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1.
Food Chem ; 305: 125500, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525593

RESUMO

The influences of folding patterns on the protein polymerization in dumpling wrappers were investigated. The dumpling dough sheet after the compounding rollers was folded with various patterns (control with no angle, 15°, 25°, 35° and 45° folding), before going through the sheeting and reduction rolls. Protein secondary structure, free sulfhydryl content, protein electrophoretic profiles, and texture of dumpling wrappers were determined. Results showed that folding could increase the proportion of α-helix conformation, and produce dumpling wrappers with enhanced toughness but reduce wrapper extensibility. The wrapper with 45° folding showed lower -SH content than the control and other folding angles. However, only a few variations in SDS band pattern and intensities were observed at the molecular weight position of around 35 kDa. Briefly, folding process could influence the gluten formation during the preparation of dumpling wrappers; the folding angle at 45° produced stronger gluten network and tougher wrappers.


Assuntos
Farinha , Alimentos , Proteínas de Plantas/química , Multimerização Proteica , Triticum/química , Glutens/química , Peso Molecular , Estrutura Quaternária de Proteína
3.
J Chem Theory Comput ; 15(11): 6358-6367, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31539250

RESUMO

Predicting the 3D structure of protein interactions remains a challenge in the field of computational structural biology. This is in part due to difficulties in sampling the complex energy landscape of multiple interacting flexible polypeptide chains. Coarse-graining approaches, which reduce the number of degrees of freedom of the system, help address this limitation by smoothing the energy landscape, allowing an easier identification of the global energy minimum. They also accelerate the calculations, allowing for modeling larger assemblies. Here, we present the implementation of the MARTINI coarse-grained force field for proteins into HADDOCK, our integrative modeling platform. Docking and refinement are performed at the coarse-grained level, and the resulting models are then converted back to atomistic resolution through a distance restraints-guided morphing procedure. Our protocol, tested on the largest complexes of the protein docking benchmark 5, shows an overall ∼7-fold speed increase compared to standard all-atom calculations, while maintaining a similar accuracy and yielding substantially more near-native solutions. To showcase the potential of our method, we performed simultaneous 7 body docking to model the 1:6 KaiC-KaiB complex, integrating mutagenesis and hydrogen/deuterium exchange data from mass spectrometry with symmetry restraints, and validated the resulting models against a recently published cryo-EM structure.


Assuntos
Simulação de Acoplamento Molecular , Proteínas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/química , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Microscopia Crioeletrônica , Estrutura Quaternária de Proteína , Termodinâmica
4.
Biophys Chem ; 253: 106226, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31376619

RESUMO

The quaternary structures of insulin glargine and glulisine under formulation conditions and upon dilution using placebo or water were investigated using synchrotron small-angle X-ray scattering. Our results revealed that insulin glulisine in Apidra® is predominantly hexameric in solution with significant fractions of dodecamers and monomers. Upon dilution with placebo, this equilibrium shifts towards monomers. Insulin glargine in Lantus® and Toujeo® is present in a stable hexamer/dimer equilibrium, which is hardly affected by dilution with water down to 1 mg/ml insulin concentration. The results provide exclusive insight into the quaternary structure and thus the association/dissociation properties of the two insulin analogues in marketed formulations.


Assuntos
Hipoglicemiantes/química , Insulina Glargina/química , Insulina/análogos & derivados , Humanos , Insulina/química , Modelos Moleculares , Estrutura Quaternária de Proteína
5.
Genes Dev ; 33(19-20): 1355-1360, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31439631

RESUMO

GIGYF (Grb10-interacting GYF [glycine-tyrosine-phenylalanine domain]) proteins coordinate with 4EHP (eIF4E [eukaryotic initiation factor 4E] homologous protein), the DEAD (Asp-Glu-Ala-Asp)-box helicase Me31B/DDX6, and mRNA-binding proteins to elicit transcript-specific repression. However, the underlying molecular mechanism remains unclear. Here, we report that GIGYF contains a motif necessary and sufficient for direct interaction with Me31B/DDX6. A 2.4 Å crystal structure of the GIGYF-Me31B complex reveals that this motif arranges into a coil connected to a ß hairpin on binding to conserved hydrophobic patches on the Me31B RecA2 domain. Structure-guided mutants indicate that 4EHP-GIGYF-DDX6 complex assembly is required for tristetraprolin-mediated down-regulation of an AU-rich mRNA, thus revealing the molecular principles of translational repression.


Assuntos
Proteínas de Transporte/química , RNA Helicases DEAD-box/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação da Expressão Gênica/genética , Modelos Moleculares , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular , Drosophila melanogaster/genética , Células HEK293 , Humanos , Ligação Proteica , Estrutura Quaternária de Proteína
6.
Acta Crystallogr D Struct Biol ; 75(Pt 7): 618-627, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31282471

RESUMO

Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic acid residues, resulting in a low-pH `locked' dimer stabilized by symmetric electrostatic interactions at the poles of the dimer. The detailed molecular events during this transition are still unresolved. Here, a 2.1 Šresolution crystal structure of an NT T61A mutant in an alternative, asymmetric, dimer form in which the electrostatic interactions at one of the poles are dramatically different from those in symmetrical dimers is presented. A similar asymmetric dimer structure from dragline silk of Nephila clavipes has previously been described. It is suggested that asymmetric dimers represent a conserved intermediate state in spider silk formation, and a revised `lock-and-trigger' mechanism for spider silk formation is presented.


Assuntos
Aracnídeos/metabolismo , Fibroínas/química , Proteínas Recombinantes/química , Animais , Cristalização/métodos , Escherichia coli/genética , Fibroínas/genética , Modelos Moleculares , Estrutura Molecular , Mutação , Domínios Proteicos , Multimerização Proteica , Estrutura Quaternária de Proteína , Eletricidade Estática
7.
Nat Commun ; 10(1): 2697, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217444

RESUMO

Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.


Assuntos
Complexos Multienzimáticos/ultraestrutura , Estrutura Quaternária de Proteína , Aminopeptidases/química , Aminopeptidases/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Peso Molecular , Complexos Multienzimáticos/química , Pyrococcus horikoshii
8.
Biochemistry (Mosc) ; 84(5): 453-463, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234761

RESUMO

Amino acids undergo many covalent modifications, but only few amino acid repair enzymes have been identified. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), also known as L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (PCMT), methylates covalently modified isoaspartate (isoAsp) residues accumulated in proteins via Asn deamidation and Asp hydrolysis. This cytoplasmic reaction occurs through the formation of succinimide cyclical intermediate and generates either isoAsp or Asp from succinimide. Succinimide conversion into Asp is spontaneous, while isoAsp is restored by PIMT using S-adenosylmethionine as a methyl donor. PIMT transforms isoAsp into succinimide, thereby creating an opportunity for the later to be converted into Asp. Apart from normal cell physiology, formation of isoAsp in proteins is promoted by various stress conditions. The resulting isoAsp can form a kink or bend in the protein backbone thus making the protein conformationally and functionally distorted. Many PIMT-interacting proteins (proteins with isoAsp residues) have been reported in eukaryotes, but only few of them have been found in prokaryotes. Extensive studies in mice have shown the importance of PIMT in neurodegeneration. Detail elucidation of PIMT function can create a platform for addressing various disorders such as Alzheimer's disease and cancer.


Assuntos
Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Animais , Ácido Aspártico/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Ácido Isoaspártico/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/química , Estrutura Quaternária de Proteína , S-Adenosilmetionina/metabolismo
9.
Nat Commun ; 10(1): 2399, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160585

RESUMO

Manganese superoxide dismutase (MnSOD) functions as a tumor suppressor; however, once tumorigenesis occurs, clinical data suggest MnSOD levels correlate with more aggressive human tumors, implying a potential dual function of MnSOD in the regulation of metabolism. Here we show, using in vitro transformation and xenograft growth assays that the MnSOD-K68 acetylation (Ac) mimic mutant (MnSODK68Q) functions as a tumor promoter. Interestingly, in various breast cancer and primary cell types the expression of MnSODK68Q is accompanied with a change of MnSOD's stoichiometry from a known homotetramer complex to a monomeric form. Biochemical experiments using the MnSOD-K68Q Ac-mimic, or physically K68-Ac (MnSOD-K68-Ac), suggest that these monomers function as a peroxidase, distinct from the established MnSOD superoxide dismutase activity. MnSODK68Q expressing cells exhibit resistance to tamoxifen (Tam) and cells selected for Tam resistance exhibited increased K68-Ac and monomeric MnSOD. These results suggest a MnSOD-K68-Ac metabolic pathway for Tam resistance, carcinogenesis and tumor progression.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Resistencia a Medicamentos Antineoplásicos/genética , Superóxido Dismutase/genética , Acetilação , Animais , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Técnicas In Vitro , Lisina/metabolismo , Células MCF-7 , Camundongos , Mutação , Transplante de Neoplasias , Peroxidase/metabolismo , Estrutura Quaternária de Proteína/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Tamoxifeno/uso terapêutico , Proteínas Supressoras de Tumor
10.
PLoS Comput Biol ; 15(6): e1006886, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31158218

RESUMO

The self-assembly of proteins into protein quaternary structures is of fundamental importance to many biological processes, and protein misassembly is responsible for a wide range of proteopathic diseases. In recent years, abstract lattice models of protein self-assembly have been used to simulate the evolution and assembly of protein quaternary structure, and to provide a tractable way to study the genotype-phenotype map of such systems. Here we generalize these models by representing the interfaces as mutable binary strings. This simple change enables us to model the evolution of interface strengths, interface symmetry, and deterministic assembly pathways. Using the generalized model we are able to reproduce two important results established for real protein complexes: The first is that protein assembly pathways are under evolutionary selection to minimize misassembly. The second is that the assembly pathway of a complex mirrors its evolutionary history, and that both can be derived from the relative strengths of interfaces. These results demonstrate that the generalized lattice model offers a powerful new idealized framework to facilitate the study of protein self-assembly processes and their evolution.


Assuntos
Evolução Molecular , Estrutura Quaternária de Proteína , Proteínas , Algoritmos , Biologia Computacional , Ligação Proteica , Estrutura Quaternária de Proteína/genética , Estrutura Quaternária de Proteína/fisiologia , Proteínas/química , Proteínas/genética
11.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 536-544, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205016

RESUMO

The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon Hyperthermus butylicus is presented at 1.8 Šresolution. Previous structures of archaeal Rubisco have been found to assemble into decamers, and this oligomerization was thought to be required for a highly thermally stable enzyme. In the current study, H. butylicus Rubisco is shown to exist as a dimer in solution, yet has a thermal denaturation midpoint of 114°C, suggesting that high thermal stability can be achieved without an increased oligomeric state. This increased thermal stability appears to be due to an increased number of electrostatic interactions within the monomeric subunit. As such, H. butylicus Rubisco presents a well characterized system in which to investigate the role of assembly and thermal stability in enzyme function.


Assuntos
Proteínas Arqueais/química , Modelos Moleculares , Pyrodictiaceae/enzimologia , Ribulose-Bifosfato Carboxilase/química , Cristalização , Cristalografia por Raios X/métodos , Estabilidade Enzimática , Estrutura Quaternária de Proteína , Eletricidade Estática
12.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 545-553, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205017

RESUMO

Spermidine N-acetyltransferase (SpeG) transfers an acetyl group from acetyl-coenzyme A to an N-terminal amino group of intracellular spermidine. This acetylation inactivates spermidine, reducing the polyamine toxicity that tends to occur under certain chemical and physical stresses. The structure of the SpeG protein from Vibrio cholerae has been characterized: while the monomer possesses a structural fold similar to those of other Gcn5-related N-acetyltransferase superfamily members, its dodecameric structure remains exceptional. In this paper, structural analyses of SpeG isolated from Escherichia coli are described. Like V. cholerae SpeG, E. coli SpeG forms dodecamers, as revealed by two crystal structures of the ligand-free E. coli SpeG dodecamer determined at 1.75 and 2.9 Šresolution. Although both V. cholerae SpeG and E. coli SpeG can adopt an asymmetric open dodecameric state, solution analysis showed that the oligomeric composition of ligand-free E. coli SpeG differs from that of ligand-free V. cholerae SpeG. Based on these data, it is proposed that the equilibrium balance of SpeG oligomers in the absence of ligands differs from one species to another and thus might be important for SpeG function.


Assuntos
Acetiltransferases/química , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/química , Modelos Moleculares , Estrutura Quaternária de Proteína , Cristalização , Cristalografia por Raios X/métodos
13.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 564-577, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205019

RESUMO

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.


Assuntos
Proteínas de Bactérias/química , N-Acilneuraminato Citidililtransferase/química , Vibrio cholerae/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Domínio Catalítico , Cristalização , Cristalografia por Raios X/métodos , Cistina Difosfato/química , Cistina Difosfato/metabolismo , Ácido N-Acetilneuramínico Citidina Monofosfato/química , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , N-Acilneuraminato Citidililtransferase/farmacologia , N-Acilneuraminato Citidililtransferase/fisiologia , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Ácidos Siálicos/metabolismo
14.
Nat Commun ; 10(1): 2635, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201302

RESUMO

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Antibacterianos/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Tomografia com Microscopia Eletrônica/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/ultraestrutura , Microscopia Intravital/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Peptidoglicano/metabolismo , Periplasma/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos
15.
Nat Commun ; 10(1): 2653, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201319

RESUMO

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.


Assuntos
Sítio Alostérico/genética , Proteínas de Bactérias/genética , Ribonucleotídeo Redutases/genética , Regulação Alostérica/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Evolução Molecular , Modelos Moleculares , Estrutura Quaternária de Proteína/genética , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Ribonucleotídeo Redutases/ultraestrutura , Ribonucleotídeos/metabolismo , Espalhamento a Baixo Ângulo
16.
Chem Biol Interact ; 309: 108698, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31176713

RESUMO

Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 Å resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature-related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under conditions approaching physiological temperature.


Assuntos
Acetilcolinesterase/química , Cristalografia por Raios X , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Aminacrina/química , Aminacrina/metabolismo , Sítios de Ligação , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Dimerização , Humanos , Simulação de Dinâmica Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura Ambiente
17.
Biochemistry (Mosc) ; 84(Suppl 1): S193-S205, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31213202

RESUMO

Cytokines of the IL-17 family play a key role in the host organism defense against bacterial and fungal infections. At the same time, upregulated synthesis of IL-17 cytokines is associated with immunoinflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and others. The members of this family are important therapeutic targets in the treatment of various human chronic inflammatory disorders. Elucidation of signaling pathways involving IL-17 family proteins and analysis of the structure of cytokine complexes with specific antibodies, inhibitors, and receptors are essential for the development of new drugs for the therapy of immunoinflammatory rheumatic diseases.


Assuntos
Doenças Autoimunes/imunologia , Interleucina-17 , Terapia de Alvo Molecular , Linfócitos T/imunologia , Anticorpos Monoclonais/farmacologia , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/química , Interleucina-17/fisiologia , Estrutura Quaternária de Proteína , Transdução de Sinais
18.
Molecules ; 24(12)2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31208071

RESUMO

Amyloid-ß oligomers (AßOs) self-assemble into polymorphic species with diverse biological activities that are implicated causally to Alzheimer's disease (AD). Synaptotoxicity of AßO species is dependent on their quaternary structure, however, low-abundance and environmental sensitivity of AßOs in vivo have impeded a thorough assessment of structure-function relationships. We developed a simple biochemical assay to quantify the relative abundance and morphology of cross-linked AßOs. We compared oligomers derived from synthetic Aß40 (wild-type (WT) Aß40) and a recombinant source, called Aß(M1-40). Both peptides assemble into oligomers with common sizes and morphology, however, the predominant quaternary structures of Aß(M1-40) oligomeric states were more diverse in terms of dispersity and morphology. We identified self-assembly conditions that stabilize high-molecular weight oligomers of Aß(M1-40) with apparent molecular weights greater than 36 kDa. Given that mixtures of AßOs derived from both peptides have been shown to be potent neurotoxins that disrupt long-term potentiation, we anticipate that the diverse quaternary structures reported for Aß(M1-40) oligomers using the assays reported here will facilitate research efforts aimed at isolating and identifying common toxic species that contribute to synaptic dysfunction.


Assuntos
Peptídeos beta-Amiloides/química , Multimerização Proteica , Estrutura Quaternária de Proteína , Peptídeos beta-Amiloides/genética , Humanos , Mutação , Eletroforese em Gel de Poliacrilamida Nativa , Agregados Proteicos , Agregação Patológica de Proteínas , Dobramento de Proteína , Proteínas Recombinantes , Relação Estrutura-Atividade
19.
Methods Mol Biol ; 1966: 125-135, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041743

RESUMO

Steroid receptors are members of a subfamily of the nuclear receptor superfamily. They play a dual role of steroid hormone receptors and transcription factors. Actually, these receptors are steroid-activated transcription factors. Classical soluble receptors exist as oligomeric complexes with the Hsp90-based chaperone machinery. The steroid receptor field was born and developed along with the molecular chaperone field. Chaperones are not exclusive partners associated to these receptors, but also comprise a large variety of heterocomplexes with other proteins involved in signal transduction. By using the glucocorticoid receptor (GR) as a standard model for most Hsp90-client proteins, in this chapter we describe the functional GR·Hsp90 heterocomplex assembly system from reticulocyte lysate or purified proteins.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Técnicas In Vitro/métodos , Multimerização Proteica , Receptores de Glucocorticoides/metabolismo , Animais , Estrutura Quaternária de Proteína , Coelhos , Receptores de Glucocorticoides/química , Reticulócitos/metabolismo
20.
Eur Biophys J ; 48(6): 569-577, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31134309

RESUMO

Microtubule mechanical properties are essential for understanding basic cellular processes, including cell motility and division, but the forces that result in microtubule rupture or breakage have not yet been measured directly. These forces are essential to understand the mechanical properties of the cytoskeleton and responses by cells to both normal conditions and stress caused by injury or disease. Here we estimate the force required to rupture a microtubule by analyzing kinesin-14 Ncd motor-induced microtubule breakage in ensemble motility assays. We model the breakage events as caused by Ncd motors pulling or pushing on single microtubules that are clamped at one end by other motors attached to the glass surface. The number of pulling or pushing Ncd motors is approximated from the length of the microtubule bound to the surface and the forces produced by the pulling or pushing motors are estimated from forces produced by the Ncd motor in laser-trap assays, reported by others. Our analysis provides an estimate, to the first approximation, of ~ 500 pN for the minimal force required to rupture a 13-pf microtubule. The value we report is close to the forces estimated from microtubule stretching/fragmentation experiments and overlaps with the forces applied by AFM in microtubule indentation assays that destabilize microtubules and break microtubule protofilaments. It is also consistent with the forces required to disrupt protein noncovalent bonds in force spectroscopy experiments. These findings are relevant to microtubule deformation and breakage caused by cellular tension in vivo.


Assuntos
Fenômenos Mecânicos , Microtúbulos/metabolismo , Fenômenos Biomecânicos , Ligações de Hidrogênio , Cinesina/metabolismo , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Tubulina (Proteína)/química
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