RESUMEN
Previous work showed that matrix metalloproteinase-7 (MMP-7) regulates colon cancer activities through an interaction with syndecan-2 (SDC-2) and SDC-2-derived peptide that disrupts this interaction and exhibits anticancer activity in colon cancer. Here, to identify potential anticancer agents, a library of 1,379 Food and Drug Administration (FDA)-approved drugs that interact with the MMP-7 prodomain were virtually screened by protein-ligand docking score analysis using the GalaxyDock3 program. Among five candidates selected based on their structures and total energy values for interacting with the MMP-7 prodomain, the known mechanistic target of rapamycin kinase (mTOR) inhibitor, everolimus, showed the highest binding affinity and the strongest ability to disrupt the interaction of the MMP-7 prodomain with the SDC-2 extracellular domain in vitro. Everolimus treatment of the HCT116 human colon cancer cell line did not affect the mRNA expression levels of MMP-7 and SDC-2 but reduced the adhesion of cells to MMP-7 prodomain-coated plates and the cell-surface localization of MMP-7. Thus, everolimus appears to inhibit the interaction between MMP-7 and SDC-2. Everolimus treatment of HCT116 cells also reduced their gelatin-degradation activity and anticancer activities, including colony formation. Interestingly, cells treated with sirolimus, another mTOR inhibitor, triggered less gelatin-degradation activity, suggesting that this inhibitory effect of everolimus was not due to inhibition of the mTOR pathway. Consistently, everolimus inhibited the colony-forming ability of mTOR-resistant HT29 cells. Together, these data suggest that, in addition to inhibiting mTOR signaling, everolimus exerts anticancer activity by interfering with the interaction of MMP-7 and SDC-2, and could be a useful therapeutic anticancer drug for colon cancer.NEW & NOTEWORTHY The utility of cancer therapeutics targeting the proteolytic activities of MMPs is limited because MMPs are widely distributed throughout the body and involved in many different aspects of cell functions. This work specifically targets the activation of MMP-7 through its interaction with syndecan-2. Notably, everolimus, a known mTOR inhibitor, blocked this interaction, demonstrating a novel role for everolimus in inhibiting mTOR signaling and impairing the interaction of MMP-7 with syndecan-2 in colon cancer.
Asunto(s)
Neoplasias del Colon , Everolimus , Humanos , Everolimus/farmacología , Sindecano-2/genética , Sindecano-2/metabolismo , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Gelatina , Sirolimus/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Serina-Treonina Quinasas TORRESUMEN
Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer's disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hß2AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.
Asunto(s)
Galanina/química , Proteínas de Unión al GTP Heterotriméricas , Receptor de Galanina Tipo 2/química , Microscopía por Crioelectrón , Galanina/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Ligandos , Receptor de Galanina Tipo 2/metabolismoRESUMEN
We previously showed that a synthetic peptide (S2-P) corresponding to a portion of the human syndecan-2 (SDC2) sequence can bind to the pro-domain of matrix metalloproteinase-7 (MMP-7) to inhibit colon cancer activities. Since S2-P had a relatively weak binding affinity for the MMP-7 pro-domain, we herein modified the amino acid sequence of S2-P to improve the anticancer potential. On the basis of the interaction structure of S2-P and MMP-7, four peptides were generated by replacing amino acids near Tyr 51, which is critical for the interaction. The SDC2-mimetic peptides harboring an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-D) or with an Ala-to-Phe substitution at the N-terminal side of Tyr 51 and an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-FE) showed improved interaction affinities for the MMP-7 pro-domain. Compared to S2-P, S2-FE was better able to inhibit the SDC2-MMP-7 interaction, the cell surface localization of MMP-7, the gelatin degradation activity of MMP-7, and the cancer activities (cell migration, invasion, and colony-forming activity) of human HCT116 colon cancer cells in vitro. In vivo, S2-FE inhibited the primary tumor growth and lung metastasis of CT26 mouse colon cancer cells in a xenograft mouse model. Together, these data suggest that S2-FE could be useful therapeutic anticancer peptides for colon cancer.
Asunto(s)
Neoplasias del Colon , Sindecano-2 , Animales , Movimiento Celular , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Humanos , Metaloproteinasa 7 de la Matriz/metabolismo , Ratones , Péptidos/farmacología , Sindecano-2/metabolismoRESUMEN
Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.
Asunto(s)
Ácidos y Sales Biliares , Microscopía por Crioelectrón , Virus de la Hepatitis B , Transportadores de Anión Orgánico Sodio-Dependiente , Receptores Virales , Simportadores , Anticuerpos , Ácidos y Sales Biliares/metabolismo , Virus de la Hepatitis B/metabolismo , Hepatocitos/metabolismo , Humanos , Transportadores de Anión Orgánico Sodio-Dependiente/química , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/ultraestructura , Receptores Virales/química , Receptores Virales/metabolismo , Receptores Virales/ultraestructura , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestructuraRESUMEN
Metallo-ß-lactamase (MBL) superfamily proteins have a common αß/ßα sandwich fold and perform a variety of functions through metal-mediated catalysis. However, because of the enormous scale of this superfamily, only a small percentage of the proteins belonging to the superfamily have been annotated structurally or functionally to date. Therefore, much remains unknown about the MBL superfamily proteins. Here, TW9814, a hypothetical MBL superfamily protein, was structurally and functionally investigated. Guided by the crystal structure of dimeric TW9814, it was demonstrated that TW9814 functions as a phosphodiesterase (PDE) in the presence of divalent metal ions such as manganese(II) or nickel(II). A docking model between TW9814 and the substrate bis(p-nitrophenyl)phosphate (bpNPP) showed the importance of the dimerization of TW9814 for its bpNPP-hydrolyzing activity and for the interaction between the enzyme and the substrate. TW9814 showed outstanding catalytic efficiency (kcat/Km) under alkaline conditions compared with other PDEs. The activity of TW9814 appears to be regulated through a disulfide bond, which is a feature that is not present in other MBL superfamily members. This study provides a platform for the functional characterization of other hypothetical proteins of the MBL or other superfamilies.
Asunto(s)
Hidrolasas Diéster Fosfóricas , beta-Lactamasas , Catálisis , Metales/metabolismo , Hidrolasas Diéster Fosfóricas/química , beta-Lactamasas/químicaRESUMEN
Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.
Asunto(s)
Arabidopsis , Fosfolipasas A1 , Proteínas de Plantas , Arabidopsis/enzimología , Capsicum/enzimología , Dimerización , Ésteres , Fosfolipasas A1/química , Proteínas de Plantas/químicaRESUMEN
Somatostatin is a peptide hormone that regulates endocrine systems by binding to G-protein-coupled somatostatin receptors. Somatostatin receptor 2 (SSTR2) is a human somatostatin receptor and is highly implicated in hormone disorders, cancers, and neurological diseases. Here, we report the high-resolution cryo-EM structure of full-length human SSTR2 bound to the agonist somatostatin (SST-14) in complex with inhibitory G (Gi) proteins. Our structural and mutagenesis analyses show that seven transmembrane helices form a deep pocket for ligand binding and that SSTR2 recognizes the highly conserved Trp-Lys motif of SST-14 at the bottom of the pocket. Furthermore, our sequence analysis combined with AlphaFold modeled structures of other SSTR isoforms provide a structural basis for the mechanism by which SSTR family proteins specifically interact with their cognate ligands. This work provides the first glimpse into the molecular recognition mechanism of somatostatin receptors and a crucial resource to develop therapeutics targeting somatostatin receptors.
Asunto(s)
Receptores de Somatostatina , Somatostatina , Microscopía por Crioelectrón , Humanos , Ligandos , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismoRESUMEN
Cancer targeting nanoparticles have been extensively studied, but stable and applicable agents have yet to be developed. Here, we report stable nanoparticles based on hepatitis B core antigen (HBcAg) for cancer therapy. HBcAg monomers assemble into spherical capsids of 180 or 240 subunits. HBcAg was engineered to present an affibody for binding to human epidermal growth factor receptor 1 (EGFR) and to present histidine and tyrosine tags for binding to gold ions. The HBcAg engineered to present affibody and tags (HAF) bound specifically to EGFR and exterminated the EGFR-overexpressing adenocarcinomas under alternating magnetic field (AMF) after binding with gold ions. Using cryogenic electron microscopy (cryo-EM), we obtained the molecular structures of recombinant HAF and found that the overall structure of HAF was the same as that of HBcAg, except with the affibody on the spike. Therefore, HAF is viable for cancer therapy with the advantage of maintaining a stable capsid form. If the affibody in HAF is replaced with a specific sequence to bind to another targetable disease protein, the nanoparticles can be used for drug development over a wide spectrum.
Asunto(s)
Adenocarcinoma/metabolismo , Antígenos del Núcleo de la Hepatitis B/química , Nanopartículas/química , Microscopía por Crioelectrón , Receptores ErbB/metabolismo , Oro/química , Células HT29 , Humanos , Nanopartículas/ultraestructura , Unión Proteica , Proteínas Recombinantes/químicaRESUMEN
Telomeric repeat binding factor a (Terfa) derived from zebrafish is a homologous protein with human telomeric repeat binding factor 2 (TRF2). Terfa is known as a senescence-associated biomarker in various research through the zebrafish animal model. In addition, according to the findings so far, it has been confirmed that human or plant telomere binding proteins bind to telomeric DNA specialized for each species, but, in our result, Terfa shows it strongly binds to both human or plant type telomeric DNA. Here we characterized the DNA binding properties and demonstrate the solution structure of Terfa and identified residues participating in the interaction with both human and plant telomeric DNA. In DNA recognition of human and plant telomere binding proteins, the N-terminal loop and the α-helix 3 part of Myb domain were bound majorly, whereas, in the case of Terfa, the N-terminal loop, the α-helix 1-2 loop, and α-helix 2 of the Myb domain were dominantly bound. Therefore, when Terfa recognizes DNA, it was found that the binding module differs from previously known telomere binding proteins. The comparison of the structure of the telomere binding proteins provides an opportunity to understand more specifically how the structural properties of each telomere binding protein are associated with telomeric DNA binding from an evolutionary point of view.
Asunto(s)
ADN de Plantas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Telómero/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Unión Proteica , Dominios Proteicos , SolucionesRESUMEN
Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , Rodopsinas Microbianas/metabolismo , Cationes Monovalentes/metabolismo , Canales de Cloruro/aislamiento & purificación , Canales de Cloruro/efectos de la radiación , Canales de Cloruro/ultraestructura , Cristalografía/métodos , Radiación Electromagnética , Rayos Láser , Simulación de Dinámica Molecular , Nocardioides , Conformación Proteica en Hélice alfa/efectos de la radiación , Estructura Terciaria de Proteína/efectos de la radiación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Proteínas Recombinantes/ultraestructura , Retinaldehído/metabolismo , Retinaldehído/efectos de la radiación , Rodopsinas Microbianas/aislamiento & purificación , Rodopsinas Microbianas/efectos de la radiación , Rodopsinas Microbianas/ultraestructura , Agua/metabolismoRESUMEN
Despite the recent advances in cancer therapeutics, highly aggressive cancer forms, such as glioblastoma (GBM), still have very low survival rates. The intracellular scaffold protein syntenin, comprising two postsynaptic density protein-95/discs-large/zona occludens-1 (PDZ) domains, has emerged as a novel therapeutic target in highly malignant phenotypes including GBM. Here, we report the development of a novel, highly potent, and metabolically stable peptide inhibitor of syntenin, KSL-128114, which binds the PDZ1 domain of syntenin with nanomolar affinity. KSL-128114 is resistant toward degradation in human plasma and mouse hepatic microsomes and displays a global PDZ domain selectivity for syntenin. An X-ray crystal structure reveals that KSL-128114 interacts with syntenin PDZ1 in an extended noncanonical binding mode. Treatment with KSL-128114 shows an inhibitory effect on primary GBM cell viability and significantly extends survival time in a patient-derived xenograft mouse model. Thus, KSL-128114 is a novel promising candidate with therapeutic potential for highly aggressive tumors, such as GBM.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Péptidos/química , Péptidos/farmacología , Sinteninas/efectos de los fármacos , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Ratones , Microsomas/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Difracción de Rayos X , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The BRG1-associated factor 60A (BAF60A), an SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1, has been known to be important for transcriptional activation and inhibition through the alteration of the DNA nucleosome. Although the association between BAF60A and p53 plays a critical role in tumor suppression, the interaction mode is still unclear. Here, we report the detailed interactions between BAF60A and p53 by both NMR spectroscopy and pull-down analysis. Both N-terminal region (BAF60ANR) and the SWIB domain (BAF60ASWIB) of BAF60A directly interact with the tetramerization domain of p53 (p53TET). NMR data show that Ile315, Met366, Ala388, and Tyr390 of BAF60ASWIB are mostly involved in p53TET binding. The calculated dissociation constant (KD) value between BAF60ASWIB and p53TET revealed relatively weak binding affinity, at approximately 0.3 ± 0.065 mM. Our data will enhance detailed interaction mechanism to elucidate the molecular basis of p53-mediated integration via BAF60A interaction.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Sitios de Unión , Proteínas Cromosómicas no Histona/genética , Humanos , Simulación del Acoplamiento Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The SWI/SNF chromatin remodeling complex plays important roles in gene regulation and it is classified as the SWI/SNF complex in yeast and BAF complex in vertebrates. BAF57, one of the subunits that forms the chromatin remodeling complex core, is well conserved in the BAF complex of vertebrates, which is replaced by bap111 in the Drosophila BAP complex and does not have a counterpart in the yeast SWI/SNF complex. This suggests that BAF57 is a key component of the chromatin remodeling complex in higher eukaryotes. BAF57 contains a HMG domain, which is widely distributed among various proteins and functions as a DNA binding motif. Most proteins with HMG domain bind to four-way junction (4WJ) DNA. Here, we report the crystal structure of the HMG domain of BAF57 (BAF57HMG) at a resolution of 2.55 Å. The structure consists of three α-helices and adopts an L-shaped form. The overall structure is stabilized by a hydrophobic core, which is formed by hydrophobic residues. The binding affinity between BAF57HMG and 4WJ DNA is determined as a 295.83 ± 1.05 nM using a fluorescence quenching assay, and the structure revealed 4WJ DNA binding site of BAF57HMG. Our data will serve structural basis in understanding the roles of BAF57 during chromatin remodeling process.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Proteínas de Unión al ADN/química , ADN/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , ADN Cruciforme/química , ADN Cruciforme/genética , ADN Cruciforme/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dominios HMG-Box , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , Espectrometría de Fluorescencia , Electricidad EstáticaRESUMEN
NADPH oxidases 1 (NOX1) derived reactive oxygen species (ROS) play an important role in the progression of cancer through signaling pathways. Therefore, in this paper, we demonstrate the effect of cold atmospheric plasma (CAP) on the structural changes of Noxa1 SH3 protein, one of the regulatory subunits of NOX1. For this purpose, firstly we purified the Noxa1 SH3 protein and analyzed the structure using X-ray crystallography, and subsequently, we treated the protein with two types of CAP reactors such as pulsed dielectric barrier discharge (DBD) and Soft Jet for different time intervals. The structural deformation of Noxa1 SH3 protein was analyzed by various experimental methods (circular dichroism, fluorescence, and NMR spectroscopy) and by MD simulations. Additionally, we demonstrate the effect of CAP (DBD and Soft Jet) on the viability and expression of NOX1 in A375 cancer cells. Our results are useful to understand the structural modification/oxidation occur in protein due to reactive oxygen and nitrogen (RONS) species generated by CAP.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , NADPH Oxidasa 1/química , Estrés Oxidativo/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Adaptadoras del Transporte Vesicular/química , Secuencia de Aminoácidos/genética , Animales , Línea Celular Tumoral , Biología Computacional , Humanos , Melanoma/enzimología , Melanoma/genética , Melanoma/patología , NADPH Oxidasa 1/genética , Oxidación-Reducción/efectos de los fármacos , Gases em Plasma/farmacología , Unión Proteica/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Understanding the structure and functional mechanisms of cyanobacterial halorhodopsin has become increasingly important, given the report that Synechocystis halorhodopsin (SyHR), a homolog of the cyanobacterial halorhodopsin from Mastigocladopsis repens (MrHR), can take up divalent ions, such as SO42-, as well as chloride ions. Here, the crystal structure of MrHR, containing a unique "TSD" chloride ion conduction motif, was determined as a homotrimer at a resolution of 1.9â¯Å. The detailed structure of MrHR revealed a unique trimeric topology of the light-driven chloride pump, with peculiar coordination of two water molecules and hydrogen-mediated bonds near the TSD motif, as well as a short B-C loop. Structural and functional analyses of MrHR revealed key residues responsible for the anion selectivity of cyanobacterial halorhodopsin and the involvement of two chloride ion-binding sites in the ion conduction pathway. Alanine mutant of Asn63, Pro118, and Glu182 locating in the anion inlet induce multifunctional uptake of chloride, nitrate, and sulfate ions. Moreover, the structure of N63A/P118A provides information on how SyHR promotes divalent ion transport. Our findings significantly advance the structural understanding of microbial rhodopsins with different motifs. They also provide insight into the general structural framework underlying the molecular mechanisms of the cyanobacterial chloride pump containing SyHR, the only molecule known to transport both sulfate and chloride ions.
Asunto(s)
Proteínas de Transporte de Anión/química , Proteínas Bacterianas/química , Cianobacterias/química , Proteínas de Transporte de Anión/metabolismo , Aniones/metabolismo , Proteínas Bacterianas/metabolismo , Cloruros/metabolismo , Cristalografía por Rayos X , Cianobacterias/metabolismo , Halorrodopsinas/química , Halorrodopsinas/metabolismo , Transporte Iónico , Modelos Moleculares , Conformación ProteicaRESUMEN
Human mitochondrial pyruvate carriers (hMPCs), which are required for the uptake of pyruvate into mitochondria, are associated with several metabolic diseases, including type 2 diabetes and various cancers. Yeast MPC was recently demonstrated to form a functional unit of heterodimers. However, human MPC-1 (hMPC-1) and MPC-2 (hMPC-2) have not yet been individually isolated for their detailed characterization, in particular in terms of their structural and functional properties, namely, whether they exist as homo- or heterodimers. In this study, hMPC-1 and hMPC-2 were successfully isolated in micelles and they formed stable homodimers. However, the heterodimer state was found to be dominant when both hMPC-1 and hMPC-2 were present. In addition, as heterodimers, the molecules exhibited a higher binding capacity to both substrates and inhibitors, together with a larger structural stability than when they existed as homodimers. Taken together, our results demonstrated that the hetero-dimerization of hMPCs is the main functional unit of the pyruvate metabolism, providing a structural insight into the transport mechanisms of hMPCs.
Asunto(s)
Enfermedades Metabólicas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/química , Transportadores de Ácidos Monocarboxílicos/química , Multimerización de Proteína , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Humanos , Enfermedades Metabólicas/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Modelos Moleculares , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Homología de Secuencia de Aminoácido , Células Sf9 , SpodopteraRESUMEN
Human SNF5 and BAF155 constitute the core subunit of multi-protein SWI/SNF chromatin-remodeling complexes that are required for ATP-dependent nucleosome mobility and transcriptional control. Human SNF5 (hSNF5) utilizes its repeat 1 (RPT1) domain to associate with the SWIRM domain of BAF155. Here, we employed X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and various biophysical methods in order to investigate the detailed binding mechanism between hSNF5 and BAF155. Multi-angle light scattering data clearly indicate that hSNF5171-258 and BAF155SWIRM are both monomeric in solution and they form a heterodimer. NMR data and crystal structure of the hSNF5171-258/BAF155SWIRM complex further reveal a unique binding interface, which involves a coil-to-helix transition upon protein binding. The newly formed αN helix of hSNF5171-258 interacts with the ß2-α1 loop of hSNF5 via hydrogen bonds and it also displays a hydrophobic interaction with BAF155SWIRM. Therefore, the N-terminal region of hSNF5171-258 plays an important role in tumorigenesis and our data will provide a structural clue for the pathogenesis of Rhabdoid tumors and malignant melanomas that originate from mutations in the N-terminal loop region of hSNF5.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Mutación , Nucleosomas/genética , Proteína SMARCB1/genética , Factores de Transcripción/genética , Sitios de Unión/genética , Dicroismo Circular , Cristalografía por Rayos X , Regulación de la Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Nucleosomas/metabolismo , Unión Proteica , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patología , Proteína SMARCB1/química , Proteína SMARCB1/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Ionic liquids (ILs) are known to provide stability to biomolecules. ILs are also widely used in the fields of chemical engineering, biological engineering, chemistry, and biochemistry because they facilitate enzyme catalyzed reactions and enhance their conversion rate. In this work, we have evaluated the influence of alkyl chain substitution of ammonium ILs such as diethylammonium dihydrogen phosphate (DEAP) and triethylammonium hydrogen phosphate (TEAP) for the stability and activity of the tobacco etch virus (TEV) protease. Further, we performed molecular dynamics (MD) simulations to calculate the RMSD (root mean square deviation) for TEV and TEV + ILs. Experimental and simulations results show that TEV is more stable in the presence of TEAP than DEAP. Whereas, TEV protease activity for the cleavage of fusion proteins is preserved in the presence of DEAP while lost in the presence of TEAP. Hence, DEAP IL can serve as alternative solvents for the stability of the TEV protease with preserved activity. To the best of our knowledge, this is first study to show that ILs can stabilize and maintain the TEV protease cleavage activity.
Asunto(s)
Compuestos de Amonio/química , Endopeptidasas/química , Endopeptidasas/metabolismo , Líquidos Iónicos/química , Estabilidad de Enzimas , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Solventes/químicaRESUMEN
A newly identified microbial rhodopsin, NM-R3, from the marine flavobacterium Nonlabens marinus, was recently shown to drive chloride ion uptake, extending our understanding of the diversity of mechanisms for biological energy conversion. To clarify the mechanism underlying its function, we characterized the crystal structures of NM-R3 in both the dark state and early intermediate photoexcited states produced by laser pulses of different intensities and temperatures. The displacement of chloride ions at five different locations in the model reflected the detailed anion-conduction pathway, and the activity-related key residues-Cys105, Ser60, Gln224, and Phe90-were identified by mutation assays and spectroscopy. Comparisons with other proteins, including a closely related outward sodium ion pump, revealed key motifs and provided structural insights into light-driven ion transport across membranes by the NQ subfamily of rhodopsins. Unexpectedly, the response of the retinal in NM-R3 to photostimulation appears to be substantially different from that seen in bacteriorhodopsin.
Asunto(s)
Proteínas Bacterianas/química , Canales de Cloruro/química , Luz , Rodopsina/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cloruros/química , Activación del Canal Iónico , Modelos Moleculares , Conformación Proteica , Rodopsina/genética , Rodopsina/metabolismo , Relación Estructura-Actividad , Agua/químicaRESUMEN
BACKGROUND: Vitamin C (also known as L-ascorbic acid) plays a critical role in reactive oxygen species (ROS) reduction and cell regeneration by protecting cell from oxidative stress. Although vitamin C is widely used in cosmetic and therapeutic markets, there is considerable evidence that vitamin C easily undergoes oxidation by air, pH, temperature, and UV light upon storage. This deficiency of vitamin C decreases its potency as an antioxidant and reduces the shelf-life of products containing vitamin C as its ingredient. To overcome the deficiency of vitamin C, we have developed Aptamin C, an innovative DNA aptamer maximizing the antioxidant efficacy of vitamin C by binding to the reduced form of vitamin C and delaying its oxidation. METHODS: Binding of Aptamin C with vitamin C was determined using ITC analysis. ITC experiment was performed 0.2 mmol/L vitamin C that was injected 25 times in 2 µL aliquots into the 1.8 mL sample cell containing the Aptamin C at a concentration of 0.02 mmol/L. The data were fitted to a one-site binding isotherm using with origin program for ITC v.5.0. RESULTS: To investigate the effect of Aptamin C and vitamin C complex in human skins, both in vitro and clinical tests were performed. We observed that the complex of Aptamin C and vitamin C was significantly effective in wrinkle improvement, whitening effect, and hydration increase. In the clinical test, subjects treated with the complex showed dramatic improvement in skin irritation and itching. No adverse reaction was presented by Aptamin C complex in the test. CONCLUSION: Taken together, these results showed that Aptamin C, an innovative novel compound, should potentially be served as a key cosmeceutical ingredient for a range of skin conditions.