RESUMO
Pulsed dipolar spectroscopy, such as double electron-electron resonance (DEER), has been underutilized in protein structure determination, despite its ability to provide valuable spatial information. In this study, we present DEERefiner, a user-friendly MATLAB-based GUI program that enables the modeling of protein structures by combining an initial structure and DEER distance restraints. We illustrate the effectiveness of DEERefiner by successfully modeling the ligand-dependent conformational changes of the proton-drug antiporter LmrP to an extracellular-open-like conformation with an impressive precision of 0.76 Å. Additionally, DEERefiner was able to uncover a previously hypothesized but experimentally unresolved proton-dependent conformation of LmrP, characterized as an extracellular-closed/partially intracellular-open conformation, with a precision of 1.16 Å. Our work not only highlights the ability of DEER spectroscopy to model protein structures but also reveals the potential of DEERefiner to advance the field by providing an accessible and applicable tool for precise protein structure modeling, thereby paving the way for deeper insights into protein function.
Assuntos
Proteínas de Membrana Transportadoras , Prótons , Espectroscopia de Ressonância de Spin EletrônicaRESUMO
BsYetJ is a bacterial homolog of transmembrane BAX inhibitor-1 motif-containing 6 (TMBIM6) membrane protein that plays a key role in the control of calcium homeostasis. However, the BsYetJ (or TMBIM6) structure embedded in a lipid bilayer is uncharacterized, let alone the molecular mechanism of the calcium transport activity. Herein, we report structures of BsYetJ in lipid nanodiscs identified by double electron-electron resonance spectroscopy. Our results reveal that BsYetJ in lipid nanodiscs is structurally different from those crystallized in detergents. We show that BsYetJ conformation is pH-sensitive in apo state (lacking calcium), whereas in a calcium-containing solution it is stuck in an intermediate, inert to pH changes. Only when the transmembrane calcium gradient is established can the calcium-release activity of holo-BsYetJ occur and be mediated by pH-dependent conformational changes, suggesting a dual gating mechanism. Conformational substates involved in the process and a key residue D171 relevant to the gating of calcium are identified. Our study suggests that BsYetJ/TMBIM6 is a pH-dependent, voltage-gated calcium channel.
Assuntos
Bacillus subtilis/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Bicamadas Lipídicas/química , Nanoestruturas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Sequência Conservada , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica , Reprodutibilidade dos Testes , Marcadores de SpinRESUMO
Nanodiscs are broadly used for characterization of membrane proteins as they are generally assumed to provide a near-native environment. In fact, it is an open question whether the physical properties of lipids in nanodiscs and membrane vesicles of the same lipid composition are identical. Here, we investigate the properties of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, and their mixtures) in two different sample types, nanodiscs and multilamellar vesicles, by means of spin-label electron spin resonance techniques. Our results provide a quantitative description of lipid dynamics and ordering, elucidating the molecular details of how lipids in the two sample types behave differently in response to temperature and lipid composition. We show that the properties of lipids are altered in nanodiscs such that the dissimilarity of the fluid and gel lipid phases is reduced, and the first-order phase transitions are largely abolished in nanodiscs. We unveil that the ensemble of lipids in the middle of a nanodisc bilayer, as probed by the end-chain spin-label 16-PC, is promoted to a state close to a miscibility critical point, thereby rendering the phase transitions continuous. Critical phenomena have recently been proposed to explain features of the heterogeneity in native cell membranes. Our results lay the groundwork for how to establish a near-native environment in nanodiscs with simple organization of lipid components.
Assuntos
Bicamadas Lipídicas , Nanoestruturas , Bicamadas Lipídicas/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana , TemperaturaRESUMO
While activation of BAX is required for initiating mitochondria-mediated apoptosis, the underlying mechanisms remain unsettled. We studied conformations of BAX protein using pressure- and temperature-resolved ESR techniques and obtained the thermodynamic properties of the conformations. We show that inactive BAX is structurally heterogeneous and exists in equilibrium between two major populations of the conformations, UM and UM', of which the former is thermodynamically favored at room temperature. An increase in the population of UM', induced by either pressure or point mutations of BAX, renders BAX susceptible to oligomerization, which leads to cell death. This study uncovers the biological significance of BAX conformations and shows that the pro-apoptotic activity of BAX can be triggered by altering the equilibrium between the two states. It suggests that therapeutic intervention may focus on shifting the balance in the conformational heterogeneity.
Assuntos
Apoptose/genética , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Animais , Camundongos , Mutação/genética , Pressão , Conformação Proteica , Temperatura , Proteína X Associada a bcl-2/metabolismoRESUMO
Double electron-electron resonance (DEER) is a powerful technique for studying protein conformations. To preserve the room-temperature ensemble, proteins are usually shock-frozen in liquid nitrogen prior to DEER measurements. The use of cryoprotectant additives is, therefore, necessary to ensure the formation of a vitrified state. Here, we present a simple modification of the freezing process using a flexible fused silica microcapillary, which increases the freezing rates and thus enables DEER measurement without the use of cryoprotectants. The Bid protein, which is highly sensitive to cryoprotectant additives, is used as a model. We show that DEER with the simple modification can successfully reveal the cold denaturation of Bid, which was not possible with the conventional DEER preparations. The DEER result reveals the nature of Bid folding. Our method advances DEER for capturing the chemically and thermally induced conformational changes of a protein in a cryoprotectant-free medium.
Assuntos
Crioprotetores , Crioprotetores/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Congelamento , Conformação Proteica , Marcadores de SpinRESUMO
The present Letter identified 2'-hydroxy-2,3,4',6'-tetramethoxychalcone (HTMC) as a potent in vitro cytotoxic agent with selective activity against cell lines derived from human lung cancer. In A549 lung adenocarcinoma cells, HTMC caused G1 phase cell-cycle arrest. HTMC treatment also led to an inhibition of cell-cycle regulatory proteins phosphorylation of cdc2 (Tyr(15) and Tyr(161)) and Rb (Ser(795) and Ser(807/811)), which was accompanied by the accumulation of tumor suppressor genes p53 and p21. In addition, in vivo data demonstrated that HTMC act as a tumor growth suppressing agent.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Genes p53 , Neoplasias Pulmonares/patologia , Adenocarcinoma/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , FosforilaçãoRESUMO
High telomerase activity (TA) is detected in most cancer cells; and thus, TA inhibition by drug or dietary food components is a new strategy for cancer prevention. In this report, we examined the effects of fourteen natural or synthetic compounds on TA in human lung adenocarcinoma A549 cells. The results demonstrated that some of the tested compounds inhibited TA, being 2'-hydroxy-2,3,4',6'-tetramethoxychalcone (HTMC) was the most potent among tested. In A549 cells, HTMC also inhibited the cell proliferation, decreased the expression of human telomerase reverse transcriptase (hTERT) and sequentially reduced the hTERT promoter. In soft agar assay HTMC treatment reduced the colony formation of A549 cells. Cellular fractionation and immunofluorescence assay demonstrated that there was no translocation of hTERT from nuclei to cytoplasm. Further studies revealed that the release of Ca(2+) was the underlying mechanism of suppressed TA and hTERT transcription in A549 cells exposed to HTMC. These in vitro data support the development of HTMC as a therapeutic agent for cancer complications.
Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Neoplasias Pulmonares/metabolismo , Telomerase/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Adenocarcinoma/enzimologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Neoplasias Pulmonares/enzimologia , Estrutura Molecular , Peso Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Relação Estrutura-Atividade , Telomerase/genética , Telomerase/metabolismoRESUMO
CONTEXT: Tianhua (TH-R) is extracted from Trichosanthes kirilowii Maxim (Cucurbitaceae) containing trichosanthin, a traditional Chinese medicine, which has been locally reported to have good anticancer effects in vivo in both animal and human models. However, there have been several reports that trichosanthin has an anticancer effect involving apoptosis. OBJECTIVE: To investigate other anticancer effects of TH-R, various tumorigenesis parameters were verified. MATERIALS AND METHODS: Telomerase activity, anti-apoptosis, anti-migration and immunomodulatory activity were estimated by telomeric repeat amplification protocol assay (TRAP), flow cytometry, Boyden chamber assay and ELISA assay, respectively. RESULTS: In our studies, we are the first to find that TH-R had a cytotoxic effect on lung cancer cells in MTS assays; it could change the cell cycle distribution of human lung cancer cells (A549 cell line) and induce apoptosis. Further anti-telomerase effects in human lung adenocarcinoma A549 cells using the TRAP assay were noted. TH-R also had an aggregation effect on peripheral blood lymphocytes, but no effect on stimulating peripheral lymphocytes to produce human interferon-γ(IFN-γ). TH-R could inhibit the migration, or metastatic ability, of A549 cells by Boyden chamber assay. In the oral feeding therapy of an in vivo mouse model, there was an initial inhibition of A549 cancer cell growth, but no statistical difference after one month of therapy. DISCUSSION AND CONCLUSION: It has been proven that medicinal herbs such as Tianhua have positive effects against cancer through preventing or inhibiting the process of lung tumorigenesis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Fatores Imunológicos/farmacologia , Extratos Vegetais/farmacologia , Tricosantina/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Inibição de Migração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Interferon gama/biossíntese , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Telomerase/metabolismo , Transplante HeterólogoRESUMO
The YtfE protein catalyzes the reduction of NO to N2O, protecting iron-sulfur clusters from nitrosylation. The structure of YtfE has a two-domain architecture, with a diiron-containing C-terminal domain linked to an N-terminal domain, in which the function of the latter is enigmatic. Here, by using electron spin resonance (ESR) spectroscopy, we show that YtfE exists in two conformational states, one of which has not been reported. Under high osmotic stress, YtfE adopts a homogeneous conformation (C state) similar to the known crystal structure. In a regular buffer, the N-terminal domain switches between the C state and a previously unidentified conformation (C' state), the latter of which has more space at the domain interface to allow the trafficking of NO molecules and thus is proposed to be a functionally active state. The conformational switch between the C and C' states is pivotal for facilitating NO access to the diiron core.
RESUMO
BCL-2, a key protein in inhibiting apoptosis, has a 65-residue-long highly flexible loop domain (FLD) located on the opposite side of its ligand-binding groove. In vivo phosphorylation of the FLD enhances the affinity of BCL-2 for pro-apoptotic ligands, and consequently anti-apoptotic activity. However, it remains unknown as to how the faraway, unstructured FLD modulates the affinity. Here we investigate the protein-ligand interactions by fluorescence techniques and monitor protein dynamics by DEER and NMR spectroscopy tools. We show that phosphomimetic mutations on the FLD lead to a reduction in structural flexibility, hence promoting ligand access to the groove. The bound pro-apoptotic ligands can be displaced by the BCL-2-selective inhibitor ABT-199 efficiently, and thus released to trigger apoptosis. We show that changes in structural flexibility on an unstructured loop can activate an allosteric protein that is otherwise structurally inactive.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação Alostérica/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Humanos , Ligantes , Simulação de Dinâmica Molecular , Fosforilação , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfonamidas/farmacologiaRESUMO
BACKGROUND: The aim of this study was to estimate the relationship of glutathione S-transferases (GST)P1, GSTA1, GSTM1, and GSTT1 gene polymorphisms to oral cancer risk. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to measure these 4 gene polymorphisms in 274 controls and 164 oral cancer patients. RESULTS: Individuals with at least 1 varied G allele of GSTP1 had a 1.53-fold risk (95% confidence interval [CI] = 1.01-2.31) of developing oral cancer compared with patients with wild-type A/A homozygotes. Oral cancer patients with at least 1 varied T allele of GSTA1 gene had a 0.42-fold risk (95% CI = 0.18-0.95) of having a tumor size >2 cm compared with patients with C/C homozygotes. CONCLUSIONS: The varied G allele of GSTP1 may be considered as a factor contributing to increased susceptibility, whereas the T allele of GSTA1 could be a protective factor for tumor size progression in Taiwanese with oral cancer.
Assuntos
Glutationa S-Transferase pi/genética , Glutationa Transferase/genética , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Adulto , Idoso , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/enzimologia , Estadiamento de Neoplasias , Polimorfismo Genético/genética , TaiwanRESUMO
The incidence and mortality of oral cancer in Taiwan have been increased during the last decade, which could be mainly resulted from the difficulty in treatment related to metastasis. As a potential and popular folk medicine, Terminalia catappa leaves have been proven to possess various biological benefits including anti-cancer activities. However, the detailed effects and molecular mechanisms of T. catappa leaves on the metastasis of oral cancer cells were still unclear. Thus, SCC-4 oral cancer cells were subjected to a treatment with ethanol extracts of T. catappa leaves (TCE) and then analyzed for the effect of TCE on the migration and invasion. Modified Boyden chamber assays revealed that TCE treatment significantly inhibited the cell migration/invasion capacities of SCC-4 cells. Furthermore, results of zymography and western blotting showed that activities and protein levels of MMP-2, MMP-9 and u-PA were all inhibited by TCE. Further studies indicated that TCE may inhibit phosphorylation of ERK1/2, JNK1/2 and Akt while the expression of nuclear protein NF-kappaB, c-Jun and c-Fos were inhibited as well. EMSA assay revealed that the DNA-binding activity with AP-1 and NF-kappaB was also decreased by TCE. In conclusion, TCE may serve as a powerful chemopreventive agent against oral cancer metastasis.