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1.
Nature ; 606(7916): 1027-1031, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35580630

RESUMO

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.


Assuntos
Ácidos e Sais Biliares , Microscopia Crioeletrônica , Vírus da Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Receptores Virais , Simportadores , Anticorpos , Ácidos e Sais Biliares/metabolismo , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Receptores Virais/química , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestrutura
2.
PLoS Pathog ; 20(8): e1012474, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39186780

RESUMO

The bacterium Vibrio vulnificus causes fatal septicemia in humans. Previously, we reported that an extracellular metalloprotease, vEP-45, secreted by V. vulnificus, undergoes self-proteolysis to generate a 34 kDa protease (vEP-34) by losing its C-terminal domain to produce the C-ter100 peptide. Moreover, we revealed that vEP-45 and vEP-34 proteases induce blood coagulation and activate the kallikrein/kinin system. However, the role of the C-ter100 peptide fragment released from vEP-45 in inducing inflammation is still unclear. Here, we elucidate, for the first time, the effects of C-ter100 on inducing inflammation and activating host innate immunity. Our results showed that C-ter100 could activate NF-κB by binding to the receptor TLR4, thereby promoting the secretion of inflammatory cytokines and molecules, such as TNF-α and nitric oxide (NO). Furthermore, C-ter100 could prime and activate the NLRP3 inflammasome (NLRP3, ASC, and caspase 1), causing IL-1ß secretion. In mice, C-ter100 induced the recruitment of immune cells, such as neutrophils and monocytes, along with histamine release into the plasma. Furthermore, the inflammatory response induced by C-ter100 could be effectively neutralized by an anti-C-ter100 monoclonal antibody (C-ter100Mab). These results demonstrate that C-ter100 can be a pathogen-associated molecular pattern (PAMP) that activates an innate immune response during Vibrio infection and could be a target for the development of antibiotics.


Assuntos
Imunidade Inata , Inflamação , Vibrio vulnificus , Animais , Camundongos , Inflamação/imunologia , Inflamação/metabolismo , Vibrio vulnificus/imunologia , Vibrioses/imunologia , Camundongos Endogâmicos C57BL , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/imunologia
3.
PLoS Biol ; 20(8): e3001714, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35913979

RESUMO

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.


Assuntos
Galanina/química , Proteínas Heterotriméricas de Ligação ao GTP , Receptor Tipo 2 de Galanina/química , Microscopia Crioeletrônica , Galanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Receptor Tipo 2 de Galanina/metabolismo
4.
Am J Physiol Cell Physiol ; 326(4): C1067-C1079, 2024 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-38314724

RESUMO

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.


Assuntos
Neoplasias do Colo , Everolimo , Humanos , Everolimo/farmacologia , Sindecana-2/genética , Sindecana-2/metabolismo , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Gelatina , Sirolimo/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Serina-Treonina Quinases TOR
5.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33753488

RESUMO

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.


Assuntos
Canais de Cloreto/metabolismo , Cloretos/metabolismo , Rodopsinas Microbianas/metabolismo , Cátions Monovalentes/metabolismo , Canais de Cloreto/isolamento & purificação , Canais de Cloreto/efeitos da radiação , Canais de Cloreto/ultraestrutura , Cristalografia/métodos , Radiação Eletromagnética , Lasers , Simulação de Dinâmica Molecular , Nocardioides , Conformação Proteica em alfa-Hélice/efeitos da radiação , Estrutura Terciária de Proteína/efeitos da radiação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Proteínas Recombinantes/ultraestrutura , Retinaldeído/metabolismo , Retinaldeído/efeitos da radiação , Rodopsinas Microbianas/isolamento & purificação , Rodopsinas Microbianas/efeitos da radiação , Rodopsinas Microbianas/ultraestrutura , Água/metabolismo
6.
Int J Mol Sci ; 23(11)2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35682569

RESUMO

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.


Assuntos
Neoplasias do Colo , Sindecana-2 , Animais , Movimento Celular , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Humanos , Metaloproteinase 7 da Matriz/metabolismo , Camundongos , Peptídeos/farmacologia , Sindecana-2/metabolismo
7.
Molecules ; 27(7)2022 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-35408716

RESUMO

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.


Assuntos
Arabidopsis , Fosfolipases A1 , Proteínas de Plantas , Arabidopsis/enzimologia , Capsicum/enzimologia , Dimerização , Ésteres , Fosfolipases A1/química , Proteínas de Plantas/química
8.
Biochem Biophys Res Commun ; 559: 252-258, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33984809

RESUMO

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.


Assuntos
DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Telômero/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Ligação Proteica , Domínios Proteicos , Soluções
9.
Biochem Biophys Res Commun ; 534: 815-821, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33168186

RESUMO

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.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Humanos , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteína Supressora de Tumor p53/genética
10.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502049

RESUMO

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.


Assuntos
Adenocarcinoma/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/química , Nanopartículas/química , Microscopia Crioeletrônica , Receptores ErbB/metabolismo , Ouro/química , Células HT29 , Humanos , Nanopartículas/ultraestrutura , Ligação Proteica , Proteínas Recombinantes/química
11.
J Biol Chem ; 294(3): 794-804, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30455349

RESUMO

Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.


Assuntos
Actinomycetales/química , Canais de Cloreto/química , Rodopsinas Microbianas/química , Cristalografia por Raios X , Domínios Proteicos
12.
Biochem Biophys Res Commun ; 533(4): 919-924, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33010889

RESUMO

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.


Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , DNA/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , DNA Cruciforme/química , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Domínios HMG-Box , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Espectrometria de Fluorescência , Eletricidade Estática
13.
Protein Expr Purif ; 167: 105530, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31698036

RESUMO

Human serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide content hamper the expression and production of rHSA using bacterial hosts. Hence, a strategy that uses a fusion technique and engineered Escherichia coli strains was employed to improve the expression of soluble rHSA in the bacterial cytoplasm. The solubilities of the b'a' domain of human protein disulphide isomerase (PDIb'a')- and maltose-binding protein (MBP)-tagged rHSA expressed in Origami 2 at 18 °C were notably increased by up to 90.1% and 96%, respectively. A simple and efficient protocol for rHSA purification was established and approximately 9.46 mg rHSA was successfully obtained from a 500-mL culture at 97% purity. However, rHSA was mostly obtained in soluble oligomeric form. By introducing a simple refolding and size-exclusion chromatography step, monomeric rHSA was obtained at 34% yield. Native polyacrylamide gel electrophoresis confirmed the similarity in the molecular weights between E. coli-derived monomeric rHSA and commercial monomeric HSA.


Assuntos
Albumina Sérica Humana/biossíntese , Cromatografia em Gel , Escherichia coli/genética , Escherichia coli/metabolismo , Etiquetas de Sequências Expressas/metabolismo , Humanos , Proteínas Ligantes de Maltose/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/isolamento & purificação , Albumina Sérica Humana/metabolismo , Solubilidade
14.
EMBO Rep ; 19(12)2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30413483

RESUMO

RAS proteins play critical roles in various cellular processes, including growth and transformation. RAS proteins are subjected to protein stability regulation via the Wnt/ß-catenin pathway, and glycogen synthase kinase 3 beta (GSK3ß) is a key player for the phosphorylation-dependent RAS degradation through proteasomes. GSK3ß-mediated RAS degradation does not occur in cells that express a nondegradable mutant (MT) ß-catenin. Here, we show that ß-catenin directly interacts with RAS at the α-interface region that contains the GSK3ß phosphorylation sites, threonine 144 and threonine 148 residues. Exposure of these sites by prior ß-catenin degradation is required for RAS degradation. The introduction of a peptide that blocks the ß-catenin-RAS interaction by binding to ß-catenin rescues the GSK3ß-mediated RAS degradation in colorectal cancer (CRC) cells that express MT ß-catenin. The coregulation of ß-catenin and RAS stabilities by the modulation of their interaction provides a mechanism for Wnt/ß-catenin and RAS-ERK pathway cross-talk and the synergistic transformation of CRC by both APC and KRAS mutations.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células HEK293 , Humanos , Camundongos Nus , Modelos Biológicos , Modelos Moleculares , Mutação/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/química , beta Catenina/genética
15.
Int J Mol Sci ; 21(9)2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32403431

RESUMO

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.


Assuntos
Doenças Metabólicas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Transportadores de Ácidos Monocarboxílicos/química , Multimerização Proteica , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Humanos , Doenças Metabólicas/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Modelos Moleculares , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Homologia de Sequência de Aminoácidos , Células Sf9 , Spodoptera
16.
Int J Mol Sci ; 21(7)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32244797

RESUMO

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.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Mutação , Nucleossomos/genética , Proteína SMARCB1/genética , Fatores de Transcrição/genética , Sítios de Ligação/genética , Dicroísmo Circular , Cristalografia por Raios X , Regulação da Expressão Gênica , Humanos , Espectroscopia de Ressonância Magnética , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Nucleossomos/metabolismo , Ligação Proteica , Tumor Rabdoide/genética , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patologia , Proteína SMARCB1/química , Proteína SMARCB1/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
17.
J Neurosci ; 38(42): 9001-9018, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30185465

RESUMO

Emerging evidences suggest that intraneuronal Aß correlates with the onset of Alzheimer's disease (AD) and highly contributes to neurodegeneration. However, critical mediator responsible for Aß uptake in AD pathology needs to be clarified. Here, we report that FcγRIIb2, a variant of Fcγ-receptor IIb (FcγRIIb), functions in neuronal uptake of pathogenic Aß. Cellular accumulation of oligomeric Aß1-42, not monomeric Aß1-42 or oligomeric Aß1-40, was blocked by Fcgr2b knock-out in neurons and partially in astrocytes. Aß1-42 internalization was FcγRIIb2 di-leucine motif-dependent and attenuated by TOM1, a FcγRIIb2-binding protein that repressed the receptor recycling. TOM1 expression was downregulated in the hippocampus of male 3xTg-AD mice and AD patients, and regulated by miR-126-3p in neuronal cells after exposure to Aß1-42 In addition, memory impairments in male 3xTg-AD mice were rescued by the lentiviral administration of TOM1 gene. Augmented Aß uptake into lysosome caused its accumulation in cytoplasm and mitochondria. Moreover, neuronal accumulation of Aß in both sexes of 3xTg-AD mice and memory deficits in male 3xTg-AD mice were ameliorated by forebrain-specific expression of Aß-uptake-defective Fcgr2b mutant. Our findings suggest that FcγRIIb2 is essential for neuropathic uptake of Aß in AD.SIGNIFICANCE STATEMENT Accumulating evidences suggest that intraneuronal Aß is found in the early step of AD brain and is implicated in the pathogenesis of AD. However, the critical mediator involved in these processes is uncertain. Here, we describe that the FcγRIIb2 variant is responsible for both neuronal uptake and intraneuronal distribution of pathogenic Aß linked to memory deficits in AD mice, showing a pathologic significance of the internalized Aß. Further, Aß internalization is attenuated by TOM1, a novel FcγRIIb2-binding protein. Together, we provide a molecular mechanism responsible for neuronal uptake of pathogenic Aß found in AD.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Receptores de IgG/metabolismo , Animais , Astrócitos/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos Knockout , MicroRNAs/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de IgG/genética
18.
Int J Mol Sci ; 20(14)2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336822

RESUMO

In the past 10 years, the world has witnessed the revolutionary development of X-ray free electron lasers (XFELs) and their applications in many scientific disciplinaries [...].


Assuntos
Cristalografia por Raios X , Lasers , Modelos Moleculares , Proteínas/química , Elétrons , Conformação Proteica
19.
Int J Mol Sci ; 20(8)2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31010024

RESUMO

HIV-1 integrase (HIV-1 IN) is an enzyme produced by the HIV-1 virus that integrates genetic material of the virus into the DNA of infected human cells. HIV-1 IN acts as a key component of the Retroviral Pre-Integration Complex (PIC). Protein dynamics could play an important role during the catalysis of HIV-1 IN; however, this process has not yet been fully elucidated. X-ray free electron laser (XFEL) together with nuclear magnetic resonance (NMR) could provide information regarding the dynamics during this catalysis reaction. Here, we report the non-cryogenic crystal structure of HIV-1 IN catalytic core domain at 2.5 Å using microcrystals in XFELs. Compared to the cryogenic structure at 2.1 Å using conventional synchrotron crystallography, there was a good agreement between the two structures, except for a catalytic triad formed by Asp64, Asp116, and Glu152 (DDE) and the lens epithelium-derived growth factor binding sites. The helix III region of the 140-153 residues near the active site and the DDE triad show a higher dynamic profile in the non-cryogenic structure, which is comparable to dynamics data obtained from NMR spectroscopy in solution state.


Assuntos
Domínio Catalítico , Elétrons , Integrase de HIV/química , Lasers , Cristalografia por Raios X , Estrutura Secundária de Proteína , Temperatura , Raios X
20.
Nat Chem Biol ; 12(8): 593-600, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27294323

RESUMO

Both the Wnt/ß-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both ß-catenin and Ras, via targeting the Wnt/ß-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating ß-catenin and Ras degradation through enhancement of the ß-catenin destruction complex activating GSK3ß. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both ß-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/ß-catenin and Ras pathways.


Assuntos
Proteína Axina/química , Proteína Axina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas RGS/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tioidantoínas/farmacologia , beta Catenina/metabolismo , Animais , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Genes APC , Genes ras , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas RGS/metabolismo , Tioidantoínas/síntese química , Tioidantoínas/química , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/química
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