peptides that modulate protein-protein interactions upon light activation. This approach is demonstrated to promote photomodulation of two important targets involved in apoptosis (the interactions Bcl-xL-Bak and MDM2-p53), but can be also applied to a large pool of therapeutically relevant protein-protein interactions mediated by α-helical motifs. The template can be adjusted using readily available information about hot spots (residues contributing most to the binding energy) at the protein-protein interface of interest.


Assuntos
Apoptose/efeitos dos fármacos , Nanoestruturas/química , Peptídeos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/química , Proteína bcl-X/metabolismo
6.
Chem Phys Lipids ; 218: 112-124, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30550879

RESUMO

The C-terminal helix of the Bcl-xl is known to initiate the membrane insertion of the protein by anchoring into the mitochondrial outer membrane. The C-terminal charged residues of that helix, R232 and K233, are reported to have an important structural role in the process of that insertion. The present work provides a quantitative understanding of the thermodynamic contribution of these residues on the membrane insertion energy-profile, calculated from the Adaptive Biasing Force based MD simulations of 2.67 µs altogether. Interestingly, the effect of the single neutralizing mutations at the C-terminus, i.e. K233A or R232A, is easily tolerated by the peptide without impacting the nature of insertion energy-profile, indicating the efficiency of one positively charged residue to drive the insertion. Whereas a double mutant, i.e. R232A and K233A, makes a significant impact on the energy-profile by destabilizing the membrane-associated states, as well as the membrane-embedded states. The finding provides molecular-level mechanistic insight. The water-mediated interaction formed by the peptide polar side chains within the bilayer core is found to modulate the membrane response during peptide insertion and that subsequently regulates the insertion mechanism. Mutation of the C-terminal residues eventually alters such a cascade of interactions that results in an insertion through energetically more expensive pathway. Since any one of the positively charged residues at the terminal is critical to ensure the membrane insertion, it appears that the natural selection of 'two' instead of 'one' charged residue is redundant in the context of membrane anchoring but may be important for other biochemical events.


Assuntos
Membranas Mitocondriais/metabolismo , Termodinâmica , Proteína bcl-X/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Membranas Mitocondriais/química , Modelos Moleculares , Mutação , Proteína bcl-X/química , Proteína bcl-X/genética
7.
Appl Biochem Biotechnol ; 187(3): 1061-1080, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30155742

RESUMO

Selective inhibition is a key focus in the design of chemotherapeutic compounds that can abrogate the oncogenic activities of anti-apoptotic Bcl-2 proteins. Although recent efforts have led to the development of highly selective BH3 mimetics, setbacks such as toxicities have limited their use in cancer therapy. Epigallocatechingallate (EGCG) has been widely reported to selectively inhibit Bcl-2 and Bcl-xL compared to other green tea phenols due to its gallate group. Herein, we investigate the interaction dynamics of EGCG at the hydrophobic grooves of Bcl-2 and Bcl-xL and the consequential effects on their BH4 domains. Arg143 and Asp108 (Bcl-2), and Glu96 and Tyr195 (Bcl-xL) formed high-affinity hydrogen interactions with the gallate group while non-gallate groups of EGCG formed weak interactions. EGCG-bound proteins showed systemic perturbations of BH4 domains coupled with the burial of crucial surface-exposed residues such as Lys17 (Bcl-2) and Asp11 (Bcl-xL); hence, a distortion of non-canonical domain interactions. Interactions of gallate group of EGCG with key hydrophobic groove residues underlie EGCG selectivity while concurrent BH4 domain perturbations potentiate EGCG inhibitory activities. Findings will aid the optimization and design of selective inhibitors that could suppress anti-apoptotic activities of Bcl2-family proteins with minimal toxicities.


Assuntos
Catequina/análogos & derivados , Desenho de Drogas , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Catequina/metabolismo , Catequina/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/química , Especificidade por Substrato , Termodinâmica , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/química , Proteína bcl-X/metabolismo
8.
J Chem Inf Model ; 59(1): 245-261, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30582811

RESUMO

Networks of biological molecules are key to cellular function, governing processes ranging from signal cascade propagation to metabolic pathway regulation. Genetic duplication processes give rise to sets of regulatory proteins that have evolved from a common ancestor, leading to interactomes whose dysregulation is often associated with disease. A better understanding of the determinants of specificity at interfaces shared by functionally related proteins is crucial to the rational design of novel pharmacotherapeutic agents. To this end, a comprehensive data set of drug and drug-like binders was assembled for the Bcl-xL and Bcl-2 antiapoptotic proteins-archetypal examples of regulatory systems governed by evolutionarily conserved protein-protein interactions. These were first used to derive a two-dimensional quantitative structure-activity relationship (2D QSAR) model, predicting ligand specificity for these homologous proteins. The strengths and weaknesses of high-throughput 2D QSAR were then compared and contrasted to those of theoretically rigorous thermodynamic integration calculations performed on 14 complexes of Bcl-xL-specific, Bcl-2-specific, and potent dual binders bound to the Bcl-xL and Bcl-2 proteins. We demonstrate that free energy calculations provide an added layer of essential information, which traditional QSAR cannot capture. Moreover, we show that protein energetic responses to different ligands, expressed as per-residue energy values, can be used to fingerprint the protein-ligand interaction, extending the framework of four-dimensional molecular dynamics/quantitative structure-activity relationships (4D-MD/QSAR) toward the facilitation of future drug design strategies.


Assuntos
Apoptose , Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo , Ligantes , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Relação Quantitativa Estrutura-Atividade , Termodinâmica , Proteína bcl-X/química
9.
Comput Biol Chem ; 77: 17-27, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30195235

RESUMO

The proteins of Bcl-2 family, which are promising anti-cancer-drug targets, have substantial similarity in primary sequence and share homologous domains as well as similar structural folds. In spite of similarities in sequence and structures, the members of its pro- and anti- apoptotic subgroups form complexes with different type of partners with discriminating binding affinities. Understanding the origin of this discrimination is very important for designing ligands that can either selectively target a protein or could be made broad ranged as necessary. Using principal component analysis (PCA) of the available structures and from the analysis of the evolution of the binding pocket residues, the correlation has been investigated considering two important anti-apoptotic protein Bcl-xl and Mcl-1, which serve as two ideal representatives of this family. The flexibility of the receptor enables them to discriminate between the ligands or the binding partners. It has been observed that although Bcl-xl and Mcl-1 are classified as homologous proteins, through the course of evolution the binding pocket residues are highly conserved for Bcl-xl; whereas they have been substituted frequently in Mcl-1. The investigation has revealed that the Bcl-xl can adjust the backbone conformation of the binding pocket residues to a larger extent to complement with the shape of different binding partners whereas the Mcl-1 shows more variation in the side chain conformation of binding pocket residues for the same purpose.


Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína bcl-X/química , Proteína bcl-X/metabolismo , Humanos , Ligantes , Modelos Moleculares , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Análise de Componente Principal , Conformação Proteica , Bibliotecas de Moléculas Pequenas/síntese química , Proteína bcl-X/antagonistas & inibidores
10.
Neurobiol Dis ; 116: 39-52, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29723606

RESUMO

Chronic hypoxic stress results in deposition of lipofuscin granules in the CA3 region of hippocampal neurons which contributes to neurodegeneration and accelerated neuronal aging. Oxidative stress and mitophagy during hypoxia are crucial to cause aggregation of these lipofuscin granules in hypoxic neurons. Salidroside, a glucoside derivative of ß-Tyrosol, has been reported to protect hypoxic neurons through maintenance of mitochondrial activity. The present study is aimed at investigating the potential of Salidroside in preventing mitophagy during chronic hypoxia and identification of the molecular targets and underlying signaling mechanisms. In-silico analysis for interaction of salidroside with Bcl-xL was carried out using VLife MDS software. The prophylactic efficacy of Salidroside for amelioration of global hypoxia induced neuronal aging was studied in adult male Sprague-Dawley rats exposed to hypobaric hypoxia simulating an altitude of 7600 m for 21 days. Salidroside was supplemented at a daily dose of 25 mg kg-1b.w. p.o. during hypoxic exposure. Ultra-structural and immune-histological studies were conducted to study lipofuscin aggregation and mitophagy. In-silico findings on salidroside mediated stabilization of Bcl-xL were validated by investigating its effect on downstream signaling molecules involved in mitophagy. Administration of Salidroside reduced deposition of lipofuscin in hypoxic CA3 hippocampal neurons and prevented mitophagy. Salidroside stabilizes Bcl-xL in hypoxic neurons resulting in inhibition of PGAM5 phosphatase activity and maintenance of FUNDC1 in phosphorylated state. Salidroside mediated inhibition of pFUNDC1 dephosphorylation prevents FUNDC1-LC3 II interaction which is crucial for mitophagy. The present study demonstrates potential of Salidroside in preventing lipofuscin deposition during chronic hypoxic stress.


Assuntos
Região CA3 Hipocampal/metabolismo , Glucosídeos/metabolismo , Hipóxia Encefálica/metabolismo , Neurônios/metabolismo , Fenóis/metabolismo , Proteína bcl-X/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Região CA3 Hipocampal/efeitos dos fármacos , Região CA3 Hipocampal/ultraestrutura , Glucosídeos/farmacologia , Hipóxia Encefálica/patologia , Masculino , Simulação de Acoplamento Molecular/métodos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Fenóis/farmacologia , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , Proteína bcl-X/química
11.
Biochim Biophys Acta Mol Cell Res ; 1865(7): 995-1001, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29694915

RESUMO

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-xL is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-xL, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na+/H+ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-xL deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-xL deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-xL is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-xL deamidation. The conservation of such histidines implies that human Bcl-xL is in essence "designed" to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-xL.


Assuntos
Histidina/química , Bombas de Íon/metabolismo , Proteína bcl-X/química , Proteína bcl-X/metabolismo , Células 3T3 , Animais , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , Desaminação , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Proteína bcl-X/genética
12.
Biochem Biophys Res Commun ; 495(1): 1067-1073, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29175327

RESUMO

DJ-1 is a multifunctional protein associated with Parkinson's disease (PD) and tumorigenesis. In response to ultraviolet B (UVB) irradiation, DJ-1 is translocated into the mitochondria, and its interaction with the mitochondrial protein Bcl-XL protects cells against death. In this study, we characterized the molecular interaction between DJ-1 and Bcl-XL by NMR spectroscopy. The NMR chemical shift perturbation data demonstrated that the oxidized but not the reduced form of DJ-1 binds to the predominantly hydrophobic groove surrounded by the BH1-BH3 domains in Bcl-XL. In addition, our results showed that the C-terminal α8-helix peptide (Cpep) of DJ-1 binds to the pro-apoptotic BH3 peptide-binding hydrophobic groove in Bcl-XL and, thus, acts as a Bcl-XL-binding motif. In combination with the NMR chemical shift perturbation data, a refined structural model of the Bcl-XL/DJ-1 Cpep complex revealed that the binding mode is remarkably similar to that of other Bcl-XL/pro-apoptotic BH3 peptide complexes. Taken together, our results provide a structural basis for the binding mechanism between DJ-1 and Bcl-XL, which will contribute to molecular understanding of the role of mitochondrial DJ-1 in Bcl-XL regulation in response to oxidative stress.


Assuntos
Simulação de Acoplamento Molecular/métodos , Proteína Desglicase DJ-1/química , Mapeamento de Interação de Proteínas/métodos , Proteína bcl-X/química , Proteína bcl-X/ultraestrutura , Sítios de Ligação , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Relação Estrutura-Atividade
13.
J Mol Graph Model ; 79: 166-174, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29197725

RESUMO

B-cell lymphoma 2 (Bcl-2) family proteins are potential drug targets in cancer and have a relatively flat and flexible binding site. ABT-199 is one of the most promising selective Bcl-2 inhibitors, and A-1155463 selectively inhibits Bcl-XL. Although the amino acid sequences of the binding sites of these two inhibitors are similar, the inhibitors selectively bind the target protein. In order to determine the origin of the selectivity of these inhibitors, we conducted molecular dynamics simulations using protein-inhibitor modeling. We confirmed that ASP103 of Bcl-2 is a key residue and that hydrogen bonding between ASP103 and ABT-199 confers the Bcl-2 selectivity of this inhibitor. For Bcl-XL selectivity, the secondary structure of α-helix 3 is a key factor. PHE105, SER106, and LEU108 in the loose α-helix 3 interact with A-1155463 to confer Bcl-XL selectivity. These findings provide important insights into the molecular mechanisms of selective inhibitors of Bcl-2 family proteins.


Assuntos
Antineoplásicos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína bcl-X/química , Antineoplásicos/farmacologia , Sítios de Ligação , Humanos , Ligações de Hidrogênio , Ligantes , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Proteína bcl-X/antagonistas & inibidores
14.
EMBO Rep ; 19(2): 234-243, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29233828

RESUMO

E2F1 is the main pro-apoptotic effector of the pRB-regulated tumor suppressor pathway by promoting the transcription of various pro-apoptotic proteins. We report here that E2F1 partly localizes to mitochondria, where it favors mitochondrial outer membrane permeabilization. E2F1 interacts with BCL-xL independently from its BH3 binding interface and induces a stabilization of BCL-xL at mitochondrial membranes. This prevents efficient control of BCL-xL over its binding partners, in particular over BAK resulting in the induction of cell death. We thus identify a new, non-BH3-binding regulator of BCL-xL localization dynamics that influences its anti-apoptotic activity.


Assuntos
Morte Celular , Fator de Transcrição E2F1/metabolismo , Proteína bcl-X/metabolismo , Apoptose , Linhagem Celular Tumoral , Fator de Transcrição E2F1/química , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transcrição Genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/química
15.
J Phys Chem B ; 121(39): 9160-9168, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28903561

RESUMO

Antiapoptotic Bcl-xL plays central roles in regulating programed cell death. Partial unfolding of Bcl-xL has been observed at the interface upon specific binding to the pro-apoptotic BH3-only protein PUMA, which in turn disrupts the interaction of Bcl-xL with tumor suppressor p53 and promotes apoptosis. Previous analysis of existing Bcl-xL structures and atomistic molecular dynamics (MD) simulations have suggested that substantial intrinsic structure heterogeneity exists at the BH3-only protein binding interface of Bcl-xL to facilitate its conformational transitions upon binding. In this study, enhanced sampling is applied to further characterize the interfacial conformations of unbound Bcl-xL in explicit solvent. Extensive replica exchange with solute tempering (REST) simulations, with a total accumulated time of 16 µs, were able to cover much wider conformational spaces for the interfacial region of Bcl-xL. The resulting structural ensembles are much better converged, with local and long-range structural features that are highly consistent with existing NMR data. These simulations further demonstrate that the BH3-only protein binding interface of Bcl-xL is intrinsically disordered and samples many rapidly interconverting conformations. Intriguingly, all previously observed conformers are well represented in the unbound structure ensemble. Such intrinsic structural heterogeneity and flexibility may be critical for Bcl-xL to undergo partial unfolding induced by PUMA binding, and likely provide a robust basis that allows Bcl-xL to respond sensitively to binding of various ligands in cellular signaling and regulation.


Assuntos
Proteína bcl-X/química , Apoptose , Humanos , Antígenos de Histocompatibilidade Menor/química , Ligação Proteica , Conformação Proteica , Desdobramento de Proteína
16.
PLoS One ; 12(5): e0177413, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28481930

RESUMO

An interesting feature of Bcl-xL protein is the presence of an unstructured loop domain between α1 and α2 helices, a domain not essential for its anti-apoptotic function and absent in CED-9 protein. Within this domain, Bcl-xL undergoes dynamic phosphorylation and dephosphorylation at Ser49 and Ser62 during G2 and mitosis in human cells. Studies have revealed that when these residues are mutated, cells harbour mitotic defects, including chromosome mis-attachment, lagging, bridging and mis-segregation with, ultimately, chromosome instability and aneuploidy. We undertook genetic experiments in Caenorhabditis elegans to understand the importance of Bcl-xL (Ser49) and (Ser62) in vivo. Transgenic worms carrying single-site S49A, S62A, S49D, S62D and dual site S49/62A mutants were generated and their effects were analyzed in germlines of young adult worms. Worms expressing Bcl-xL variants showed decreased egg-laying and hatching potency, variations in the length of their mitotic regions but not of their transition zones, appearance of chromosomal abnormalities at their diplotene stages, and increased germline apoptosis, with the exception of the S62D variants. Some of these transgenic strains, particularly the Ser to Ala variants, also showed slight modulations of lifespan compared to their controls. In addition, RNAi experiments silencing expression of the various Bcl-xL variants reversed their effects in vivo. Our in vivo observations confirmed the importance of Ser49 and Ser62 within Bcl-xL loop domain in maintaining chromosome stability.


Assuntos
Aneuploidia , Caenorhabditis elegans/genética , Mutação em Linhagem Germinativa , Serina/genética , Proteína bcl-X/genética , Animais , Animais Geneticamente Modificados , Humanos , Proteína bcl-X/química
17.
Proteins ; 85(8): 1567-1579, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28486788

RESUMO

Bcl-xl protein has a long unstructured loop attached to its structured region which joins two helices. The necessity to have this unstructured segment in Bcl-xl is not yet well understood. To what extent the unstructured segment can influence the dynamics of the structured region of protein, with potential to influence the function, has been investigated in this work. Molecular dynamics simulation and principal component analysis show how the loop affects the internal motions of the protein, particularly its ligand binding pocket. Generally an unstructured region in the structure would promote flexibility resulting entropic stability but in contrary, here it narrows down the conformational space of the structured region of protein that could be hypothesized to impact the functional precision. Effects of the loop propagate to the binding pocket through structural rearrangements of polar side chains. The immediate suspicion of possible impact of phosphorylation to modulate the function of the protein is proven to be a fact, as the phosphorylated S49 and S62 located on the large unstructured region are seen to perturb the electrostatic network of the structure; an observation that validates and clarifies the role of loop as a modulator through biophysical and biochemical mechanisms. Proteins 2017; 85:1567-1579. © 2017 Wiley Periodicals, Inc.


Assuntos
Simulação de Dinâmica Molecular , Proteína bcl-X/química , Sítios de Ligação , Humanos , Ligantes , Fosforilação , Análise de Componente Principal , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Eletricidade Estática , Termodinâmica , Proteína bcl-X/metabolismo
18.
Chem Biol Interact ; 268: 53-67, 2017 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-28235427

RESUMO

The limited efficacy of marketed anticancer agents demands the design of novel target-specific hybrid molecules incorporating multiple bioactive pharmacores to combat cancer. In the present study, a one-pot simple and efficient T3P® mediated procedure for the preparation of twelve new 3-(substituted- [1,2,4]triazolo[3,4-b] [1,3,4]thiadiazolo)-1H-indoles with short reaction times, easy workup procedure, good yields, and purity of products is described. Cytotoxicity assay (MTT), flow-cytometric univariate cell cycle analysis, Annexin V-FITC staining and DNA fragmentation for cell death mechanism suggested that compound 3d with chloro-substituted phenyl ring induced enhanced cytotoxicity by an apoptotic pathway with high differential toxicity to breast adenocarcinoma cells (MCF-7) when compared with normal human dermal fibroblast cells. Additionally, the interaction between the BH3 domain of anti-apoptotic proteins Bcl-2 and Bcl-xL with the pharmacophore 3d was examined by molecular docking simulations to assess its potential to induce apoptosis. The docking solutions were proposed to explain the observed selectivity of 3d to Bcl-xL protein. From the present findings, the lead compound, 3d exhibited better anticancer activity when related to the other synthesized molecules with specific action on MCF-7 cells and hence can be considered as a plausible candidate chemo-therapeutic agent, although this warrants further experimentation.


Assuntos
Adenocarcinoma/tratamento farmacológico , Anidridos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Indóis/farmacologia , Organofosfonatos/química , Tiadiazóis/farmacologia , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Fragmentação do DNA/efeitos dos fármacos , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Química Verde , Humanos , Indóis/síntese química , Indóis/química , Ligantes , Células MCF-7/efeitos dos fármacos , Simulação de Acoplamento Molecular , Proteínas Proto-Oncogênicas c-bcl-2/química , Relação Estrutura-Atividade , Tiadiazóis/síntese química , Tiadiazóis/química , Triazóis/síntese química , Triazóis/química , Proteína bcl-X/química
19.
J Biochem ; 161(3): 291-296, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28003430

RESUMO

Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína bcl-X/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Sítios de Ligação , Células CHO , Cricetulus , Células HEK293 , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Proteína bcl-X/química
20.
Angew Chem Int Ed Engl ; 55(36): 10746-50, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27351143

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non-natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope-labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions. Using a biradical-labeled ligand, we were able to direct the hyperpolarization to the protein of interest, maintaining comparable signal enhancement with about 400-fold less radicals than conventionally used. We could selectively filter out our target protein directly from crude cell lysate obtained from only 8 mL of fully isotope-enriched cell culture. Our approach offers effective means to study proteins with atomic resolution in increasingly native concentrations and environments.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Isótopos de Carbono/química , Óxidos N-Cíclicos/química , Marcação por Isótopo , Polietilenoglicóis/química , Propanóis/química , Estrutura Secundária de Proteína , Proteínas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/química , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA