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1.
ACS Chem Biol ; 16(7): 1191-1200, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34161732

RESUMO

Intrinsically disordered regions in proteins often function as binding motifs in protein-protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Precursor de Proteína beta-Amiloide/síntese química , Precursor de Proteína beta-Amiloide/genética , Animais , Caderinas/síntese química , Caderinas/genética , Proteínas de Transporte/síntese química , Proteínas de Transporte/genética , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/síntese química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Mutação , Proteínas do Tecido Nervoso/síntese química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Domínios Proteicos/genética , Engenharia de Proteínas , Dobramento de Proteína , Ratos , Termodinâmica
2.
Curr Pharm Biotechnol ; 22(7): 878-891, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32838715

RESUMO

In recent years, extensive attention has been given to the generation of new classes of ligand- specific binding proteins to supplement monoclonal antibodies. A combination of protein engineering and display technologies has been used to manipulate non-human antibodies for humanization and stabilization purposes or even the generation of new binding proteins. Engineered protein scaffolds can now be directed against therapeutic targets to treat cancer and immunological disorders. Although very few of these scaffolds have successfully passed clinical trials, their remarkable properties such as robust folding, high solubility, and small size motivate their employment as a tool for biology and applied science studies. Here, we have focused on the generation of new non-Ig binding proteins and single domain antibody manipulation, with a glimpse of their applications.


Assuntos
Proteínas de Transporte/síntese química , Proteínas de Transporte/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Humanos , Biblioteca de Peptídeos , Ligação Proteica/fisiologia , Engenharia de Proteínas/tendências , Estrutura Secundária de Proteína
3.
ACS Chem Biol ; 15(9): 2395-2405, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32835479

RESUMO

Vaccines based on isolated polysaccharides successfully protect humans from bacterial pathogens such as Streptococcus pneumoniae. Because polysaccharide production and isolation can be technically challenging, glycoconjugates containing synthetic antigens are an attractive alternative. Typically, the shortest possible oligosaccharide antigen is preferable as syntheses of longer structures are more difficult and time-consuming. Combining several protective epitopes or polysaccharide repeating units as blocks by bonds other than glycosidic linkages would greatly reduce the synthetic effort if the immunological response to the polysaccharide could be retained. To explore this concept, we bridged the well-understood and immunologically potent RU of S. pneumoniae serotype 14 (ST14) with an aliphatic spacer and conjugated it to the carrier protein CRM197. Mice immunized with the spacer-bridged glycan conjugates produced high levels of specific antibodies after just one or two vaccine doses, while the tetrasaccharide repeating unit alone required three doses. The antibodies recognized specifically ST14 CPS, while no significant antibody levels were raised against the spacer or unrelated CPS. Synthetic vaccines generated antibodies with opsonic activity. Mimicking polysaccharides by coupling repeating unit antigens via an aliphatic spacer may prove useful also for the development of other glycoconjugate vaccine candidates, thereby reducing the synthetic complexity while enhancing a faster immune response.


Assuntos
Glicoconjugados/farmacologia , Oligossacarídeos/farmacologia , Vacinas Estreptocócicas/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Animais , Sequência de Carboidratos , Proteínas de Transporte/síntese química , Proteínas de Transporte/imunologia , Proteínas de Transporte/farmacologia , Epitopos/química , Epitopos/imunologia , Feminino , Glicoconjugados/síntese química , Glicoconjugados/imunologia , Células HL-60 , Humanos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Oligossacarídeos/síntese química , Oligossacarídeos/imunologia , Sorogrupo , Vacinas Estreptocócicas/síntese química , Vacinas Estreptocócicas/imunologia , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/farmacologia
4.
Probiotics Antimicrob Proteins ; 12(4): 1582-1593, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32445120

RESUMO

Lipopolysaccharide (LPS) is a toxic and immunogenic agent for human. Additionally, LPS is a good target for some antimicrobial compounds, including antimicrobial peptides (AMPs). LPS-binding peptides (LBPs) can recognize and neutralize LPS. Rabbit and human cathelicidins are AMPs with LPS-binding activity. In this study, we designed and synthesized two new truncated LBPs from rabbit and human CAP18 peptides by in silico methods. After synthesis of peptides, the antimicrobial properties and LPS-binding activity of these peptides were evaluated. The parental rabbit and human CAP18 peptides were selected as positive controls. Next, the changes in the secondary structure of these peptides before and after treatment with LPS were measured by circular dichroism (CD). Human cytotoxicity of the peptides was evaluated by MTT and red blood cells (RBCs) hemolysis assays. Finally, field emission scanning electron microscopy (FE-SEM), confocal microscopy, and flow cytometry were performed to study the action mechanism of these peptides. Results indicated that the hCap18 and rCap18 had antibacterial activity (at a MIC of 4-128 µg/mL). The results of the quantitative LAL test demonstrated that LPS-binding activity of hCap18 peptide was better than rCap18, while rCap18 peptide had better antimicrobial properties. Furthermore, rCap18 had less cytotoxicity than hCap18. However, both peptides were nontoxic for normal human skin fibroblast cell in MIC range. In conclusion, rCap18 has good antibacterial properties, while hCap18 can be tested as a diagnostic molecule in our future studies.


Assuntos
Proteínas de Fase Aguda/síntese química , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Proteínas de Transporte/síntese química , Desenho de Fármacos , Lipopolissacarídeos/antagonistas & inibidores , Glicoproteínas de Membrana/síntese química , Proteínas de Fase Aguda/metabolismo , Proteínas de Fase Aguda/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Testes de Sensibilidade Microbiana , Engenharia de Proteínas/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Coelhos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Relação Estrutura-Atividade
5.
Anal Chem ; 92(2): 1963-1971, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31854989

RESUMO

High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.


Assuntos
Proteínas de Transporte/análise , Proteínas de Escherichia coli/análise , Glicoproteínas/análise , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Actinobacillus pleuropneumoniae/enzimologia , Proteínas de Transporte/síntese química , Proteínas de Transporte/genética , Escherichia coli/química , Proteínas de Escherichia coli/síntese química , Proteínas de Escherichia coli/genética , Glicoproteínas/síntese química , Glicoproteínas/genética , Glicosilação , Glicosiltransferases/química , Mutação , Biblioteca de Peptídeos , Estudo de Prova de Conceito , Proteínas Recombinantes/análise , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética
6.
Arch Virol ; 164(5): 1259-1269, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30903291

RESUMO

The long-term administration of acyclovir (ACV) for therapy against herpes simplex virus type 1 (HSV-1) infections can result in the emergence of ACV-resistant HSV strains. It is therefore urgent to develop new anti-herpetic compounds with mechanisms that differ from that of ACV. Cyanovirin-N (CV-N) is an antiviral agent that has an inhibitory effect on HSV-1 infections, and PEGylation of CV-N is potentially useful for pharmaceutical applications. Here, a (Gly4Ser)3 linker molecule was attached to the N-terminus of CV-N, and the resulting compound, linker-CV-N (LCV-N), was produced on a pilot scale with purity up to 95%. Then, PEG10k-LCV-N was synthesized by modifying at the α-amine group of the N-terminus of LCV-N with 10-kDa polyethylene glycol propionaldehyde (mPEG-ALD). CV-N, LCV-N and PEG10k-LCV-N were all found to have potent inhibitory activity against ACV-resistant HSV strains with IC50 values in the nM range. LCV-N was the most potent of these three compounds against both normal and ACV-resistant HSV strains. Although PEG10k-LCV-N showed less antiviral activity than CV-N and LCV-N, it still exhibited significant and universal virucidal activity against drug-resistant viruses. The toxicity and immunogenicity of PEG10k-LCV-N were dramatically lower than those of CV-N and LCV-N. In conclusion, we suggest that LCV-N and PEG10k-LCV-N are promising and safe microbicides for the control and/or treatment of ACV-resistant HSV infection.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/uso terapêutico , Proteínas de Transporte/química , Proteínas de Transporte/uso terapêutico , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1/efeitos dos fármacos , Polietilenoglicóis/química , Aciclovir/farmacologia , Animais , Proteínas de Bactérias/síntese química , Proteínas de Transporte/síntese química , Linhagem Celular , Chlorocebus aethiops , Farmacorresistência Viral/genética , Feminino , Herpesvirus Humano 1/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-Dawley , Células Vero
7.
Chembiochem ; 20(1): 40-45, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30137694

RESUMO

Proteins containing intrinsic disorder often form secondary structure upon interaction with a binding partner. Modulating such structures presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E-BP1 is an example of an intrinsically disordered protein that forms an α-helix upon binding to its protein ligand, eIF4E. Current biophysical methods for analyzing binding-induced structural changes are low-throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E-BP1 peptide that reports structural changes of its helix in high-throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the α-helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high-throughput screening to discover ligands that alter peptide secondary structure.


Assuntos
Proteínas de Transporte/metabolismo , Corantes Fluorescentes/química , Peptídeos/metabolismo , Tioamidas/química , Sequência de Aminoácidos , Proteínas de Transporte/síntese química , Proteínas de Transporte/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Fluoresceína-5-Isotiocianato/química , Humanos , Peptídeos/síntese química , Peptídeos/química , Conformação Proteica em alfa-Hélice , Dobramento de Proteína
8.
Eur J Med Chem ; 144: 318-329, 2018 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-29275231

RESUMO

A new series of blood-brain barrier permeable molecules designed to mimic the activity of Pleiotrophin in the CNS has been designed and synthesized. These compounds exert their action by interacting with the intracellular domain PD1 of the Protein Tyrosine-Phosphatase Receptor Z1 (PTPRZ1), and inhibiting its tyrosine phosphatase activity. The most potent compounds 10a and 12b (IC50 = 0,1 µM) significantly increase the phosphorylation of key tyrosine residues of PTPRZ1 substrates involved in neuronal survival and differentiation, and display protective effects against amphetamine-induced toxicity. Docking and molecular dynamics experiments have been used to analyze the binding mode and to explain the observed selectivity against PTP1B. An In vivo experiment has demonstrated that 10a can cross the BBB, thus promoting the possibility of moving forward these candidates for the development of drugs for the treatment of CNS disorders, such as drug addiction and neurodegenerative diseases.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteínas de Transporte/farmacologia , Doenças do Sistema Nervoso Central/tratamento farmacológico , Citocinas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Proteínas de Transporte/síntese química , Proteínas de Transporte/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doenças do Sistema Nervoso Central/metabolismo , Citocinas/síntese química , Citocinas/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Ratos , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Relação Estrutura-Atividade
9.
J Pept Sci ; 23(4): 282-293, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28185350

RESUMO

The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Alérgenos/química , Proteínas de Transporte/síntese química , Prunus persica/química , Proteínas de Transporte/química , Humanos , Estrutura Molecular
10.
J Pept Sci ; 22(9): 577-91, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27440580

RESUMO

The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Astrócitos/metabolismo , Proteínas de Transporte/síntese química , Portadores de Fármacos/síntese química , Células Endoteliais/metabolismo , Biblioteca de Peptídeos , Peptídeos/síntese química , Animais , Astrócitos/citologia , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Bovinos , Técnicas de Cocultura , Técnicas de Química Combinatória , Cultura em Câmaras de Difusão , Portadores de Fármacos/isolamento & purificação , Portadores de Fármacos/metabolismo , Células Endoteliais/citologia , Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Membranas Artificiais , Modelos Biológicos , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Permeabilidade , Cultura Primária de Células , Estabilidade Proteica , Ratos
11.
Immunol Res ; 64(4): 887-900, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27138940

RESUMO

Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations.


Assuntos
Proteína do Homeodomínio de Antennapedia/imunologia , Proteínas de Transporte/imunologia , Células Dendríticas/imunologia , Ovalbumina/imunologia , Vacinas/imunologia , Animais , Apresentação de Antígeno , Artrópodes/imunologia , Proteínas de Transporte/síntese química , Peptídeos Penetradores de Células , Células Cultivadas , Sistemas de Liberação de Medicamentos , Epitopos de Linfócito T/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/síntese química
12.
Chem Commun (Camb) ; 51(88): 15898-901, 2015 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-26391199

RESUMO

Quantitative cysteine-independent ligation of a Gd(3+) tag to genetically encoded p-azido-L-phenylalanine via Cu(I)-catalyzed click chemistry is shown to deliver an exceptionally powerful tool for Gd(3+)-Gd(3+) distance measurements by double electron-electron resonance (DEER) experiments, as the position of the Gd(3+) ion relative to the protein can be predicted with high accuracy.


Assuntos
Proteínas de Transporte/síntese química , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/química , Gadolínio , Glutamatos/química , Fenilalanina/análogos & derivados , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Azidas/química , Proteínas de Transporte/química , Química Click , Mutagênese Sítio-Dirigida , Fenilalanina/química , Fenilalanina/genética , Marcadores de Spin
13.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 7): 901-5, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26144236

RESUMO

Human hydroxysteroid dehydrogenase-like protein 2 (HSDL2) is a member of the short-chain dehydrogenase/reductase (SDR) subfamily of oxidoreductases and contains an N-terminal catalytic domain and a C-termianl sterol carrier protein type 2 (SCP-2) domain. In this study, the C-terminal SCP-2 domain of human HSDL2, including residues Lys318-Arg416, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 2.10 Šresolution. The crystal belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 70.4, c = 60.6 Å, α = ß = 90, γ = 120°. Two protein molecules are present in the asymmetric unit, resulting in a Matthews coefficient of 2.16 Å(3) Da(-1) and an approximate solvent content of 43%.


Assuntos
Proteínas de Transporte/síntese química , Proteínas de Transporte/isolamento & purificação , Hidroxiesteroide Desidrogenases/síntese química , Hidroxiesteroide Desidrogenases/isolamento & purificação , Sequência de Aminoácidos , Proteínas de Transporte/genética , Cristalização , Cristalografia por Raios X/métodos , Humanos , Hidroxiesteroide Desidrogenases/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína
14.
J Leukoc Biol ; 97(2): 341-50, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25412625

RESUMO

CAP37, a protein constitutively expressed in human neutrophils and induced in response to infection in corneal epithelial cells, plays a significant role in host defense against infection. Initially identified through its potent bactericidal activity for Gram-negative bacteria, it is now known that CAP37 regulates numerous host cell functions, including corneal epithelial cell chemotaxis. Our long-term goal is to delineate the domains of CAP37 that define these functions and synthesize bioactive peptides for therapeutic use. We report the novel finding of a multifunctional domain between aa 120 and 146. Peptide analogs 120-146 QR, 120-146 QH, 120-146 WR, and 120-146 WH were synthesized and screened for induction of corneal epithelial cell migration by use of the modified Boyden chamber assay, antibacterial activity, and LPS-binding activity. In vivo activity was demonstrated by use of mouse models of sterile and infected corneal wounds. The identity of the amino acid at position 132 (H vs. R) was important for cell migration and in vivo corneal wound healing. All analogs demonstrated antimicrobial activity. However, analogs containing a W at position 131 showed significantly greater antibacterial activity against the Gram-negative pathogen Pseudomonas aeruginosa. All analogs bound P. aeruginosa LPS. Topical administration of analog 120-146 WH, in addition to accelerating corneal wound healing, effectively cleared a corneal infection as a result of P. aeruginosa. In conclusion, we have identified a multifunctional bioactive peptide, based on CAP37, that induces cell migration, possesses antibacterial and LPS-binding activity, and is effective at healing infected and noninfected corneal wounds in vivo.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas Sanguíneas/farmacologia , Proteínas de Transporte/farmacologia , Lesões da Córnea/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/imunologia , Cicatrização/efeitos dos fármacos , Animais , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Proteínas Sanguíneas/síntese química , Proteínas Sanguíneas/química , Proteínas de Transporte/síntese química , Proteínas de Transporte/química , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Lesões da Córnea/imunologia , Lesões da Córnea/patologia , Feminino , Células HEK293 , Humanos , Camundongos , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/patologia , Cicatrização/imunologia
15.
J Org Chem ; 79(18): 8550-6, 2014 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-25147913

RESUMO

We report the X-ray crystal structure of a site-selective peptide catalyst moiety and teicoplanin A2-2 complex. The expressed protein ligation technique was used to couple T4 lysozyme (T4L) and a synthetic peptide catalyst responsible for the selective phosphorylation of the N-acetylglucosamine sugar in a teicoplanin A2-2 derivative. The T4L-Pmh-dPro-Aib-dAla-dAla construct was crystallized in the presence of teicoplanin A2-2. The resulting 2.3 Å resolution protein-peptide-teicoplanin complex crystal structure revealed that the nucleophilic nitrogen of N-methylimidazole in the Pmh residue is in closer proximity (7.6 Å) to the N-acetylglucosamine than the two other sugar rings present in teicoplanin (9.3 and 20.3 Å, respectively). This molecular arrangement is consistent with the observed selectivity afforded by the peptide-based catalyst when it is applied to a site-selective phosphorylation reaction involving a teicoplanin A2-2 derivative.


Assuntos
Acetilglucosamina/química , Antibacterianos/síntese química , Proteínas de Transporte/síntese química , Teicoplanina/análogos & derivados , Sequência de Aminoácidos , Antibacterianos/química , Sítios de Ligação , Proteínas de Transporte/química , Catálise , Cristalografia por Raios X , Conformação Molecular , Fosforilação , Teicoplanina/síntese química , Teicoplanina/química
16.
J Am Chem Soc ; 135(36): 13464-72, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-24001318

RESUMO

Although animal lectins usually show a high degree of specificity for glycan structures, their single-site binding affinities are typically weak, a drawback which is often compensated in biological systems by an oligovalent presentation of carbohydrate epitopes. For the design of monovalent glycomimetics, structural information regarding solution and bound conformation of the carbohydrate lead represents a valuable starting point. In this paper, we focus on the conformation of the trisaccharide Le(x) (Gal[Fucα(1-3)]ß(1-4)GlcNAc). Mainly because of the unfavorable tumbling regime, the elucidation of the solution conformation of Le(x) by NMR has only been partially successful so far. Le(x) was therefore attached to a (13)C,(15)N-labeled protein. (13)C,(15)N-filtered NOESY NMR techniques at ultrahigh field allowed increasing the maximal NOE enhancement, resulting in a high number of distance restraints per glycosidic bond and, consequently, a well-defined structure. In addition to the known contributors to the conformational restriction of the Le(x) structure (exoanomeric effect, steric compression induced by the NHAc group adjacent to the linking position of L-fucose, and the hydrophobic interaction of L-fucose with the ß-face of D-galactose), a nonconventional C-H···O hydrogen bond between H-C(5) of L-fucose and O(5) of D-galactose was identified. According to quantum mechanical calculations, this C-H···O hydrogen bond is the most prominent factor in stabilization, contributing 40% of the total stabilization energy. We therefore propose that the nonconventional hydrogen bond contributing to a reduction of the conformational flexibility of the Le(x) core represents a novel element of the glycocode. Its relevance to the stabilization of related branched oligosaccharides is currently being studied.


Assuntos
Oligossacarídeos/química , Proteínas de Transporte/síntese química , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Glicosilação , Ligação de Hidrogênio , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Teoria Quântica
17.
Molecules ; 17(12): 13740-58, 2012 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-23174893

RESUMO

Within this study, a unique 3D structure-based pharmacophore model of the enzyme glyoxalase-1 (Glo-1) has been revealed. Glo-1 is considered a zinc metalloenzyme in which the inhibitor binding with zinc atom at the active site is crucial. To our knowledge, this is the first pharmacophore model that has a selective feature for a "zinc binding group" which has been customized within the structure-based pharmacophore model of Glo-1 to extract ligands that possess functional groups able to bind zinc atom solely from database screening. In addition, an extensive 2D similarity search using three diverse similarity techniques (Tanimoto, Dice, Cosine) has been performed over the commercially available "Zinc Clean Drug-Like Database" that contains around 10 million compounds to help find suitable inhibitors for this enzyme based on known inhibitors from the literature. The resultant hits were mapped over the structure based pharmacophore and the successful hits were further docked using three docking programs with different pose fitting and scoring techniques (GOLD, LibDock, CDOCKER). Nine candidates were suggested to be novel Glo-1 inhibitors containing the "zinc binding group" with the highest consensus scoring from docking.


Assuntos
Proteínas de Transporte , Lactoilglutationa Liase , Relação Estrutura-Atividade , Zinco/química , Algoritmos , Sítios de Ligação , Proteínas de Transporte/síntese química , Proteínas de Transporte/química , Domínio Catalítico , Bases de Dados Factuais , Humanos , Lactoilglutationa Liase/antagonistas & inibidores , Lactoilglutationa Liase/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica
19.
Methods Enzymol ; 503: 157-88, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22230569

RESUMO

Anticalins are a novel class of small, robust proteins with designed ligand-binding properties derived from the natural lipocalin scaffold. Due to their compact molecular architecture, comprising a single polypeptide chain, they provide several benefits as protein therapeutics, such as high target specificity, good tissue penetration, low immunogenicity, tunable plasma half-life, efficient Escherichia coli expression, and suitability for furnishing with additional effector functions via genetic fusion or chemical conjugation. The lipocalins are a widespread family of proteins that naturally serve in many organisms, including humans, for the transport, storage, or sequestration of small biological compounds like vitamins and hormones. Their fold is dominated by an eight-stranded antiparallel ß-barrel, which is open to the solvent at one end. There, four loops connect the ß-strands in a pairwise manner and, altogether, they form the entry to a ligand-binding site. This loop region can be engineered via site-directed random mutagenesis in combination with genetic library selection techniques to yield "Anticalins" with exquisite specificities-and down to picomolar affinities-for prescribed molecular targets of either hapten or antigen type. Several Anticalins directed against medically relevant disease targets have been successfully engineered and can be applied, for example, for the blocking of soluble signaling factors or cell surface receptors or for tissue-specific drug targeting. While natural lipocalins were already subject to clinical studies in the past, a first Anticalin has completed Phase I trials in 2011, thus paving the way for the broad application of Anticalins as a promising novel class of biopharmaceuticals.


Assuntos
Proteínas de Fase Aguda/química , Proteínas de Transporte/química , Sistemas de Liberação de Medicamentos/métodos , Lipocalinas/química , Biblioteca de Peptídeos , Proteínas Proto-Oncogênicas/química , Proteínas de Fase Aguda/síntese química , Proteínas de Fase Aguda/uso terapêutico , Animais , Proteínas de Transporte/síntese química , Proteínas de Transporte/uso terapêutico , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/química , Vetores Genéticos/química , Humanos , Lipocalina-2 , Lipocalinas/síntese química , Lipocalinas/uso terapêutico , Mutagênese Sítio-Dirigida/métodos , Plasmídeos/química , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas/síntese química , Proteínas Proto-Oncogênicas/uso terapêutico , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Especificidade por Substrato
20.
Methods Enzymol ; 503: 223-51, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22230571

RESUMO

Cystine-knot miniproteins, also known as knottins, contain a conserved core of three tightly woven disulfide bonds which impart extraordinary thermal and proteolytic stability. Interspersed between their conserved cysteine residues are constrained loops that possess high levels of sequence diversity among knottin family members. Together these attributes make knottins promising molecular scaffolds for protein engineering and translational applications. While naturally occurring knottins have shown potential as both diagnostic agents and therapeutics, protein engineering is playing an important and increasing role in creating designer molecules that bind to a myriad of biomedical targets. Toward this goal, rational and combinatorial approaches have been used to engineer knottins with novel molecular recognition properties. Here, methods are described for creating and screening knottin libraries using yeast surface display and fluorescence-activated cell sorting. Protocols are also provided for producing knottins by synthetic and recombinant methods, and for measuring the binding affinity of knottins to target proteins expressed on the cell surface.


Assuntos
Proteínas de Transporte/química , Miniproteínas Nó de Cistina/química , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Marcadores de Afinidade/química , Animais , Proteínas de Transporte/síntese química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Cisteína/química , Miniproteínas Nó de Cistina/síntese química , Miniproteínas Nó de Cistina/genética , Miniproteínas Nó de Cistina/isolamento & purificação , DNA/química , DNA/genética , Citometria de Fluxo , Corantes Fluorescentes/química , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Fases de Leitura Aberta , Pichia/química , Plasmídeos/química , Ligação Proteica , Dobramento de Proteína , Receptores de Superfície Celular/química , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Técnicas de Síntese em Fase Sólida , Especificidade por Substrato , Leveduras/química
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