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1.
Chem Commun (Camb) ; 55(59): 8556-8559, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31271173

RESUMO

Non-hydrated organic solutions of a diphenylalanine amphiphile blocked at the C-terminus with a fluorenylmethyl ester and stabilized at the N-terminus with a trifluoroacetate have been used to prepare amyloid fibrils. The solvent used to prepare the stock solution together with the co-solvent added enables regulation of the characteristics of the fibrils, which is important for their use in technological applications.


Assuntos
Amiloide/síntese química , Proteínas Amiloidogênicas/química , Dipeptídeos/química , Tensoativos/química , Teoria da Densidade Funcional , Dimetilformamida/química , Metanol/química , Propanóis/química , Conformação Proteica em Folha beta , Multimerização Proteica , Teoria Quântica
2.
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
3.
Chem Commun (Camb) ; 55(59): 8564-8566, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31271158

RESUMO

Diphenylalanine (FF), as the smallest unit and core recognition motif of ß-amyloid (Aß), could self-assemble into nanofibers, which induces an early onset of Alzheimer's disease (AD). Green/near-infrared fluorescent BODIPY probes were designed and synthesized to detect FF-assembly, providing unique insights into the chemical and molecular mechanism of Aß aggregation and drug development for AD.


Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Fenilalanina/análogos & derivados , Técnicas Biossensoriais/métodos , Compostos de Boro/síntese química , Corantes Fluorescentes/síntese química , Nanofibras/química , Fenilalanina/análise , Fenilalanina/química , Multimerização Proteica
4.
J Chem Theory Comput ; 15(8): 4673-4686, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31265271

RESUMO

The time step of atomistic molecular dynamics (MD) simulations is determined by the fastest motions in the system and is typically limited to 2 fs. An increasingly popular approach is to increase the mass of the hydrogen atoms to ∼3 amu and decrease the mass of the parent atom by an equivalent amount. This approach, known as hydrogen-mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simulation stability. While HMR has been applied in many published studies to date, it has not been extensively tested for membrane-containing systems. Here, we compare the results of simulations of a variety of membranes and membrane-protein systems run using a 2 fs time step and a 4 fs time step with HMR. For pure membrane systems, we find almost no difference in structural properties, such as area-per-lipid, electron density profiles, and order parameters, although there are differences in kinetic properties such as the diffusion constant. Conductance through a porin in an applied field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane mixing show very little dependence on HMR and the time step. We also tested a 9 Å cutoff as compared to the standard CHARMM cutoff of 12 Å, finding significant deviations in many properties tested. We conclude that HMR is a valid approach for membrane systems, but a 9 Å cutoff is not.


Assuntos
Membrana Celular/química , Hidrogênio/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Difusão , Glicoforina/química , Humanos , Movimento (Física) , Peptídeos/química , Fosfatidilcolinas/química , Multimerização Proteica , Receptores Acoplados a Proteínas-G/química , Termodinâmica
5.
Nat Commun ; 10(1): 2765, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235691

RESUMO

G protein-coupled receptors (GPCRs) can integrate extracellular signals via allosteric interactions within dimers and higher-order oligomers. However, the structural bases of these interactions remain unclear. Here, we use the GABAB receptor heterodimer as a model as it forms large complexes in the brain. It is subjected to genetic mutations mainly affecting transmembrane 6 (TM6) and involved in human diseases. By cross-linking, we identify the transmembrane interfaces involved in GABAB1-GABAB2, as well as GABAB1-GABAB1 interactions. Our data are consistent with an oligomer made of a row of GABAB1. We bring evidence that agonist activation induces a concerted rearrangement of the various interfaces. While the GB1-GB2 interface is proposed to involve TM5 in the inactive state, cross-linking of TM6s lead to constitutive activity. These data bring insight for our understanding of the allosteric interaction between GPCRs within oligomers.


Assuntos
Modelos Moleculares , Domínios Proteicos/fisiologia , Multimerização Proteica/fisiologia , Receptores de GABA-B/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/fisiologia , Reagentes para Ligações Cruzadas/química , Agonistas dos Receptores de GABA-B/farmacologia , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Domínios Proteicos/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo
6.
Nat Chem ; 11(7): 605-614, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209296

RESUMO

Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns in fractal-shaped branched objects, such as trees, lungs and sponges, results in a high surface area to volume ratio, which provides key functional advantages including molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies could provide access to functional biomaterials. Here we describe a computational design approach for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Guided by atomic-resolution models, we develop fusions of Src homology 2 (SH2) domain or a phosphorylatable SH2-binding peptide, respectively, to two symmetric, homo-oligomeric proteins. Mixing the two designed components resulted in a variety of dendritic, hyperbranched and sponge-like topologies that are phosphorylation-dependent and self-similar over three decades (~10 nm-10 µm) of length scale, in agreement with models from multiscale computational simulations. Designed assemblies perform efficient phosphorylation-dependent capture and release of cargo proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Fractais , Agregados Proteicos , Proteínas Recombinantes de Fusão/metabolismo , Algoritmos , Proteínas de Bactérias/genética , Escherichia coli/química , Humanos , Modelos Químicos , Modelos Moleculares , Fosforilação , Engenharia de Proteínas/métodos , Multimerização Proteica , Proteínas Recombinantes de Fusão/genética , Domínios de Homologia de src/genética , Quinases da Família src/metabolismo
7.
Chem Commun (Camb) ; 55(54): 7752-7755, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31204733

RESUMO

Metal-binding peptides are versatile building blocks in supramolecular chemistry. We recently reported a class of crystalline materials formed through a combination of coiled-coil peptide self-association and metal coordination. Here, we probe the serendipitously discovered metal binding motif that drives the assembly and apply these insights to exert rational control over structure and morphology in the materials.


Assuntos
Metaloproteínas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Cobre/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Metaloproteínas/síntese química , Engenharia de Proteínas/métodos , Multimerização Proteica , Piridinas/química
8.
Inorg Chem ; 58(13): 8800-8819, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247881

RESUMO

Very few inorganic antineoplastic drugs have entered the clinic in the last decades, mainly because of toxicity issues. Because copper is an essential trace element of ubiquitous occurrence, decreased side effects could be expected in comparison with the widely used platinum anticancer compounds. In the present work, two novel hydrazonic binucleating ligands and their µ-hydroxo dicopper(II) complexes were prepared and fully characterized. They differ by the nature of the aromatic group present in their aroylhydrazone moieties: while H3L1 and its complex, 1, possess a thiophene ring, H3L2 and 2 contain the more polar furan heterocycle. X-ray diffraction indicates that both coordination compounds are very similar in structural terms and generate dimeric arrangements in the solid state. Positive-ion electrospray ionization mass spectrometry analyses confirmed that the main species present in a 10% dimethyl sulfoxide (DMSO)/water solution should be [Cu2(HL)(OH)]+ and the DMSO-substituted derivative [Cu2(L)(DMSO)]+. Scattering techniques [dynamic light scattering (DLS) and small-angle X-ray scattering] suggest that the complexes and their free ligands interact with bovine serum albumin (BSA) in a reversible manner. The binding constants to BSA were determined for the complexes through fluorescence spectroscopy. Moreover, to gain insight into the mechanism of action of the compounds, calf thymus DNA binding studies by UV-visible and DLS measurements using plasmid pBR322 DNA were also performed. For the complexes, DLS data seem to point to the occurrence of DNA cleavage to Form III (linear). Both ligands and their dicopper(II) complexes display potent antiproliferative activity in a panel of four cancer cell lines, occasionally even in the submicromolar range, with the complexes being more potent than the free ligands. Our data on cellular models correlate quite well with the DNA interaction experiments. The results presented herein show that aroylhydrazone-derived binucleating ligands, as well as their dinuclear µ-hydroxodicopper(II) complexes, may represent a promising structural starting point for the development of a new generation of highly active potential antitumor agents.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Hidrazonas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Bovinos , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Cobre/química , DNA/química , Clivagem do DNA/efeitos dos fármacos , Cães , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Hidrazonas/toxicidade , Isomerismo , Ligantes , Células Madin Darby de Rim Canino , Camundongos , Plasmídeos/química , Multimerização Proteica/efeitos dos fármacos , Soroalbumina Bovina/metabolismo
9.
Nat Commun ; 10(1): 2532, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182717

RESUMO

Targeted inhibition of the ERK-MAPK pathway, upregulated in a majority of human cancers, has been hindered in the clinic by drug resistance and toxicity. The MRAS-SHOC2-PP1 (SHOC2 phosphatase) complex plays a key role in RAF-ERK pathway activation by dephosphorylating a critical inhibitory site on RAF kinases. Here we show that genetic inhibition of SHOC2 suppresses tumorigenic growth in a subset of KRAS-mutant NSCLC cell lines and prominently inhibits tumour development in autochthonous murine KRAS-driven lung cancer models. On the other hand, systemic SHOC2 ablation in adult mice is relatively well tolerated. Furthermore, we show that SHOC2 deletion selectively sensitizes KRAS- and EGFR-mutant NSCLC cells to MEK inhibitors. Mechanistically, SHOC2 deletion prevents MEKi-induced RAF dimerization, leading to more potent and durable ERK pathway suppression that promotes BIM-dependent apoptosis. These results present a rationale for the generation of SHOC2 phosphatase targeted therapies, both as a monotherapy and to widen the therapeutic index of MEK inhibitors.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Quinases raf/metabolismo , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos Knockout , Camundongos Nus , Mutação , Transplante de Neoplasias , Multimerização Proteica , Quinases raf/antagonistas & inibidores , Quinases raf/genética , Proteínas ras/metabolismo
10.
Eur J Med Chem ; 177: 247-258, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158742

RESUMO

Alzheimer's disease (AD) is a chronic, fatal and complex neurodegenerative disorder, which is characterized by cholinergic system dysregulation, metal dyshomeostasis, amyloid-ß (Aß) aggregation, etc. Therefore in most cases, single-target or single-functional agents are insufficient to achieve the desirable effect against AD. Multi-Target-Directed Ligand (MTDL), which is rationally designed to simultaneously hit multiple targets to improve the pharmacological profiles, has been developed as a promising approach for drug discovery against AD. To identify the multifunctional agents for AD, we developed an innovative method to successfully conceal the metal chelator into acetylcholinesterase (AChE) inhibitor. Briefly, the "hidden" agents first cross the Blood Brain Barrier (BBB) to inhibit the function of AChE, and the metal chelator will then be released via the enzymatic hydrolysis by AChE. Therefore, the AChE inhibitor, in this case, is not only a single-target agent against AD, but also a carrier of the metal chelator. In this study a total of 14 quinoline derivatives were synthesized and biologically evaluated. Both in vitro and in vivo results demonstrated that compound 9b could cross the BBB efficiently, then release 8a, the metabolite of 9b, into brain. In vitro, 9b had a potent AChE inhibitory activity, while 8a displayed a significant metal ion chelating function, therefore in combination, both 9b and 8a exhibited a considerable inhibition of Aß aggregation, one of the observations that plays important roles in the pathogenesis of AD. The efficacy of 9b against AD was further investigated in both a zebrafish model and two different mice models.


Assuntos
Quelantes/farmacologia , Inibidores da Colinesterase/farmacologia , Nootrópicos/farmacologia , Quinolinas/farmacologia , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Peptídeos beta-Amiloides/química , Animais , Barreira Hematoencefálica/metabolismo , Quelantes/síntese química , Quelantes/farmacocinética , Quelantes/toxicidade , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/farmacocinética , Inibidores da Colinesterase/toxicidade , Desenho de Drogas , Canal de Potássio ERG1/antagonistas & inibidores , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Nootrópicos/síntese química , Nootrópicos/farmacocinética , Nootrópicos/toxicidade , Fragmentos de Peptídeos/química , Multimerização Proteica/efeitos dos fármacos , Quinolinas/síntese química , Quinolinas/farmacocinética , Quinolinas/toxicidade , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Peixe-Zebra
11.
Eur J Med Chem ; 177: 414-424, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158754

RESUMO

Due to the role of butyrylcholinesterase (BChE) in acetylcholine hydrolysis in the late stages of the Alzheimer's disease (AD), inhibitors of butyrylcholinesterase (BChE) have been recently envisaged, besides acetylcholinesterase (AChE) inhibitors, as candidates for treating mild-to-moderate AD. Herein, synthesis and AChE/BChE inhibition activity of some twenty derivatives of 1,2,3,4,5,6-hexahydroazepino[4,3-b]indole (HHAI) is reported. Most of the newly synthesized HHAI derivatives achieved the inhibition of both ChE isoforms with IC50s in the micromolar range, with a structure-dependent selectivity toward BChE. Apparently, molecular volume and lipophilicity do increase selectivity toward BChE, and indeed the N2-(4-phenylbutyl) HHAI derivative 15d, which behaves as a mixed-type inhibitor, resulted the most potent (IC50 0.17 µM) and selective (>100-fold) inhibitor toward either horse serum and human BChE. Moreover, 15d inhibited in vitro self-induced aggregation of neurotoxic amyloid-ß (Aß) peptide and displayed neuroprotective effects in neuroblastoma SH-SY5Y cell line, significantly recovering (P < 0.001) cell viability when impaired by Aß1-42 and hydrogen peroxide insults. Overall, this study highlighted HHAI as useful and versatile scaffold for developing new small molecules targeting some enzymes and biochemical pathways involved in the pathogenesis of AD.


Assuntos
Azepinas/farmacologia , Inibidores da Colinesterase/farmacologia , Indóis/farmacologia , Fármacos Neuroprotetores/farmacologia , Peptídeos beta-Amiloides/metabolismo , Azepinas/síntese química , Azepinas/química , Azepinas/metabolismo , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Relação Dose-Resposta a Droga , Desenho de Drogas , Depuradores de Radicais Livres/síntese química , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/metabolismo , Depuradores de Radicais Livres/farmacologia , Humanos , Indóis/síntese química , Indóis/química , Indóis/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Relação Estrutura-Atividade
12.
J Chem Theory Comput ; 15(8): 4318-4331, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31241940

RESUMO

The relative prevalence of native protein-protein interactions (PPIs) are the cornerstone for understanding the structure, dynamics and mechanisms of function of protein complexes. In this study, we develop a scheme for scaling the protein-water interaction in the CHARMM36 force field, in order to better fit the solvation free energy of amino acids side-chain analogues. We find that the molecular dynamics simulation with the scaled force field, CHARMM36s, as well as a recently released version, CHARMM36m, effectively improve on the overly sticky association of proteins, such as ubiquitin. We investigate the formation of a heterodimer protein complex between the SAM domains of the EphA2 receptor and the SHIP2 enzyme by performing a combined total of 48 µs simulations with the different potential functions. While the native SAM heterodimer is only predicted at a low rate of 6.7% with the original CHARMM36 force field, the yield is increased to 16.7% with CHARMM36s, and to 18.3% with CHARMM36m. By analyzing the 25 native SAM complexes formed in the simulations, we find that their formation involves a preorientation guided by Coulomb interactions, consistent with an electrostatic steering mechanism. In 12 cases, the complex could directly transform to the native protein interaction surfaces with only small adjustments in domain orientation. In the other 13 cases, orientational and/or translational adjustments are needed to reach the native complex. Although the tendency for non-native complexes to dissociate has nearly doubled with the modified potential functions, a dissociation followed by a reassociation to the correct complex structure is still rare. Instead, the remaining non-native complexes undergo configurational changes/surface searching, which, however, rarely leads to native structures on a time scale of 250 ns. These observations provide a rich picture of the mechanisms of protein-protein complex formation and suggest that computational predictions of native complex PPIs could be improved further.


Assuntos
Mapas de Interação de Proteínas , Proteínas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas/química , Receptor EphA2/química , Receptor EphA2/metabolismo , Eletricidade Estática , Termodinâmica , Ubiquitina/química , Ubiquitina/metabolismo , Água/metabolismo
13.
Phys Chem Chem Phys ; 21(24): 12806-12817, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31165827

RESUMO

We present a study of the combined effects of natural cosolvents (TMAO, glycine, urea) and pressure on the activity of the tetrameric enzyme lactate dehydrogenase (LDH). To this end, high-pressure stopped-flow methodology in concert with fast UV/Vis spectroscopic detection of product formation was applied. To reveal possible pressure effects on the stability and dynamics of the enzyme, FTIR spectroscopic and neutron scattering measurements were carried out. In neat buffer solution, the catalytic turnover number of the enzyme, kcat, increases up to 1000 bar, the pressure range where dissociation of the tetrameric species to dimers sets in. Accordingly, we obtain a negative activation volume, ΔV# = -45.3 mL mol-1. Further, the enzyme substrate complex has a larger volume compared to the enzyme and substrate in the unbound state. The neutron scattering data show that changes in the fast internal dynamics of the enzyme are not responsible for the increase of kcat upon compression. Whereas the magnitude of kcat is similar in the presence of the osmolytes, the pressure of deactivation is modulated by the addition of cosolvents. TMAO and glycine increase the pressure of deactivation, and in accordance with the observed stabilizing effect both cosolvents exhibit against denaturation and/or dissociation of proteins. While urea does not markedly affect the magnitude of the Michaelis constant, KM, both 1 M TMAO and 1 M glycine exhibit smaller KM values of about 0.07 mM and 0.05 mM below about 1 kbar. Such positive effect on the substrate affinity could be rationalized by the effect the two cosolutes impose on the thermodynamic activities of the reactants, which reflect changes in water-mediated intermolecular interactions. Our data show that the intracellular milieu, i.e., the solution conditions that have evolved, may be sufficient to maintain enzymatic activity under extreme environmental conditions, including the whole pressure range encountered on Earth.


Assuntos
L-Lactato Desidrogenase/química , Solventes/química , Glicina/química , Cinética , Metilaminas/química , Modelos Moleculares , Pressão , Desnaturação Proteica , Dobramento de Proteína , Multimerização Proteica , Termodinâmica , Ureia/química , Água/química
14.
Nat Commun ; 10(1): 2607, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197133

RESUMO

Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.


Assuntos
Sítio Alostérico/efeitos dos fármacos , Antineoplásicos/farmacologia , Desenho de Drogas , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Repetição de Anquirina , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Histidina/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/genética , Neoplasias/patologia , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
16.
J Chem Phys ; 150(22): 225102, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31202237

RESUMO

A majority of cellular proteins function as part of multimeric complexes of two or more subunits. Multimer formation requires interactions between protein surfaces that lead to closed structures, such as dimers and tetramers. If proteins interact in an open-ended way, uncontrolled growth of fibrils can occur, which is likely to be detrimental in most cases. We present a statistical physics model that allows aggregation of proteins as either closed dimers or open fibrils of all lengths. We use pairwise amino-acid contact energies to calculate the energies of interacting protein surfaces. The probabilities of all possible aggregate configurations can be calculated for any given sequence of surface amino acids. We link the statistical physics model to a population genetics model that describes the evolution of the surface residues. When proteins evolve neutrally, without selection for or against multimer formation, we find that a majority of proteins remain as monomers at moderate concentrations, but strong dimer-forming or fibril-forming sequences are also possible. If selection is applied in favor of dimers or in favor of fibrils, then it is easy to select either dimer-forming or fibril-forming sequences. It is also possible to select for oriented fibrils with protein subunits all aligned in the same direction. We measure the propensities of amino acids to occur at interfaces relative to noninteracting surfaces and show that the propensities in our model are strongly correlated with those that have been measured in real protein structures. We also show that there are significant differences between amino acid frequencies at isologous and heterologous interfaces in our model, and we observe that similar effects occur in real protein structures.


Assuntos
Evolução Molecular , Modelos Biológicos , Agregados Proteicos , Multimerização Proteica , Proteínas/química , Aminoácidos/química , Cadeias de Markov , Método de Monte Carlo , Termodinâmica
17.
J Chem Phys ; 150(22): 225101, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31202253

RESUMO

Understanding the key factors that govern the rate of protein aggregation is of immense interest since protein aggregation is associated with a number of neurodegenerative diseases. Previous experimental and theoretical studies have revealed that the hydrophobicity, charge, and population of the fibril-prone monomeric state control the fibril formation rate. Because the fibril structures consist of cross beta sheets, it is widely believed that those sequences that have a high beta content (ß) in the monomeric state should have high aggregation rates as the monomer can serve as a template for fibril growth. However, this important fact has never been explicitly proven, motivating us to carry out this study. Using replica exchange molecular dynamics simulation with implicit water, we have computed ß of 19 mutations of amyloid beta peptide of 42 residues (Aß42) for which the aggregation rate κ has been measured experimentally. We have found that κ depends on ß in such a way that the higher the propensity to aggregation, the higher the beta content in the monomeric state. Thus, we have solved a long-standing problem of the dependence of fibril formation time of the ß-structure on a quantitative level.


Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Multimerização Proteica , Peptídeos beta-Amiloides/genética , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutação , Fragmentos de Peptídeos/genética , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Termodinâmica
18.
Phys Chem Chem Phys ; 21(25): 13545-13554, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31172995

RESUMO

Human aldehyde oxidase (hAOX1) is a molybdenum dependent enzyme that plays an important role in the metabolism of various compounds either endogenous or xenobiotics. Due to its promiscuity, hAOX1 plays a major role in the pharmacokinetics of many drugs and therefore has gathered a lot of attention from the scientific community and, particularly, from the pharmaceutical industry. In this work, homology modelling, molecular docking and molecular dynamics simulations were used to study the structure of the monomer and dimer of human AOX. The results with the monomer of hAOX1 allowed to shed some light on the role played by thioridazine and two malonate ions that are co-crystalized in the recent X-ray structure of hAOX1. The results show that these molecules endorse several conformational rearrangements in the binding pocket of the enzyme and these changes have an impact in the active site topology as well as in the stability of the substrate (phthalazine). The results show that the presence of both molecules open two gates located at the entrance of the binding pocket, from which results the flooding of the active site. They also endorse several modifications in the shape of the binding pocket (namely the position of Lys893) that, together with the presence of the solvent molecules, favour the release of the substrate to the solvent. Further insights were also obtained with the assembled homodimer of hAOX1. The allosteric inhibitor (THI) binds closely to the region where the dimerization of both monomers occur. These findings suggest that THI can interfere with protein dimerization.


Assuntos
Aldeído Oxidase/química , Domínio Catalítico , Cristalização , Humanos , Cinética , Malonatos/química , Modelos Moleculares , Ftalazinas/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Solventes , Tioridazina/química
19.
Soft Matter ; 15(21): 4326-4333, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31070654

RESUMO

A persistent problem in the studies of membrane-active peptides, including antimicrobial peptides and pathogenic amyloidal peptides, is the lack of methods for investigating their molecular configurations in membranes. These peptides spontaneously bind to membranes from solutions, and often form oligomers that induce changes of membrane permeability. For antimicrobials, such actions appear to relate to the antimicrobial mechanisms, but for amyloidal peptides, the oligomerization has been linked to neurodegenerative diseases. In many cases, no further understanding of such oligomerization has been achieved due to the lack of structural information. In this article, we will demonstrate a method of trapping such peptide oligomers in a rhombohedral (R) phase of lipid so that the oligomers can be subjected to 3D diffraction analysis. The conditions for forming the R phase and the electron density distribution in the rhombohedral unit cell provide information about peptide-lipid interactions and the molecular size of the trapped oligomer. Such information cannot be obtained from membranes in the planar configuration. For illustration, we apply this method to daptomycin, an FDA-approved antibiotic that attacks membranes containing phosphatidylglycerol, in the presence of calcium ions. We have successfully used the brominated phosphatidylglycerol to perform bromine-atom anomalous diffraction in the rhombohedral phase containing daptomycin and calcium ions. The preliminary results apparently exhibit diffraction data related to daptomycin oligomers. We believe that this method will also be applicable to the difficult problems related to amyloidal peptides, such as amyloid beta of Alzheimer's disease.


Assuntos
Membrana Celular/química , Daptomicina/química , Multimerização Proteica , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Daptomicina/metabolismo , Estrutura Quaternária de Proteína , Água/química
20.
Nat Commun ; 10(1): 2320, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127104

RESUMO

The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration. Inhibition of Hh receptor Patched (Ptch) by the Hh ligands relieves suppression of signaling cascades. Here, we report the cryo-EM structure of tetrameric Ptch1 in complex with the palmitoylated N-terminal signaling domain of human Sonic hedgehog (ShhNp) at a 4:2 stoichiometric ratio. The structure shows that four Ptch1 protomers are organized as a loose dimer of dimers. Each dimer binds to one ShhNp through two distinct inhibitory interfaces, one mainly through the N-terminal peptide and the palmitoyl moiety of ShhNp and the other through the Ca2+-mediated interface on ShhNp. Map comparison reveals that the cholesteryl moiety of native ShhN occupies a recently identified extracellular steroid binding pocket in Ptch1. Our structure elucidates the tetrameric assembly of Ptch1 and suggests an asymmetric mode of action of the Hh ligands for inhibiting the potential cholesterol transport activity of Ptch1.


Assuntos
Proteínas Hedgehog/ultraestrutura , Receptor Patched-1/ultraestrutura , Domínios Proteicos , Colesterol/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Proteínas Hedgehog/química , Proteínas Hedgehog/isolamento & purificação , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Lipoilação , Modelos Moleculares , Receptor Patched-1/isolamento & purificação , Receptor Patched-1/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
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