RESUMO
Fatty acids (FAs) are one of the essential energy sources for physiological processes, and they play a vital role in regulating immune and inflammatory responses, promoting cell differentiation and apoptosis, and inhibiting tumor growth. These functions are carried out by FA binding proteins (FABPs) that recognize and transport FAs. Although the crystal structure of the FA-FABPs complex has long been characterized, the mechanism behind FA binding and dissociation from FABP remains unclear. This study employed conventional MD simulations and enhanced sampling technologies to investigate the atomic-scale complexes of heart fatty acid binding proteins and stearic acid (SA). The results revealed two primary pathways for the binding or dissociation of the flexible long-chain ligand, with the orientation of the SA carboxyl head during dissociation determining the chosen path. Conformational changes in the portal region of FABP during the ligand binding/unbinding were found to be trivial, and the overturn of the â³capâ³ or the unfolding of the α2 helix was not required. This study resolves the long-standing debate on the binding mechanism of SA with the long-flexible tail to FABP, which significantly improves the understanding of the transport mechanism of FABPs and the development of related therapeutic agents.
Assuntos
Proteínas de Ligação a Ácido Graxo , Proteínas de Neoplasias , Proteínas de Ligação a Ácido Graxo/química , Ligantes , Proteínas de Neoplasias/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Ligação ProteicaRESUMO
Fatty acid-binding proteins (FABPs) are chaperones that facilitate the transport of long-chain fatty acids within the cell and can provide cargo-dependent localization to specific cellular compartments. Understanding the nature of this transport is important because lipid signaling functions are associated with metabolic pathways impacting disease pathologies including cancer, autism, and schizophrenia. FABPs often associate with cell membranes to acquire and deliver their bound cargo as part of transport. We focus on brain FABP (FABP7), which demonstrates localization to the cytoplasm and nucleus, influencing transcription and fatty acid metabolism. We use a combined biophysical-computational approach to elucidate the interaction between FABP7 and model membranes. Specifically, we use multiple experiments to demonstrate that FABP7 can bind oleic acid and docosahexaenoic acid micelles. Data from NMR and multiscale molecular dynamics simulations reveal that the interaction with micelles is through FABP7's portal region residues. Simulations suggest that binding to membranes occurs through the same residues as micelles. Simulations also capture binding events where fatty acids dissociate from the membrane and enter FABP7's binding pocket. Overall, our data shed light on the interactions between FABP7 and OA or DHA micelles and provide insight into the transport of long-chain fatty acids.
Assuntos
Ácidos Graxos , Neoplasias , Humanos , Ácidos Graxos/metabolismo , Micelas , Proteínas de Ligação a Ácido Graxo/química , Neoplasias/metabolismo , Membrana Celular/metabolismo , Proteína 7 de Ligação a Ácidos Graxos/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3-VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells.
Assuntos
Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Itraconazol/farmacologia , Membrana Nuclear/metabolismo , Proteínas de Transporte Vesicular/metabolismo , proteínas de unión al GTP Rab7/metabolismo , Transporte Ativo do Núcleo Celular , Antifúngicos/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Colestenonas/farmacologia , Endocitose , Endossomos/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Humanos , Cetoconazol/farmacologia , Modelos Moleculares , Saponinas/farmacologia , Proteínas de Transporte Vesicular/química , proteínas de unión al GTP Rab7/químicaRESUMO
Thermal shift assay (TSA) is a widely used method in discovering potential compounds (e.g., ligands, inhibitors, and other additives) to the target protein for structural genomics and drug screening in both academia and industry. The presence of sensitive fluorescent dye enables to monitor thermal stability of protein and compounds affecting this stability. By using a conventional real-time PCR instrument, it is determined as a low-cost and high efficacy experiment applied to identify optimal conditions for ligand binds to protein. Fatty acid-binding proteins (FABPs) are small molecular proteins in transporting fatty acids and other lipophilic substances in physiological and pathological responses. This chapter presents a comprehensive workflow to monitor recombinant FABP-compound interactions for an initial screening for inhibitors using TSA with SYPRO Orange dye.
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Corantes Fluorescentes/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Antineoplásicos/farmacologia , Linhagem Celular , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Proteínas de Ligação a Ácido Graxo/química , Humanos , Ligantes , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/metabolismo , Temperatura de TransiçãoRESUMO
Oxysterol-binding protein (OSBP) and its related protein (ORP) constitute a conserved family of lipid transfer proteins (LTPs). ORPs have been implicated as intracellular lipid exchanger and sensor in recent years, which regulate the lipid homeostasis and signal pathway. OSBP-related protein 3 plays key role in controlling cell adhesion and migration and could be developed as the drug target for cancer therapy. Here, we report the crystal structures of human ORP3 ORD to 2.1 Å and ORD-PI4P complex to 3.2 Å. The binding assay in vitro confirms the ORP3 has the capability of PI4P binding. This study further verifies that the PI4P is the common ligand of all ORPs and ORPs should be the lipid exchanger in membrane contact sites(MCS).
Assuntos
Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Homologia Estrutural de ProteínaRESUMO
This chapter presents the different techniques to purify the native forms of Fasciola hepatica fatty acid-binding protein (Fh12) using size exclusion chromatography and isoelectric focusing (IEF). Also, it presents the procedure to study the immunological effect of the purified protein Fh12 using monocyte-derived macrophages (MDM) obtained from healthy human donors. For this purpose, I present the procedure to isolate and culture peripheral blood mononuclear cells (PBMCs) to generate alternatively activated macrophages (AAMΦ) by in vitro exposure to Fh12.
Assuntos
Fasciola hepatica/química , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Macrófagos/parasitologia , Animais , Fasciolíase/parasitologia , Humanos , Focalização Isoelétrica/métodos , Leucócitos Mononucleares/parasitologia , Monócitos/parasitologiaRESUMO
The fatty acid-binding protein (FABP) gene family, which encodes a group of fatty acid-trafficking molecules that affect cellular functions, has been studied extensively in mammals. However, little is known about the gene structure, expression profile, and regulatory mechanism of the gene family in chickens. In the present study, bioinformatics-based methods were used to identify the family members and investigate their evolutionary history and features of gene structure. Real-time PCR combined with in vivo and in vitro experiments were used to examine the spatiotemporal expression pattern, and explore the regulatory mechanism of FABP genes. The results show that nine members of the FABP gene family, which branched into two clusters and shared a conserved FATTYACIDBP domain, exist in the genome of chickens. Of these, seven FABP genes, including FABP1, FABP3-7, and FABP10 were abundantly expressed in the liver of hens. The expression levels of FABP1, FABP3, and FABP10 were significantly increased, FABP5 and FABP7 were significantly decreased, and FABP4 and FABP6 remained unchanged in hens at the peak laying stage in comparison to those at the pre-laying stage. Transcription of FABP1 and FABP3 were activated by estrogen via estrogen receptor (ER) α, whilst FABP10 was activated by estrogen via ERß. Meanwhile, the expression of FABP1 was regulated by peroxisome proliferator activated receptor (PPAR) isoforms, of which tested PPARα and PPARß agonists significantly inhibited the expression of FABP1, while tested PPARγ agonists significantly increased the expression of FABP1, but downregulated it when the concentration of the PPARγ agonist reached 100 nM. The expression of FABP3 was upregulated via tested PPARß and PPARγ agonists, and the expression of FABP7 was selectively promoted via PPARγ. The expression of FABP10 was activated by all of the three tested PPAR agonists, but the expression of FABP4-6 was not affected by any of the PPAR agonists. In conclusion, members of the FABP gene family in chickens shared similar functional domains, gene structures, and evolutionary histories with mammalian species, but exhibited varying expression profiles and regulatory mechanisms. The results provide a valuable resource for better understanding the biological functions of individual FABP genes in chickens.
Assuntos
Biologia Computacional/métodos , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Animais , Linhagem Celular , Galinhas , Evolução Molecular , Proteínas de Ligação a Ácido Graxo/química , Feminino , Regulação da Expressão Gênica , Fígado/metabolismo , Família Multigênica , Regiões Promotoras Genéticas , Domínios Proteicos , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Distribuição Tecidual , Ativação TranscricionalRESUMO
1. Adipocyte fatty acid binding protein (A-FABP) plays a key role in fatty acid uptake and intracellular transport. The objective of the present study was to identify and characterise the A-FABP gene in Xupu goose.2. The full-length cDNA of goose A-FABP gene was cloned from the liver tissue using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The distribution of the goose A-FABP in different tissues was determined by quantitative real-time PCR (qRT-PCR).3. The results showed that the full-length cDNA sequence of goose A-FABP was 657 bp, containing a 5'-UTR of 52 bp, a 3'-UTR of 206 bp and an open reading frame (ORF) of 399 bp, which encoded a polypeptide of 132 amino acids (AA).4. The AA sequence of goose A-FABP showed 76.52%, 75.00%, 93.18% and 99.24% identities with previously described homologues from humans (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), and duck (Anas platyrhynchos), respectively, and phylogenetic analysis revealed a close relationship among them. The transcript of Xupu goose A-FABP was ubiquitously expressed in all tested tissues, and showed a high-level expression in abdominal fat, sebum and liver.5. A significant positive correlation was identified between A-FABP mRNA abundance in the three adipose tissues and liver weight, ratio of liver to body weight, TG content, and VLDL concentration in the plasma of Xupu goose. A significant negative correlation was observed between the mRNA level of A-FABP and HDL concentration in the plasma of Xupu goose.6. These findings provide a foundation for further research on the function and mechanism of A-FABP in the fat deposition process.
Assuntos
DNA Complementar/química , Proteínas de Ligação a Ácido Graxo/genética , Gansos/genética , Gordura Abdominal/química , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , China , Clonagem Molecular , DNA Complementar/biossíntese , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Gansos/classificação , Gansos/metabolismo , Perfilação da Expressão Gênica/veterinária , Fígado/química , Masculino , Filogenia , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Sebo/química , Alinhamento de Sequência/veterináriaRESUMO
CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic pulmonary surfactant currently under clinical development for the treatment of Respiratory Distress Syndrome in premature infants. The product is composed of phospholipids in liposomal organization, together with two peptide analogues of human surfactant proteins B and C. Phospholipids in liposomes can undergo oxidation of unsaturated lipids and hydrolysis, with formation of fatty acids and lysolipids, both affecting the physico-chemical properties of the formulation. We exploited two fluorescence probes, Prodan and ADIFAB, to evaluate the stability of the phospholipid components of CHF5633. While Prodan enters the phospholipid bilayer and probes the polarity of this environment, ADIFAB binds free fatty acids in the aqueous phase, allowing to determine their concentration. Changes of Prodan fluorescence emission indicated an increase in the polarity of the phospholipid bilayer as a function of time. This behavior is coupled with an increase in fatty acids concentration in the aqueous phase, as determined by ADIFAB, and an increase in lysolipids concentration, as determined by HPLC-MS. Prodan and ADIFAB resulted efficient probes to monitor phospholipids hydrolysis in liposomes, reporting an increased stability of CHF5633 at pH values higher than 6.5.
Assuntos
Fragmentos de Peptídeos/química , Fosfatidilcolinas/química , Fosfolipídeos/química , Proteína B Associada a Surfactante Pulmonar/química , Proteína C Associada a Surfactante Pulmonar/química , Surfactantes Pulmonares/química , Espectrometria de Fluorescência/métodos , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Cromatografia Líquida de Alta Pressão/métodos , Estabilidade de Medicamentos , Proteínas de Ligação a Ácido Graxo/química , Ácidos Graxos/química , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Lipossomos , Espectrometria de Massas/métodos , Proteínas Recombinantes/químicaRESUMO
Schistosomiasis is a fatal disease that has a negative impact on health and economics. Praziquantel (PZQ) is the drug of choice for schistosomiasis treatment, but it has no prophylactic effect; therefore, vaccination is an essential requirement in schistosomiasis control. This work was carried out to investigate the possible effect of DNA vaccination against Schistosoma mansoni infection using recombinant S. mansoni fatty acid binding protein (rsmFABP). The smFABP gene was cloned into the eukaryotic expression vector pcDNAI/Amp in order to obtain an smFABP-pcDNAI recombinant plasmid (DNA vaccine) and was used for the intramuscular DNA vaccination of out-bread Swiss albino mice prior to infection with S. mansoni cercariae. Infected groups, either DNA vaccinated or unvaccinated, were treated with PZQ at week 6 post-infection. After 8 weeks post-infection, all mouse groups were sacrificed and parasitological, immunological and histopathological parameters were studied. DNA vaccinated mice showed a high titer of anti-smFABP-IgG antibodies and acquired significant protection (74.2%, pâ¯<â¯0.01) against S. mansoni infection, with a reduction in ova and granuloma counts. DNA vaccinated and PZQ treated animals had higher titers of anti-smFABP-IgG antibodies and decreased (87%, P < 0.001) parenchymal granulomas compared to the DNA vaccinated PZQ untreated group. Infected mice, either non DNA vaccinated or vaccinated, had very high collagen content and fibrous granulomas (74%) compared to the PZQ treated group (10.3% fibrous granuloma) and PZQ treated + DNA vaccinated group (0% fibrous granuloma). In conclusion, DNA vaccination had protective and anti-pathological effects in naive mice and greatly improved the pathological status in PZQ-treated animals, suggesting an immunological and pathological modulating effect of PZQ treatment.
Assuntos
Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Helminto/genética , Schistosoma mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Vacinas de DNA , Sequência de Aminoácidos , Animais , Biomphalaria , Cricetinae , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/imunologia , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Fígado/patologia , Mesocricetus , Camundongos , Vacinas de DNA/administração & dosagemRESUMO
Designing highly selective inhibitors of fatty acid binding proteins 4 and 5 (FABP4 and FABP5) is of importance for treatment of some diseases related with inflammation, metabolism, and tumor growth. In this study, molecular dynamics (MD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) method were performed to probe binding selectivity of three inhibitors (5M7, 65X, and 65Z) to FABP4/FABP5 with Ki values of 0.022/0.50 µM, 0.011/0.086 µM, and 0.016/0.12 µM, respectively. The results not only suggest that all inhibitors associate more tightly with FABP4 than FABP5, but also prove that the main forces driving the selective bindings of inhibitors to FABP4 and FABP5 stem from the difference in the van der Waals interactions and polar interactions of inhibitors with two proteins. Meanwhile, a residue-based free energy decomposition method was applied to reveal molecular basis that inhibitors selectively interact with individual residues of two different proteins. The calculated results show that the binding difference of inhibitors to the residues (Phe16, Phe19), (Ala33, Gly36), (Phe57, Leu60), (Ala75, Ala78), (Arg126, Arg129), and (Tyr128, Tyr131) in (FABP4, FABP5) drive the selectivity of inhibitors toward FABP4 and FABP5. This study will provide great help for further design of effective drugs to protect against a series of metabolic diseases, arteriosclerosis, and inflammation.
Assuntos
Anti-Inflamatórios/química , Proteínas de Ligação a Ácido Graxo/química , Piperidinas/química , Quinolinas/química , Sequência de Aminoácidos , Anti-Inflamatórios/síntese química , Sítios de Ligação , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Piperidinas/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quinolinas/síntese química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two ß-sheets of the ß-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.
Assuntos
Proteínas de Ligação a Ácido Graxo/química , Receptores do Ácido Retinoico/química , Tretinoína/química , Água/química , Apoproteínas/química , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
Calnexin is a type 1 integral endoplasmic reticulum membrane molecular chaperone with an endoplasmic reticulum luminal chaperone domain and a highly conserved C-terminal domain oriented to the cytoplasm. Fabp5 is a cytoplasmic protein that binds long-chain fatty acids and other lipophilic ligands. Using a yeast two-hybrid screen, immunoprecipitation, microscale thermophoresis analysis and cellular fractionation, we discovered that Fabp5 interacts with the calnexin cytoplasmic C-tail domain at the endoplasmic reticulum. These observations identify Fabp5 as a previously unrecognized calnexin binding partner.
Assuntos
Calnexina/química , Calnexina/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Fibroblastos/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Citoplasma/química , Retículo Endoplasmático/química , Proteínas de Ligação a Ácido Graxo/química , Fibroblastos/química , Camundongos , Proteínas de Neoplasias/química , Ligação Proteica , Domínios ProteicosRESUMO
Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule. Results demonstrated that Fh15 suppresses the expression of IL-1ß and TNFα in murine macrophages and THP1 Blue CD14 cells. Additionally, Fh15 suppress the LPS-induced TLR4 stimulation. This effect was not impaired by a thermal denaturing process or blocked by the presence of anti-Fh12 antibodies. Fh15 also suppressed the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti-inflammatory drug in preclinical studies in the near future.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Fasciola hepatica/química , Proteínas de Ligação a Ácido Graxo/farmacologia , Proteínas de Helminto/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Receptor 4 Toll-Like/imunologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Anticorpos Neutralizantes/farmacologia , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fasciola hepatica/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Expressão Gênica , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Proteínas de Helminto/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células THP-1 , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Human FABP5 and FABP7 are intracellular endocannabinoid transporters. SBFI-26 is an α-truxillic acid 1-naphthyl monoester that competitively inhibits the activities of FABP5 and FABP7 and produces antinociceptive and anti-inflammatory effects in mice. The synthesis of SBFI-26 yields several stereoisomers, and it is not known how the inhibitor binds the transporters. Here we report co-crystal structures of SBFI-26 in complex with human FABP5 and FABP7 at 2.2 and 1.9 Å resolution, respectively. We found that only (S)-SBFI-26 was present in the crystal structures. The inhibitor largely mimics the fatty acid binding pattern, but it also has several unique interactions. Notably, the FABP7 complex corroborates key aspects of the ligand binding pose at the canonical site previously predicted by virtual screening. In FABP5, SBFI-26 was unexpectedly found to bind at the substrate entry portal region in addition to binding at the canonical ligand-binding pocket. Our structural and binding energy analyses indicate that both R and S forms appear to bind the transporter equally well. We suggest that the S enantiomer observed in the crystal structures may be a result of the crystallization process selectively incorporating the (S)-SBFI-26-FABP complexes into the growing lattice, or that the S enantiomer may bind to the portal site more rapidly than to the canonical site, leading to an increased local concentration of the S enantiomer for binding to the canonical site. Our work reveals two binding poses of SBFI-26 in its target transporters. This knowledge will guide the development of more potent FABP inhibitors based upon the SBFI-26 scaffold.
Assuntos
Analgésicos/metabolismo , Ciclobutanos/metabolismo , Ácidos Dicarboxílicos/metabolismo , Proteína 7 de Ligação a Ácidos Graxos/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Modelos Moleculares , Proteínas Supressoras de Tumor/metabolismo , Analgésicos/química , Analgésicos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Apoproteínas/antagonistas & inibidores , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Domínio Catalítico , Biologia Computacional , Cristalografia por Raios X , Ciclobutanos/química , Ciclobutanos/farmacologia , Ácidos Dicarboxílicos/química , Ácidos Dicarboxílicos/farmacologia , Proteína 7 de Ligação a Ácidos Graxos/antagonistas & inibidores , Proteína 7 de Ligação a Ácidos Graxos/química , Proteína 7 de Ligação a Ácidos Graxos/genética , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Ligantes , Camundongos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas Recombinantes , Estereoisomerismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites.
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acil Coenzima A/química , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Coenzima A Ligases/química , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Simulação por Computador , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Mutagênese Sítio-Dirigida , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In the present study, the cDNA encoding FABP (Ov-FABP) was isolated from the adult stage of Opisthorchis viverrini and characterized. The Ov-FABP protein sequence (107 amino acids) was predicted to have a molecular mass of 12.26kDa and an isoelectric point (PI) of 6.82. This sequence had a high identity and similarity to Cs-FABP of the related opisthorchid Clonorchis sinensis. Multiple sequence alignment with FABPs from other parasitic flatworms and mammals showed a number of conserved amino acids including Phe34, Gly37, Glu38, Glu39,Val50, Iso62, Gly81, Ile84, Ser87 and Arg101. In addition, the structure of Ov-FABP was predicted to have eleven ß-sheets and one α-helix based on the known structures for FABPs from human (hL-FABP), rat and a schistosome. Phylogenetic analysis of amino acid sequence data revealed a close relationship of Ov-FABP with Cs-FABP and hL-FABP. Reverse transcription-PCR revealed that Ov-FABP was transcribed in the egg, metacercaria, juvenile and adult stages. The soluble form of recombinant Ov-FABP (rOv-FABP) was shown to specifically bind fatty acids, including oleic acid, palmitic acid and linoleic acid, as shown for other animals. Anti-serum against rOv-FABP (produced in mice) located the protein to parenchyma, egg, sucker musculature, testes and tegument of adult O. viverrini. Taken together, the findings suggest key functional roles for Ov-FABP in development, reproduction and/or host-parasite interactions.
Assuntos
Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita , Opisthorchis/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Masculino , Camundongos , Opisthorchis/genética , Especificidade de Órgãos , Filogenia , Alinhamento de SequênciaRESUMO
Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.
Assuntos
DNA de Helmintos/genética , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Helminto/genética , Tylenchoidea/genética , Animais , Sítios de Ligação , DNA de Helmintos/química , DNA de Helmintos/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/classificação , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Hibridização In Situ , Filogenia , Raízes de Plantas/parasitologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Tela Subcutânea/metabolismo , Triticum/parasitologia , Tylenchoidea/metabolismo , Vitamina A/química , Vitamina A/metabolismoRESUMO
Recently, the Markov state model has been applied for kinetic analysis of molecular dynamics simulations. However, discretization of the conformational space remains a primary challenge in model building, and it is not clear how the space decomposition by distinct clustering strategies exerts influence on the model output. In this work, different clustering algorithms are employed to partition the conformational space sampled in opening and closing of fatty acid binding protein 4 as well as inactivation and activation of the epidermal growth factor receptor. Various classifications are achieved, and Markov models are set up accordingly. On the basis of the models, the total net flux and transition rate are calculated between two distinct states. Our results indicate that geometric and kinetic clustering perform equally well. The construction and outcome of Markov models are heavily dependent on the data traits. Compared to other methods, a combination of Bayesian and hierarchical clustering is feasible in identification of metastable states.
Assuntos
Algoritmos , Cadeias de Markov , Simulação de Dinâmica Molecular , Análise por Conglomerados , Receptores ErbB/química , Receptores ErbB/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Cinética , Conformação ProteicaRESUMO
A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.