RESUMO
Cholesterol (CHL) plays an integral role in modulating the function and activity of various mammalian membrane proteins. Due to the slow dynamics of lipids, conventional computational studies of protein-CHL interactions rely on either long-time scale atomistic simulations or coarse-grained approximations to sample the process. A highly mobile membrane mimetic (HMMM) has been developed to enhance lipid diffusion and thus used to facilitate the investigation of lipid interactions with peripheral membrane proteins and, with customized in silico solvents to replace phospholipid tails, with integral membrane proteins. Here, we report an updated HMMM model that is able to include CHL, a nonphospholipid component of the membrane, henceforth called HMMM-CHL. To this end, we had to optimize the effect of the customized solvents on CHL behavior in the membrane. Furthermore, the new solvent is compatible with simulations using force-based switching protocols. In the HMMM-CHL, both improved CHL dynamics and accelerated lipid diffusion are integrated. To test the updated model, we have applied it to the characterization of protein-CHL interactions in two membrane protein systems, the human ß2-adrenergic receptor (ß2AR) and the mitochondrial voltage-dependent anion channel 1 (VDAC-1). Our HMMM-CHL simulations successfully identified CHL binding sites and captured detailed CHL interactions in excellent consistency with experimental data as well as other simulation results, indicating the utility of the improved model in applications where an enhanced sampling of protein-CHL interactions is desired.
Assuntos
Colesterol , Simulação de Dinâmica Molecular , Colesterol/química , Colesterol/metabolismo , Humanos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Ligação Proteica , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Difusão , Solventes/químicaRESUMO
The overexpression of voltage dependent anion channels (VDACs), particularly VDAC1, in cancer cells compared to normal cells, plays a crucial role in cancer cell metabolism, apoptosis regulation, and energy homeostasis. In this study, we used molecular dynamics (MD) simulations to investigate the effect of a low level of VDAC1 oxidation (induced e.g., by cold atmospheric plasma (CAP)) on the pyruvate (Pyr) uptake by VDAC1. Inhibiting Pyr uptake through VDAC1 can suppress cancer cell proliferation. Our primary target was to study the translocation of Pyr across the native and oxidized forms of hVDAC1, the human VDAC1. Specifically, we employed MD simulations to analyze the hVDAC1 structure by modifying certain cysteine residues to cysteic acids and methionine residues to methionine sulfoxides, which allowed us to investigate the effect of oxidation. Our results showed that the free energy barrier for Pyr translocation through the native and oxidized channel was approximately 4.3 ± 0.7 kJ mol-1 and 10.8 ± 1.8 kJ mol-1, respectively. An increase in barrier results in a decrease in rate of Pyr permeation through the oxidized channel. Thus, our results indicate that low levels of CAP oxidation reduce Pyr translocation, resulting in decreased cancer cell proliferation. Therefore, low levels of oxidation are likely sufficient to treat cancer cells given the inhibition of Pyr uptake.
Assuntos
Neoplasias , Ácido Pirúvico , Humanos , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Apoptose , Cisteína/química , Oxirredução , Metionina/metabolismoRESUMO
The mitochondrial voltage-dependent anion channel 1 (VDAC1) plays a central role in metabolism and apoptosis, which makes it a promising therapeutic target. Nevertheless, molecular mechanisms governing VDAC1 functioning remain unclear. Small-molecule ligands specifically interacting with the channel provide an attractive way of exploring its structure-function relationships and can possibly be used as founding stones for future drug-candidates. While around 30 VDAC1 ligands have been identified over the years, various techniques have been used by research teams, making a fair and direct comparison between compounds impossible. To tackle this issue, we performed ligand-binding assays on a representative set of seventeen known VDAC1 ligands using nano-differential scanning fluorimetry and microscale thermophoresis. While all the compounds have been confirmed as VDAC1 ligands by at least one method, combining both technologies lead to the selection of four molecules (cannabidiol, curcumin, DIDS and VBIT4) as chemical starting points for future design of VDAC1 selective ligands.
Assuntos
Canabidiol , Canal de Ânion 1 Dependente de Voltagem , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Mitocôndrias/metabolismo , Apoptose , Canabidiol/metabolismoRESUMO
The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, is responsible for the transport of all ions and metabolites into and out of mitochondria. Larger than any of the ß-barrel structures determined to date by magic-angle spinning (MAS) NMR, but smaller than the size limit of cryo-electron microscopy (cryo-EM), VDAC1's 31 kDa size has long been a bottleneck in determining its structure in a near-native lipid bilayer environment. Using a single two-dimensional (2D) crystalline sample of human VDAC1 in lipids, we applied proton-detected fast magic-angle spinning NMR spectroscopy to determine the arrangement of ß strands. Combining these data with long-range restraints from a spin-labeled sample, chemical shift-based secondary structure prediction, and previous MAS NMR and atomic force microscopy (AFM) data, we determined the channel's structure at a 2.2 Å root-mean-square deviation (RMSD). The structure, a 19-stranded ß-barrel, with an N-terminal α-helix in the pore is in agreement with previous data in detergent, which was questioned due to the potential for the detergent to perturb the protein's functional structure. Using a quintuple mutant implementing the channel's closed state, we found that dynamics are a key element in the protein's gating behavior, as channel closure leads to the destabilization of not only the C-terminal barrel residues but also the α2 helix. We showed that cholesterol, previously shown to reduce the frequency of channel closure, stabilizes the barrel relative to the N-terminal helix. Furthermore, we observed channel closure through steric blockage by a drug shown to selectively bind to the channel, the Bcl2-antisense oligonucleotide G3139.
Assuntos
Bicamadas Lipídicas/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Sítios de Ligação , Colesterol/química , Colesterol/metabolismo , Humanos , Ativação do Canal Iônico , Ligantes , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Tionucleotídeos/química , Tionucleotídeos/metabolismo , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
The voltage-dependent anion channel 1 (VDAC1) is a crucial mitochondrial transporter that controls the flow of ions and respiratory metabolites entering or exiting mitochondria. As a voltage-gated channel, VDAC1 can switch between a high-conducting "open" state and a low-conducting "closed" state emerging at high transmembrane (TM) potentials. Although cell homeostasis depends on channel gating to regulate the transport of ions and metabolites, structural hallmarks characterizing the closed states remain unknown. Here, we performed microsecond accelerated molecular dynamics to highlight a vast region of VDAC1 conformational landscape accessible at typical voltages known to promote closure. Conformers exhibiting durable subconducting properties inherent to closed states were identified. In all cases, the low conductance was due to the particular positioning of an unfolded part of the N-terminus, which obstructed the channel pore. While the N-terminal tail was found to be sensitive to voltage orientation, our models suggest that stable low-conducting states of VDAC1 predominantly take place from disordered events and do not result from the displacement of a voltage sensor or a significant change in the pore. In addition, our results were consistent with conductance jumps observed experimentally and corroborated a recent study describing entropy as a key factor for VDAC gating.
Assuntos
Ativação do Canal Iônico , Simulação de Dinâmica Molecular , Conformação Proteica , Canal de Ânion 1 Dependente de Voltagem/química , Animais , Camundongos , Modelos MolecularesRESUMO
Cholesterol is the precursor of all steroid hormones, and the entry of cholesterol into the mitochondria is the rate-limiting step of steroidogenesis. Voltage-dependent anion channel (VDAC1) is an outer mitochondrial protein part of a multiprotein complex that imports cholesterol. We previously reported that intratesticular administration of a 25 amino acid peptide blocking the interaction between 14-3-3ϵ with VDAC1 increased circulating levels of testosterone. This fusion peptide was composed of a HIV-1 transactivator of transcription (TAT) protein transduction domain cell-penetrating peptide, a glycine linker, and amino acids 159-172 of VDAC1 (TV159-172). Here, we describe the development of a family of small molecules that increase circulating testosterone levels after an oral administration. We first characterized an animal model where TV159-172 was delivered subcutaneously. This subcutaneous model allowed us to study the interactions between TV159-172 and the hypothalamus-pituitary-gonadal axis (HPG) and identify the biologically active core of TV159-172. The core consisted of the tetrapeptide RVTQ, which we used as a platform to design synthetic peptide derivatives that can be administered orally. We developed a second animal model to test various derivatives of RVTQ and found 11 active compounds. Dose-response experiments identified 4 synthetic peptides that robustly increased androgen levels in a specific manner. We selected RdVTQ as the leading VDAC1-core derivative and profiled the response across the lifespan of Brown-Norway rats. In summary, we present the development of a new class of therapeutics that act within the HPG axis to increase testosterone levels specifically. This new class of small molecules self-regulates, preventing abuse.
Assuntos
Apoptose , Canal de Ânion 1 Dependente de Voltagem , Ratos , Masculino , Animais , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Peptídeos/metabolismo , Canais de Ânion Dependentes de Voltagem , Testosterona , Administração OralRESUMO
Current cancer treatment regimens such as chemotherapy and traditional chemical drugs have adverse side effects including the appearance of drug-resistant tumor cells. For these reasons, it is imperative to find novel therapeutic agents that overcome these factors. To this end, we explored a cationic antimicrobial peptide derived from Litopenaeus vannamei hemocyanin (designated LvHemB1) that induces cancer cell death, but sparing normal cells. LvHemB1 inhibits the proliferation of human cervical (HeLa), esophageal (EC109), hepatocellular (HepG2), and bladder (EJ) cancer cell lines, but had no significant effect on normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE-3) cells). In addition to its antiproliferative effects, LvHemB1 induced apoptosis, by permeating cells and targeting mitochondrial voltage-dependent anion channel 1 (VDAC1). Colocalization studies revealed the localization of LvHemB1 in mitochondria, while molecular docking and pull-down analyses confirmed LvHemB1-VDAC1 interaction. Moreover, LvHemB1 causes loss in mitochondrial membrane potential and increases levels of reactive oxygen species (ROS) and apoptotic proteins (caspase-9, caspase-3, and Bax (Bcl-2-associated X)), which results in mitochondrial-mediated apoptosis. Thus, peptide LvHemB1 has the potential of being used as an anticancer agent due to its antiproliferation effect and targeting to VDAC1 to cause mitochondrial dysfunction in cancer cells, as well as its ability to induce apoptosis by increasing ROS levels, and the expression of proapoptotic proteins.
Assuntos
Neoplasias , Canal de Ânion 1 Dependente de Voltagem , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Hemocianinas/metabolismo , Hemocianinas/farmacologia , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
Complex formation between hexokinase-II (HKII) and the mitochondrial VDAC1 is crucial to cell growth and survival. We hypothesize that HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we devised a hybrid approach. First, we describe membrane binding of HKII with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion capturing membrane insertion of its H-anchor. The insertion depth of the H-anchor was then used to derive positional restraints in subsequent millisecond-scale Brownian dynamics (BD) simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models for the complex were further refined and their structural stability probed with additional MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1's permeation pathway, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. We also show how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and has implications in mitochondria-mediated cell death.
Assuntos
Hexoquinase/metabolismo , Proteínas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Complexos Multiproteicos/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Hexoquinase/química , Hexoquinase/genética , Humanos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Ligação Proteica , Domínios Proteicos , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.
Assuntos
Compostos de Diazônio/química , Marcadores de Fotoafinidade/química , Proteínas/química , Aminoácidos/análise , Aminoácidos/química , Sítios de Ligação , Diazometano/química , Humanos , Concentração de Íons de Hidrogênio , Conformação Proteica , Proteínas/análise , Proteoma/análise , Proteoma/química , Canal de Ânion 1 Dependente de Voltagem/químicaRESUMO
We describe approaches for the preparation of membrane proteins in detergent micelles and lipid bilayers for solution and magic angle spinning NMR studies, respectively, as exemplified by the human voltage dependent anion channel 1 (hVDAC1). Here, we report protocols for the preparation of homogenous samples of recombinant hVDAC1 in detergent micelles and lipid two-dimensional crystals yielding high resolution NMR spectra. Procedures are described for the recombinant production of stable-isotope labeled hVDAC1 in E. coli, the isolation of hVDAC1 from inclusion bodies and the refolding into detergent micelles, as well as the reconstitution of hVDAC1 into lipids to form 2D crystals.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Bicamadas Lipídicas/química , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Escherichia coli/genética , Humanos , Corpos de Inclusão/química , Marcação por Isótopo , Micelas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Redobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transformação Bacteriana , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
The voltage-dependent anion channel (VDAC) is a critical ß-barrel membrane protein of the mitochondrial outer membrane, which regulates the transport of ions and ATP between mitochondria and the cytoplasm. In addition, VDAC plays a central role in the control of apoptosis and is therefore of great interest in both cancer and neurodegenerative diseases. Although not fully understood, it is presumed that the gating mechanism of VDAC is governed by its N-terminal region which, in the open state of the channel, exhibits an α-helical structure positioned midway inside the pore and strongly interacting with the ß-barrel wall. In the present work, we performed molecular simulations with a recently developed force field for disordered systems to shed new light on known experimental results, showing that the N-terminus of VDAC is an intrinsically disordered region (IDR). First, simulation of the N-terminal segment as a free peptide highlighted its disordered nature and the importance of using an IDR-specific force field to properly sample its conformational landscape. Secondly, accelerated dynamics simulation of a double cysteine VDAC mutant under applied voltage revealed metastable low conducting states of the channel representative of closed states observed experimentally. Related structures were characterized by partial unfolding and rearrangement of the N-terminal tail, that led to steric hindrance of the pore. Our results indicate that the disordered properties of the N-terminus are crucial to properly account for the gating mechanism of VDAC.
Assuntos
Apoptose , Proteínas Intrinsicamente Desordenadas/química , Conformação Proteica , Canal de Ânion 1 Dependente de Voltagem/química , Algoritmos , Animais , Ânions , Simulação por Computador , Cristalografia por Raios X , Cisteína/química , Bicamadas Lipídicas/química , Camundongos , Mitocôndrias/metabolismo , Membranas Mitocondriais , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Neoplasias/metabolismo , Peptídeos/química , Desnaturação Proteica , Domínios Proteicos , SoftwareRESUMO
The Voltage-Dependent Anion Channel (VDAC) plays a vital role in mitochondria-mediated transport of ions and metabolites. It is well established that mitochondria are a site for production of hydrogen peroxide (H2O2). Excess production of H2O2 is toxic to the cell and causes oxidative stress. Therefore, the effect of H2O2 on the single-channel conductance of VDAC was investigated. In vitro bilayer electrophysiology experiments were performed on VDAC isolated from rat brain mitochondria, which consists predominately of the isoform VDAC1. VDAC was treated with H2O2 on a planar bilayer membrane (BLM). The conductance of VDAC increased upon H2O2 treatment, whereas the same concentration of H2O2 was unable to affect the BLM (without protein) over a long period of time. Subsequently, the sequential addition of curcumin to H2O2-treated VDAC reduced the conductance. Experimental results (bilayer electrophysiology) demonstrate the role of curcumin in the restoration of the activity of VDAC affected by H2O2. In silico docking studies enables identification of the probable binding site of H2O2 on VDAC. We further find that the oligomerization of VDAC that results in its increased conductance is an effect of lipid oxidation by H2O2.
Assuntos
Peróxido de Hidrogênio/química , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 2 Dependente de Voltagem/química , Animais , Sítios de Ligação , Encéfalo/metabolismo , Simulação por Computador , Curcumina/química , Cisteína/química , Eletrofisiologia , Ligantes , Bicamadas Lipídicas/química , Lipídeos/química , Mitocôndrias/metabolismo , Conformação Molecular , Neurônios/metabolismo , Estresse Oxidativo , Oxigênio/química , Isoformas de Proteínas , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
Quinidine is an antiarrhythmic drug commonly used for the treatment of cardiac ailments. It affects oxidative phosphorylation, calcium uptake, and ion channels of mitochondria. We have investigated the interaction of Quinidine and mitochondrial voltage-dependent anion channel (VDAC). VDAC was purified from neuronal tissue of Wistar rats and in vitro bilayer electrophysiology experiments were performed on it. 50-mM Quinidine treatment on VDAC leads to a sudden drop in its conductance. The dose of Quinidine leading to a half-maximal current through a single-channel VDAC was calculated using Quinidine at different concentrations. In silico molecular docking studies using Autodock-4.2 software indicate interaction between Quinidine and VDAC. Docking results demonstrate the interaction of Quinidine and VDAC on its Glutamic acid residue (Glu-206 of VDAC). Fluorescence spectroscopy results on Quinidine and Glutamic acid interaction show an increase in the intensity and wavelength of Quinidine fluorescence, whereas no interaction between Quinidine and Cysteine was observed. This further supports the Glutamic acid and Quinidine interaction. In conclusion, we report Quinidine partially blocks VDAC due to the interaction of Glutamic acid and Quinidine in the channel pore.
Assuntos
Ânions , Neurônios/metabolismo , Quinidina/farmacologia , Canal de Ânion 1 Dependente de Voltagem/química , Animais , Cristalografia por Raios X , Cisteína/química , Feminino , Ácido Glutâmico/química , Concentração Inibidora 50 , Masculino , Mitocôndrias/metabolismo , Simulação de Acoplamento Molecular , Fosforilação Oxidativa , Estrutura Secundária de Proteína , Ratos , Ratos Wistar , Espectrometria de Fluorescência , Canal de Ânion 2 Dependente de Voltagem/químicaRESUMO
Diabetes mellitus is a metabolic disorder approaching epidemic proportions. Non-alcoholic fatty liver disease (NAFLD) regularly coexists with metabolic disorders, including type 2 diabetes, obesity, and cardiovascular disease. Recently, we demonstrated that the voltage-dependent anion channel 1 (VDAC1) is involved in NAFLD. VDAC1 is an outer mitochondria membrane protein that serves as a mitochondrial gatekeeper, controlling metabolic and energy homeostasis, as well as crosstalk between the mitochondria and the rest of the cell. It is also involved in mitochondria-mediated apoptosis. Here, we demonstrate that the VDAC1-based peptide, R-Tf-D-LP4, affects several parameters of a NAFLD mouse model in which administration of streptozotocin (STZ) and high-fat diet 32 (STZ/HFD-32) led to both type 2 diabetes (T2D) and NAFLD phenotypes. We focused on diabetes, showing that R-Tf-D-LP4 peptide treatment of STZ/HFD-32 fed mice restored the elevated blood glucose back to close to normal levels, and increased the number and average size of islets and their insulin content as compared to untreated controls. Similar results were obtained when staining the islets for glucose transporter type 2. In addition, the R-Tf-D-LP4 peptide decreased the elevated glucose levels in a mouse displaying obese, diabetic, and metabolic symptoms due to a mutation in the obese (ob) gene. To explore the cause of the peptide-induced improvement in the endocrine pancreas phenotype, we analyzed the expression levels of the proliferation marker, Ki-67, and found it to be increased in the islets of STZ/HFD-32 fed mice treated with the R-Tf-D-LP4 peptide. Moreover, peptide treatment of STZ/HFD-32 fed mice caused an increase in the expression of ß-cell maturation and differentiation PDX1 transcription factor that enhances the expression of the insulin-encoding gene, and is essential for islet development, function, proliferation, and maintenance of glucose homeostasis in the pancreas. This increase occurred mainly in the ß-cells, suggesting that the source of their increased number after R-Tf-D-LP4 peptide treatment was most likely due to ß-cell proliferation. These results suggest that the VDAC1-based R-Tf-D-LP4 peptide has potential as a treatment for diabetes.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Peptídeos/uso terapêutico , Canal de Ânion 1 Dependente de Voltagem/química , Células 3T3-L1 , Sequência de Aminoácidos , Animais , Glicemia/análise , Glicemia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Tipo 2/induzido quimicamente , Dieta Hiperlipídica/efeitos adversos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Peptídeos/farmacologia , Estreptozocina/efeitos adversos , Resultado do Tratamento , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs.
Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanoestruturas/química , Canal de Ânion 1 Dependente de Voltagem/genética , Fenômenos Biofísicos , Membrana Celular/química , Membrana Celular/genética , Humanos , Proteínas de Membrana/genética , Fosfolipídeos/química , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Canal de Ânion 1 Dependente de Voltagem/química , Proteína bcl-X/química , Proteína bcl-X/genéticaRESUMO
The voltage-dependent anion channel (VDAC) forms the primary diffusion pore of the outer mitochondrial membrane. In its apo form, VDAC adopts an open conformation with high conductance. States of lower conductance can be induced by ligand binding or the application of voltage. Here, we clarify at the atomic level how ß-NADH binding leads to a low-conductance state and characterize the role of the VDAC N-terminal helix in voltage gating. A high-resolution NMR structure of human VDAC-1 with bound NADH, combined with molecular dynamics simulation show that ß-NADH binding reduces the pore conductance sterically without triggering a structural change. Electrophysiology recordings of crosslinked protein variants and NMR relaxation experiments probing different time scales show that increased helix dynamics is present in the open state and that motions of the N-terminal helices are involved in the VDAC voltage gating mechanism.
Assuntos
NAD/metabolismo , Canal de Ânion 1 Dependente de Voltagem/química , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
The second isoform of the human voltage dependent anion channel (VDAC2) is a mitochondrial porin that translocates calcium and other metabolites across the outer mitochondrial membrane. VDAC2 has been implicated in cardioprotection and plays a critical role in a unique apoptotic pathway in tumor cells. Despite its medical importance, there have been few biophysical studies of VDAC2 in large part due to the difficulty of obtaining homogeneous preparations of the protein for spectroscopic characterization. Here we present high resolution magic angle spinning nuclear magnetic resonance (NMR) data obtained from homogeneous preparation of human VDAC2 in 2D crystalline lipid bilayers. The excellent resolution in the spectra permit several sequence-specific assignments of the signals for a large portion of the VDAC2 N-terminus and several other residues in two- and three-dimensional heteronuclear correlation experiments. The first 12 residues appear to be dynamic, are not visible in cross polarization experiments, and they are not sufficiently mobile on very fast timescales to be visible in 13C INEPT experiments. A comparison of the NMR spectra of VDAC2 and VDAC1 obtained from highly similar preparations demonstrates that the spectral quality, line shapes and peak dispersion exhibited by the two proteins are nearly identical. This suggests an overall similar dynamic behavior and conformational homogeneity, which is in contrast to two earlier reports that suggested an inherent conformational heterogeneity of VDAC2 in membranes. The current data suggest that the sample preparation and spectroscopic methods are likely applicable to studying other human membrane porins, including human VDAC3, which has not yet been structurally characterized.
Assuntos
Bicamadas Lipídicas , Ressonância Magnética Nuclear Biomolecular/métodos , Canal de Ânion 2 Dependente de Voltagem/química , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Canal de Ânion 1 Dependente de Voltagem/químicaRESUMO
Non-alcoholic steatosis and non-alcoholic steatohepatitis (NASH) are liver pathologies characterized by severe metabolic alterations due to fat accumulation that lead to liver damage, inflammation, and fibrosis. We demonstrate that the voltage-dependent anion channel 1 (VDAC1)-based peptide R-Tf-D-LP4 arrested steatosis and NASH progression, as produced by a high-fat diet (HFD-32) in a mouse model, and reversed liver pathology to a normal-like state. VDAC1, a multi-functional mitochondrial protein, regulates cellular metabolic and energetic functions and apoptosis and interacts with many proteins. R-Tf-D-LP4 treatment eliminated hepatocyte ballooning degeneration, inflammation, and liver fibrosis associated with steatosis, NASH, and hepatocarcinoma, and it restored liver pathology-associated enzyme and glucose levels. Peptide treatment affected carbohydrate and lipid metabolism, increasing the expression of enzymes and factors associated with fatty acid transport to mitochondria, enhancing ß-oxidation and thermogenic processes, yet decreasing the expression of enzymes and regulators of fatty acid synthesis. The VDAC1-based peptide thus offers a promising therapeutic approach for steatosis and NASH.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Cirrose Hepática/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Canal de Ânion 1 Dependente de Voltagem/química , Animais , Metabolismo dos Carboidratos/efeitos dos fármacos , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Resultado do Tratamento , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
The translocator protein (TSPO), located at the outer mitochondrial membrane (OMM), serves multiple functions and contributes to numerous processes, including cholesterol import, mitochondrial metabolism, apoptosis, cell proliferation, Ca2+ signaling, oxidative stress, and inflammation. TSPO forms a complex with the voltage-dependent anion channel (VDAC), a protein that mediates the flux of ions, including Ca2+, nucleotides, and metabolites across the OMM, controls metabolism and apoptosis and interacts with many proteins. This review focuses on the two OMM proteins TSPO and VDAC1, addressing their structural interaction and associated functions. TSPO appears to be involved in the generation of reactive oxygen species, proposed to represent the link between TSPO activation and VDAC, thus playing a role in apoptotic cell death. In addition, expression of the two proteins in healthy brains and diseased states is considered, as is the relationship between TSPO and VDAC1 expression. Both proteins are over-expressed in in brains from Alzheimer's disease patients. Finally, TSPO expression levels were proposed as a biomarker of some neuropathological settings, while TSPO-interacting ligands have been considered as a potential basis for drug development.
Assuntos
Regulação da Expressão Gênica , Receptores de GABA/genética , Receptores de GABA/metabolismo , Transdução de Sinais , Canal de Ânion 1 Dependente de Voltagem/genética , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Transporte Biológico , Suscetibilidade a Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Especificidade de Órgãos , Estresse Oxidativo , Ligação Proteica , Multimerização Proteica , Receptores de GABA/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Canal de Ânion 1 Dependente de Voltagem/químicaRESUMO
Voltage-dependent anion channel-1 (VDAC1) is a mitochondrial porin that is implicated in cellular metabolism and apoptosis, and modulated by numerous small molecules including lipids. VDAC1 binds sterols, including cholesterol and neurosteroids such as allopregnanolone. Biochemical and computational studies suggest that VDAC1 binds multiple cholesterol molecules, but photolabeling studies have identified only a single cholesterol and neurosteroid binding site at E73. To identify all the binding sites of neurosteroids in VDAC1, we apply photo-affinity labeling using two sterol-based photolabeling reagents with complementary photochemistry: 5α-6-AziP which contains an aliphatic diazirine, and KK200 which contains a trifluoromethyl-phenyldiazirine (TPD) group. 5α-6-AziP and KK200 photolabel multiple residues within an E73 pocket confirming the presence of this site and mapping sterol orientation within this pocket. In addition, KK200 photolabels four other sites consistent with the finding that VDAC1 co-purifies with five cholesterol molecules. Both allopregnanolone and cholesterol competitively prevent photolabeling at E73 and three other sites indicating that these are common sterol binding sites shared by both neurosteroids and cholesterol. Binding at the functionally important residue E73 suggests a possible role for sterols in regulating VDAC1 signaling and interaction with partner proteins.