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1.
Protein Expr Purif ; 121: 31-40, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26792557

RESUMO

RhlR is a 241-residue quorum sensing receptor that controls the expression of a myriad of virulence genes in Pseudomonas aeruginosa. Here, the DNA sequence encoding the carboxi-terminal DNA-binding domain of RhlR was cloned into the pET-RP1B plasmid and expressed as an N-terminal fusion protein to the expression/purification Thio6His6 tag. The fusion construct expressed insolubly in Escherichia coli BL21 (DE3) cells. The recombinant protein was extracted from the bacterial inclusion bodies and refolded in the presence of the charged amino acids l-arginine and l-glutamate. The refolded protein was purified by a combination of Ni(+2)-affinity and size exclusion chromatography, allowing the production of 2 mg of highly purified protein (>95% purity) per 5 mg of wet cells derived from 1 L culture. (1)H 1D NMR analysis revealed that the recombinant protein is folded. Moreover, a fluorescence anisotropy DNA-binding assay showed that the refolded protein is functional, as it recognizes the rhlAB promoter. This is the first time that a domain of the quorum sensing regulator RhlR was produced in sufficient amounts for structural studies, enabling the investigation of the molecular basis for RhlR specific interaction with DNA promoters.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Percepção de Quorum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Dobramento de Proteína , Pseudomonas aeruginosa/genética
2.
Biochim Biophys Acta ; 1844(4): 837-49, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24590112

RESUMO

MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Peptídeos/química , Fosfoproteínas/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Química Encefálica , Embrião de Galinha , Dicroísmo Circular , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Substrato Quinase C Rico em Alanina Miristoilada , Peptídeos/síntese química , Peptídeos/metabolismo , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Estrutura Secundária de Proteína , Ressonância de Plasmônio de Superfície
3.
An Acad Bras Cienc ; 87(4): 2189-203, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26247154

RESUMO

Bacteria are able to synchronize the population behavior in order to regulate gene expression through a cell-to-cell communication mechanism called quorum sensing. This phenomenon involves the production, detection and the response to extracellular signaling molecules named autoinducers, which directly or indirectly regulate gene expression in a cell density-dependent manner. Quorum sensing may control a wide range of biological processes in bacteria, such as bioluminescence, virulence factor production, biofilm formation and antibiotic resistance. The autoinducers are recognized by specific receptors that can either be membrane-bound histidine kinase receptors, which work by activating cognate cytoplasmic response regulators, or cytoplasmic receptors acting as transcription factors. In this review, we focused on the cytosolic quorum sensing regulators whose three-dimensional structures helped elucidate their mechanisms of action. Structural studies of quorum sensing receptors may enable the rational design of inhibitor molecules. Ultimately, this approach may represent an effective alternative to treat infections where classical antimicrobial therapy fails to overcome the microorganism virulence.


Assuntos
Fenômenos Fisiológicos Bacterianos , Citosol/fisiologia , Percepção de Quorum/fisiologia , Transdução de Sinais/fisiologia
4.
Proc Natl Acad Sci U S A ; 107(11): 5112-7, 2010 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-20190181

RESUMO

Inhibition of blood vessel formation is a viable therapeutic approach in angiogenesis-dependent diseases. We previously used a combinatorial screening on vascular endothelial growth factor (VEGF)-activated endothelial cells to select the sequence CPQPRPLC and showed that the motif Arg-Pro-Leu targets VEGF receptor-1 and neuropilin-1. Here, we evaluated and validated (D)(LPR), a derivative molecule with strong antiangiogenesis attributes. This prototype drug markedly inhibits neovascularization in three mouse models: Matrigel-based assay, functional human/murine blood vessel formation, and retinopathy of prematurity. In addition to its systemic activity, (D)(LPR) also inhibits retinal angiogenesis when administered in an eye-drop formulation. Finally, in preliminary studies, we have showed targeted drug activity in an experimental tumor-bearing mouse model. These results show that drugs targeting extracellular domains of VEGF receptors are active, affect signal transduction, and have potential for clinical application. On a larger context, this study illustrates the power of ligand-directed selection plus retro-inversion for rapid drug discovery and development.


Assuntos
Desenho de Fármacos , Biblioteca de Peptídeos , Peptídeos/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Resistência a Medicamentos/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Neuropilina-1/metabolismo , Peptídeos/química , Peptídeos/uso terapêutico , Retina/efeitos dos fármacos , Retina/patologia , Neovascularização Retiniana/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
5.
Front Mol Biosci ; 8: 653148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34041264

RESUMO

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

6.
Structure ; 26(2): 199-208.e3, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29307486

RESUMO

Lipases and esterases constitute a group of enzymes that catalyze the hydrolysis or synthesis of ester bonds. A major biotechnological interest corresponds to thermophilic esterases, due to their intrinsic stability at high temperatures. The Pf2001 esterase from Pyrococcus furiosus reaches its optimal activity between 70°C and 80°C. The crystal structure of the Pf2001 esterase shows two different conformations: monomer and dimer. The structures reveal important rearrangements in the "cap" subdomain between monomer and dimer, by the formation of an extensive intertwined helical interface. Moreover, the dimer interface is essential for the formation of the hydrophobic channel for substrate selectivity, as confirmed by mutagenesis and kinetic analysis. We also provide evidence for dimer formation at high temperatures, a process that correlates with its enzymatic activation. Thus, we propose a temperature-dependent activation mechanism of the Pf2001 esterase via dimerization that is necessary for the substrate channel formation in the active-site cleft.


Assuntos
Ativação Enzimática/fisiologia , Esterases/metabolismo , Modelos Moleculares , Pyrococcus furiosus/metabolismo , Estabilidade Enzimática , Temperatura Alta , Conformação Proteica , Temperatura
7.
Chem Biol ; 12(10): 1075-83, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16242650

RESUMO

Vascular endothelial growth factor (VEGF) is central to the survival and development of the vascular and nervous systems. We screened phage display libraries and built a peptide-based ligand-receptor map of binding sites within the VEGF family. We then validated a cyclic peptide, CPQPRPLC, as a VEGF-mimic that binds specifically to neuropilin-1 and VEGF receptor-1. Here, we use NMR spectroscopy to understand the structural basis of the interaction between our mimic peptide and the VEGF receptors. We show that: (1) CPQPRPLC has multiple interactive conformations; (2) receptor binding is mediated by the motif Arg-Pro-Leu; and (3) the Pro residue within Arg-Pro-Leu participates in binding to neuropilin-1 but not to VEGF receptor-1, perhaps representing an evolutionary gain-of-function. Therefore, Arg-Pro-Leu is a differential ligand motif to VEGF receptors and a candidate peptidomimetic lead for VEGF pathway modulation.


Assuntos
Mimetismo Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Fatores de Crescimento do Endotélio Vascular/química , Fatores de Crescimento do Endotélio Vascular/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Neuropilina-1/metabolismo , Peptídeos Cíclicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
8.
Biomol NMR Assign ; 9(2): 281-4, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25487676

RESUMO

Human antigen R (HuR) is a ubiquitous protein that recognizes adenylate and uridylate-rich elements in mRNA, thereby interfering with the fate of protein translation. This protein plays a central role in the outcome of the inflammatory response as it may stabilize or silence mRNAs of key components of the immune system. HuR is able to interact with other RNA-binding proteins, reflecting a complex network that dictates mRNAs post-transcriptional control. HuR is composed of three functional domains, known as RNA-recognition motifs (RRM1, RRM2 and RRM3). It is known that RRM1 is the most important domain for mRNA-binding affinity. In this study, we completed the NMR chemical shift assignment of the RRM1 domain of HuR, as a first step to further establishing the structure, dynamics and function relationship for this protein.


Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Proteínas ELAV/química , Espectroscopia de Prótons por Ressonância Magnética , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
9.
Biomed Res Int ; 2014: 684506, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24783219

RESUMO

Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to "order" a "customized" enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design), as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts.


Assuntos
Biotecnologia/métodos , Lipase/química , Catálise , Relação Estrutura-Atividade
11.
PLoS One ; 3(10): e3452, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18941632

RESUMO

Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11R alpha is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11R alpha has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs.


Assuntos
Subunidade alfa de Receptor de Interleucina-11/metabolismo , Interleucina-11/metabolismo , Neoplasias/química , Fragmentos de Peptídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação/genética , Proliferação de Células , Humanos , Interleucina-11/genética , Subunidade alfa de Receptor de Interleucina-11/genética , Ligantes , Mimetismo Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/isolamento & purificação , Ligação Proteica , Fator de Transcrição STAT3/metabolismo
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