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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.
Phys Chem Chem Phys ; 21(44): 24393-24405, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31663524

RESUMO

Engineered repeat proteins have proven to be a fertile ground for studying the competition between folding, misfolding and transient aggregation of tethered protein domains. We examine the interplay between folding and inter-domain interactions of engineered FiP35 WW domain repeat proteins with n = 1 through 5 repeats. We characterize protein expression, thermal and guanidium melts, as well as laser T-jump kinetics. All experimental data is fitted by a global fitting model with two states per domain (U, N), plus a third state M to account for non-native states due to domain interactions present in all but the monomer. A detailed structural model is provided by coarse-grained simulated annealing using the AWSEM Hamiltonian. Tethered FiP35 WW domains with n = 2 and 3 domains are just slightly less stable than the monomer. The n = 4 oligomer is yet less stable, its expression yield is much lower than the monomer's, and depends on the purification tag used. The n = 5 plasmid did not express at all, indicating the sudden onset of aggregation past n = 4. Thus, tethered FiP35 has a critical nucleus size for inter-domain aggregation of n ≈ 4. According to our simulations, misfolded structures become increasingly prevalent as one proceeds from monomer to pentamer, with extended inter-domain beta sheets appearing first, then multi-sheet 'intramolecular amyloid' structures, and finally novel motifs containing alpha helices. We discuss the implications of our results for oligomeric aggregate formation and structure, transient aggregation of proteins whilst folding, as well as for protein evolution that starts with repeat proteins.


Assuntos
Proteínas/química , Cinética , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Proteínas/genética , Proteínas/metabolismo , Termodinâmica , Domínios WW
4.
BMC Bioinformatics ; 20(1): 464, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31500562

RESUMO

BACKGROUND: The function of oligomeric proteins is inherently linked to their quaternary structure. In the absence of high-resolution data, low-resolution information in the form of spatial restraints can significantly contribute to the precision and accuracy of structural models obtained using computational approaches. To obtain such restraints, chemical cross-linking coupled with mass spectrometry (XL-MS) is commonly used. However, the use of XL-MS in the modeling of protein complexes comprised of identical subunits (homo-oligomers) is often hindered by the inherent ambiguity of intra- and inter-subunit connection assignment. RESULTS: We present a comprehensive evaluation of (1) different methods for inter-residue distance calculations, and (2) different approaches for the scoring of spatial restraints. Our results show that using Solvent Accessible Surface distances (SASDs) instead of Euclidean distances (EUCs) greatly reduces the assignation ambiguity and delivers better modeling precision. Furthermore, ambiguous connections should be considered as inter-subunit only when the intra-subunit alternative exceeds the distance threshold. Modeling performance can also be improved if symmetry, characteristic for most homo-oligomers, is explicitly defined in the scoring function. CONCLUSIONS: Our findings provide guidelines for proper evaluation of chemical cross-linking-based spatial restraints in modeling homo-oligomeric protein complexes, which could facilitate structural characterization of this important group of proteins.


Assuntos
Reagentes para Ligações Cruzadas/química , Modelos Moleculares , Complexos Multiproteicos/química , Multimerização Proteica , Simulação de Acoplamento Molecular , Subunidades Proteicas/química , Solventes
5.
Nat Biotechnol ; 37(10): 1209-1216, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501561

RESUMO

Chemical and optogenetic methods for post-translationally controlling protein function have enabled modulation and engineering of cellular functions. However, most of these methods only confer single-input, single-output control. To increase the diversity of post-translational behaviors that can be programmed, we built a system based on a single protein receiver that can integrate multiple drug inputs, including approved therapeutics. Our system translates drug inputs into diverse outputs using a suite of engineered reader proteins to provide variable dimerization states of the receiver protein. We show that our single receiver protein architecture can be used to program a variety of cellular responses, including graded and proportional dual-output control of transcription and mammalian cell signaling. We apply our tools to titrate the competing activities of the Rac and Rho GTPases to control cell morphology. Our versatile tool set will enable researchers to post-translationally program mammalian cellular processes and to engineer cell therapies.


Assuntos
Proteínas/química , Proteínas/metabolismo , Animais , Linhagem Celular , Técnicas de Química Combinatória , Desenho de Drogas , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Células NIH 3T3 , Optogenética/métodos , Conformação Proteica , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Biologia Sintética/métodos
6.
J Phys Chem Lett ; 10(19): 5815-5822, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31525988

RESUMO

The topological knot is thought to play a stabilizing role in maintaining the global fold and nature of proteins with the underlying mechanism yet to be elucidated. Given that most proteins containing trefoil knots exist and function as homodimers with a large part of the dimer interface occupied by the knotted region, we reason that the knotted conformation cooperates with dimerization in protein stabilization. Here, we take YbeA from Escherichia coli as the knotted protein model, using molecular dynamics (MD) simulations to compare the stability of two pairs of dimeric proteins having the same sequence and secondary structures but differing in the presence or absence of a trefoil knot in each subunit. The dimer interface of YbeA is identified to involve favorable contacts among three α-helices (α1, α3, and α5), one of which (α5) is threaded through a loop connected with α3 to form the knot. Upon removal of the knot by appropriate change of the knot-making crossing of the polypeptide chain, relevant domains are less constrained and exhibit enhanced fluctuations to decrease contacts at the interface. Unknotted subunits are less compact and undergo structural changes to ease the dimer separation. Such a stabilizing effect is evidenced by steered MD simulations, showing that the mechanical force required for dimer separation is significantly reduced by removing the knot. In addition to the knotted conformation, dimerization further improves the protein stability by restricting the α1-α5 separation, which is defined as a leading step for protein unfolding. These results provide important insights into the structure-function relationship of dimerization in knotted proteins.


Assuntos
Simulação de Dinâmica Molecular , Multimerização Proteica , Estabilidade Proteica , Proteínas/química , Proteínas de Escherichia coli/química , Metiltransferases/química , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Termodinâmica
7.
Soft Matter ; 15(38): 7663-7671, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31490506

RESUMO

Clear understanding of the principles that control the arrangement of proteins and their self-assembly into viral shells is very important for the development of antiviral strategies. Here we consider the structural peculiarities and hidden symmetry of the anomalous bluetongue virus (BTV) capsid. Each of its three concentric shells violates the paradigmatic geometrical model of Caspar and Klug, which is otherwise well suited to describe most of the known icosahedral viral shells. As we show, three icosahedral spherical lattices, which are commensurate with each other and possess locally hexagonal (primitive or honeycomb) order, underlie the proteinaceous shells of the BTV capsid. This interpretation of the multishelled envelope allows us to discuss the so-called "symmetry mismatch" between its layers. We also analyze the structural stability of the considered spherical lattices on the basis of the classical theory of spherical packing and relate the proximity of the outer spherical lattice to destabilization with the fact that during infection of the cell VP2 trimers are detached from the surface of the BTV capsid. An electrostatic mechanism that can assist in this detachment is discussed in detail.


Assuntos
Vírus Bluetongue/química , Proteínas do Capsídeo/química , Capsídeo/química , Modelos Biológicos , Viroses/metabolismo , Vírus Bluetongue/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Concentração de Íons de Hidrogênio , Conformação Proteica , Multimerização Proteica , Eletricidade Estática , Termodinâmica
8.
Nature ; 574(7776): 132-136, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554965

RESUMO

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.


Assuntos
Rodopsinas Microbianas/química , Thermoplasmales/química , Bacteriorodopsinas/química , Sítios de Ligação , Cristalografia por Raios X , Microscopia de Força Atômica , Modelos Moleculares , Dobramento de Proteína , Multimerização Proteica , Retinaldeído/química , Rodopsinas Microbianas/ultraestrutura
9.
Nature ; 574(7777): 206-210, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31514202

RESUMO

Soluble guanylate cyclase (sGC) is the primary sensor of nitric oxide. It has a central role in nitric oxide signalling and has been implicated in many essential physiological processes and disease conditions. The binding of nitric oxide boosts the enzymatic activity of sGC. However, the mechanism by which nitric oxide activates the enzyme is unclear. Here we report the cryo-electron microscopy structures of the human sGCα1ß1 heterodimer in different functional states. These structures revealed that the transducer module bridges the nitric oxide sensor module and the catalytic module. Binding of nitric oxide to the ß1 haem-nitric oxide and oxygen binding (H-NOX) domain triggers the structural rearrangement of the sensor module and a conformational switch of the transducer module from bending to straightening. The resulting movement of the N termini of the catalytic domains drives structural changes within the catalytic module, which in turn boost the enzymatic activity of sGC.


Assuntos
Microscopia Crioeletrônica , Guanilil Ciclase Solúvel/metabolismo , Guanilil Ciclase Solúvel/ultraestrutura , Animais , Dissulfetos/química , Dissulfetos/metabolismo , Drosophila melanogaster , Ativação Enzimática , Células HEK293 , Heme/metabolismo , Humanos , Hidrazinas/farmacologia , Camundongos , Modelos Moleculares , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Domínios Proteicos , Multimerização Proteica , Guanilil Ciclase Solúvel/química , Guanilil Ciclase Solúvel/genética
10.
Int J Nanomedicine ; 14: 6179-6195, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31447559

RESUMO

Background and purpose: Gambogic acid (GA) is a natural compound that exhibited a promising multi-target antitumor activity against several types of cancer. However, the clinical application of this drug is limited due to its poor solubility and low tumor cell-specific delivery. In this study, the monomeric and dimeric Cyclo (Arg-Gly-Asp) c(RGD) tumor targeting peptides (c(RGDfK) and E-[c(RGDfK)2]) were used to modify GA loaded nanostructured lipid carriers (NLC) to reduce the limitations associated with GA and improve its antitumor activity. Methods: GA-NLC was prepared by emulsification and solvent evaporation methods and the surface of the NLC was conjugated with the c(RGD) peptides via an amide bond. The formulations were characterized for particle size, morphology and zeta potential, encapsulation efficiency and drug loading. The in-vitro cytotoxicity and cell uptake studies were conducted using 4T1 cell. Furthermore, the in-vivo antitumor activity and bio-distribution study were performed on female BALB/c nude mice. Results: The c(RGD) peptides modified GA-NLC was successfully prepared with the particles size about 20 nm. The HPLC analysis, FT-IR and 1H-NMR spectra confirmed the successful conjugation of the peptides with the NLC. The in-vitro cytotoxicity study on 4T1 cells revealed that c(RGD) peptides modified GA-NLCs showed significantly higher cytotoxicity at 0.25 and 0.5 µg/mL as compared to unmodified GA-NLC. Furthermore, the cell uptake study demonstrated that better accumulation of E-[c(RGDfK)2] peptides modified NLC in 4T1 cell after 12 h incubation. Moreover, the in-vivo study showed that c(RGD)s functionalized GA-NLC exhibited better accumulation in tumor tissue and tumor growth inhibition. In contrast to the monomeric c(RGD) peptide, the dimeric c(RGD) peptide (E-[c(RGDfK)2]) conjugated GA-NLC showed the improved antitumor activity and tumor targeting ability of GA-NLC. Conclusion: These data provide further support for the potential clinical applications of E-[c(RGDfK)2]-GA-NLC in breast cancer therapy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Lipídeos/química , Nanoestruturas/química , Oligopeptídeos/química , Xantonas/uso terapêutico , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Multimerização Proteica , Distribuição Tecidual/efeitos dos fármacos , Xantonas/farmacologia
11.
Nucleic Acids Res ; 47(16): 8888-8898, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31372631

RESUMO

DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.


Assuntos
Apoproteínas/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade
12.
Soft Matter ; 15(33): 6660-6676, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31389467

RESUMO

The dynamic behavior of monoclonal antibodies (mAbs) at high concentration provides insight into protein microstructure and protein-protein interactions (PPI) that influence solution viscosity and protein stability. At high concentration, interpretation of the collective-diffusion coefficient Dc, as determined by dynamic light scattering (DLS), is highly challenging given the complex hydrodynamics and PPI at close spacings. In contrast, self-diffusion of a tracer particle by Brownian motion is simpler to understand. Herein, we develop fluorescence correlation spectroscopy (FCS) for the measurement of the long-time self-diffusion of mAb2 over a wide range of concentrations and viscosities in multiple co-solute formulations with varying PPI. The normalized self-diffusion coefficient D0/Ds (equal to the microscopic relative viscosity ηeff/η0) was found to be smaller than η/η0. Smaller ratios of the microscopic to macroscopic viscosity (ηeff/η) are attributed to a combination of weaker PPI and less self-association. The interaction parameters extracted from fits of D0/Ds with a length scale dependent viscosity model agree with previous measurements of PPI by SLS and SAXS. Trends in the degree of self-association, estimated from ηeff/η with a microviscosity model, are consistent with oligomer sizes measured by SLS. Finally, measurements of collective diffusion and osmotic compressibility were combined with FCS data to demonstrate that the changes in self-diffusion between formulations are due primarily to changes in the protein-protein friction in these systems, and not to protein-solvent friction. Thus, FCS is a robust and accessible technique for measuring mAb self-diffusion, and, by extension, microviscosity, PPI and self-association that govern mAb solution dynamics.


Assuntos
Anticorpos Monoclonais/química , Fenômenos Biofísicos , Difusão , Fluorescência , Corantes Fluorescentes/química , Microscopia de Fluorescência , Modelos Químicos , Multimerização Proteica , Estabilidade Proteica , Soluções , Viscosidade
13.
Pharm Res ; 36(11): 152, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31463609

RESUMO

PURPOSE: To develop an analytical platform for the estimation as well as characterization of aggregates over the complete size spectrum (from invisible monomer to visible precipitates). METHODS: Two mAb samples were incubated at 30°C in different buffer systems of protein A chromatography for observing degradation due to aggregation. The aggregation in these samples was quantified by size exclusion chromatography (SEC), dynamic light scattering (DLS), and micro flow imaging (MFI). RESULTS: The results obtained from various characterization tools were analysed in various size ranges - size exclusion chromatography (SEC) (1 nm - 25 nm), dynamic light scattering (DLS) (10 nm - 5 µm), and micro flow imaging (MFI) (2 µm - 300 µm). Since each characterization tool covers a particular size range, data from multiple tools was collected in the "handover" regions to demonstrate accuracy of the platform. CONCLUSIONS: Based on the observations from the experiments, an analytical platform has been proposed covering the whole size spectrum that would be of utility to those engaged in formulation development as well as other aspects related to stability of biotherapeutic products.


Assuntos
Anticorpos Monoclonais/análise , Tampões (Química) , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Difusão Dinâmica da Luz , Nanopartículas/química , Tamanho da Partícula , Multimerização Proteica , Estabilidade Proteica
14.
Biochim Biophys Acta Bioenerg ; 1860(10): 148063, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31419396

RESUMO

Ferritin-like proteins, Dps (DNA-binding protein from starved cells), store iron and play a key role in the iron homeostasis in bacteria, yet their iron releasing machinery remains largely unexplored. The electron donor proteins that may interact with Dps and promote the mobilization of the stored iron have hitherto not been identified. Here, we investigate the binding capacity of the two atypical Dps proteins NpDps4 and NpDps5 from Nostoc punctiforme to isolated ferredoxins. We report NpDps-ferredoxin interactions by fluorescence correlation spectroscopy (FCS) and fluorescence resonance energy transfer (FRET) methods. Dynamic light scattering, size exclusion chromatography and native gel electrophoresis results show that NpDps4 forms a dodecamer at both pH 6.0 and pH 8.0, while NpDps5 forms a dodecamer only at pH 6.0. In addition, FCS data clearly reveal that the non-canonical NpDps5 interacts with DNA at pH 6.0. Our spectroscopic analysis shows that [FeS] centers of the three recombinantly expressed and isolated ferredoxins are properly incorporated and are consistent with their respective native states. The results support our hypothesis that ferredoxins could be involved in cellular iron homeostasis by interacting with Dps and assisting the release of stored iron.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DNA/metabolismo , Ferredoxinas/metabolismo , Nostoc/metabolismo , Proteínas de Bactérias/metabolismo , Cianobactérias , Proteínas de Ligação a DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Ligação Proteica , Multimerização Proteica , Espectrometria de Fluorescência
15.
Phys Chem Chem Phys ; 21(34): 18595-18604, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31414082

RESUMO

With application to the nitrite reductase hexameric protein complex of Desulfovibrio vulgaris, NrfH2A4, we suggest a strategy to compute the energy landscape of electron transfer in large systems of biochemical interest. For small complexes, the energy of all electronic configurations can be scanned completely on the level of a numerical solution of the Poisson-Boltzmann equation. In contrast, larger systems have to be treated using a pair approximation, which is verified here. Effective Coulomb interactions between neighbouring sites of excess electron localization may become as large as 200 meV, and they depend in a nontrivial manner on the intersite distance. We discuss the implications of strong Coulomb interactions on the thermodynamics and kinetics of charging and decharging a protein complex. Finally, we turn to the effect of embedding the system into a biomembrane.


Assuntos
Desulfovibrio vulgaris/enzimologia , Modelos Moleculares , Nitrito Redutases/química , Dimiristoilfosfatidilcolina/química , Transporte de Elétrons , Cinética , Membranas Artificiais , Conformação Proteica , Multimerização Proteica , Termodinâmica
16.
Cell Mol Life Sci ; 76(21): 4245-4273, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31317204

RESUMO

Molecular self-organziation, also regarded as pattern formation, is crucial for the correct distribution of cellular content. The processes leading to spatiotemporal patterns often involve a multitude of molecules interacting in complex networks, so that only very few cellular pattern-forming systems can be regarded as well understood. Due to its compositional simplicity, the Escherichia coli MinCDE system has, thus, become a paradigm for protein pattern formation. This biological reaction diffusion system spatiotemporally positions the division machinery in E. coli and is closely related to ParA-type ATPases involved in most aspects of spatiotemporal organization in bacteria. The ATPase MinD and the ATPase-activating protein MinE self-organize on the membrane as a reaction matrix. In vivo, these two proteins typically oscillate from pole-to-pole, while in vitro they can form a variety of distinct patterns. MinC is a passenger protein supposedly operating as a downstream cue of the system, coupling it to the division machinery. The MinCDE system has helped to extract not only the principles underlying intracellular patterns, but also how they are shaped by cellular boundaries. Moreover, it serves as a model to investigate how patterns can confer information through specific and non-specific interactions with other molecules. Here, we review how the three Min proteins self-organize to form patterns, their response to geometric boundaries, and how these patterns can in turn induce patterns of other molecules, focusing primarily on experimental approaches and developments.


Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Proteínas de Escherichia coli/fisiologia , Proteínas de Membrana/fisiologia , Transporte Proteico/fisiologia , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica/fisiologia , Transporte Proteico/genética
17.
Cancer Sci ; 110(10): 3340-3349, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31342590

RESUMO

Aberrant activation of the MET/hepatocyte growth factor (HGF) receptor participates in the malignant behavior of cancer cells, such as invasion-metastasis and resistance to molecular targeted drugs. Many mutations in the MET extracellular region have been reported, but their significance is largely unknown. Here, we report the dysregulation of mutant MET originally found in a lung cancer patient with Val370 to Asp370 (V370D) replacement located in the extracellular SEMA domain. MET-knockout cells were prepared and reconstituted with WT-MET or V370D-MET. HGF stimulation induced MET dimerization and biological responses in cells reconstituted with WT-MET, but HGF did not induce MET dimerization and failed to induce biological responses in V370D-MET cells. The V370D mutation abrogated HGF-dependent drug resistance of lung cancer cells to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI). Compared with WT-MET cells, V370D-MET cells showed different activation patterns in receptor tyrosine kinases upon exposure to survival/growth-stressed conditions. Surface plasmon resonance analysis indicated that affinity between the extracellular region of V370D-MET and HGF was reduced compared with that for WT-MET. Further analysis of the association between V370D-MET and the separate domains of HGF indicated that the SP domain of HGF was unchanged, but its association with the NK4 domain of HGF was mostly lost in V370D-MET. These results indicate that the V370D mutation in the MET receptor impairs the functional association with HGF and is therefore a loss-of-function mutation. This mutation may change the dependence of cancer cell growth/survival on signaling molecules, which may promote cancer cell characteristics under certain conditions.


Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/genética , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Resistencia a Medicamentos Antineoplásicos , Técnicas de Inativação de Genes , Humanos , Mutação com Perda de Função , Domínios Proteicos , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Proto-Oncogênicas c-met/metabolismo , Ativação Transcricional
18.
Comput Biol Chem ; 82: 37-43, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31255973

RESUMO

Tubulin protein is the fundamental unit of microtubules, and comprises of α and ß subunits arranged in an alternate manner forming protofilaments. These longitudinal protofilaments are made up of intra- (α-ß) and inter-dimer (ß-α) interactions. Literature review confirms that GTP hydrolysis results in considerable structural rearrangement within GTP binding site of ß-α dimer interface after the release of γ phosphate. In addition to this, the intra-dimer interface exhibits structural rigidity which needs further investigation. In this study, we explored the reasons for the flexibility and the rigidity of the ß-α dimer and the α-ß dimer respectively through molecular simulation and Anisotropic Normal Mode based analysis. As per the sequence alignment report, two glycine residues (Gly96 and Gly98) were observed in the T3 loop of the ß subunit which get substituted by Asp98 and Ala100 in the T3 loop of the α subunit. The higher mobility of glycine residues contributes to the flexibility of the T3 loop of inter-dimer when they come in direct contact with the GTPase Activating Protein (GAP) domain of the subunit. This was confirmed through RMSD, RMSF and Radius of Gyration based studies. Conversely, the intra-dimer exhibited a lower mobility in the absence of glycine residues. As per ANM based analysis, positive domain correlations were observed between T3 loop and GAP domain of intra- and inter- dimeric contact regions. However, these correlation motions were higher in the intra-dimer as compared to the inter-dimer interface. Thus on the basis of our findings, we hypothesize that the higher flexibility of T3 loop and the GAP domain of the inter-dimer is required for structural rearrangement and protofilament stability during hydrolysis. Furthermore, the slightly rigid nature of the T3 loop and the GAP domain of the intra-dimer assists in enhancing the monomer-monomer interaction through the higher positive domain correlation.


Assuntos
Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Anisotropia , Sítios de Ligação , Bovinos , Glicina/química , Simulação de Dinâmica Molecular , Mutação , Maleabilidade , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Alinhamento de Sequência , Tubulina (Proteína)/genética
19.
Chem Commun (Camb) ; 55(59): 8595-8598, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31276123

RESUMO

The amino acid sequence plays an essential role in amyloid formation. Here, using the central core recognition module of the Aß peptide and its reverse sequence, we show that although both peptides assemble into ß-sheets, their morphologies, kinetics and cell toxicities display marked differences. In addition, the native peptide, but not the reverse one, shows notable affinity towards bilayer lipid model membranes that modulates the aggregation pathways to stabilize the oligomeric intermediate states and function as the toxic agent responsible for neuronal dysfunction.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/toxicidade , Animais , Linhagem Celular Tumoral , Colesterol/química , Humanos , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fragmentos de Peptídeos/toxicidade , Fosfatidilcolinas/química , Conformação Proteica em Folha beta , Multimerização Proteica , Ratos , Esfingomielinas/química
20.
Nat Commun ; 10(1): 3070, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296852

RESUMO

CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/ultraestrutura , Guanilato Ciclase/ultraestrutura , Domínios Proteicos , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Microscopia Crioeletrônica , Guanilato Ciclase/genética , Guanilato Ciclase/imunologia , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Multimerização Proteica/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Transdução de Sinais/imunologia
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