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1.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064890

RESUMO

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the essential reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of chromene-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I, hCA II and the transmembrane hCA IX and XII. Several of the investigated derivatives showed interesting inhibition activity towards the tumor associate isoforms hCA IX and hCA XII. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.


Assuntos
Benzopiranos/química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/química , Sulfonamidas/farmacologia , Inibidores da Anidrase Carbônica/química , Domínio Catalítico , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/química
2.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34065004

RESUMO

Phosphinate pseudopeptide are analogs of peptides containing phosphinate moiety in a place of the amide bond. Due to this, the organophosphorus fragment resembles the tetrahedral transition state of the amide bond hydrolysis. Additionally, it is also capable of coordinating metal ions, for example, zinc or magnesium ions. These two properties of phosphinate pseudopeptides make them an ideal candidate for metal-related protease inhibitors. This research investigates the influence of additional residue in the P2 position on the inhibitory properties of phosphinopeptides. The synthetic strategy is proposed, based on retrosynthetic analysis. The N-C-P bond formation in the desired compounds is conveniently available from the three-component condensation of appropriate amino components, aldehydes, and hypophosphorous acid. One of the crucial synthetic steps is the careful selection of the protecting groups for all the functionals. Determination of the inhibitor activity of the obtained compounds has been done using UV-Vis spectroscopy and standard substrate L-Leu-p-nitroanilide toward the enzymes isolated from the porcine kidney (SsLAP, Sus scrofa Leucine aminopeptidase) and barley seeds (HvLAP, Hordeum vulgare Leucine aminopeptidase). An efficient procedure for the preparation of phosphinotripeptides has been performed. Activity test shown that introduction of additional residue into P2 position obtains the micromolar range inhibitors of SsLAP and HvLAP. Moreover, careful selection of the residue in the P2 position should improve its selectivity toward mammalian and plant leucyl aminopeptidases.


Assuntos
Inibidores Enzimáticos/farmacologia , Leucil Aminopeptidase/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Fosfinas/química , Animais , Inibidores Enzimáticos/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação Proteica , Suínos
3.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065957

RESUMO

The presented research concerns the triple activity of trans-cinnamic (tCA), ferulic (FA) and syringic acids (SA). They act as thyroid peroxidase (TPO) activators, lipoxygenase (LOX) inhibitors and show antiradical activity. All compounds showed a dose-dependent TPO activatory effect, thus the AC50 value (the concentration resulting in 50% activation) was determined. The tested compounds can be ranked as follows: tCA > FA > SA with AC50 = 0.10, 0.39, 0.69 mM, respectively. Strong synergism was found between FA and SA. The activatory effects of all tested compounds may result from interaction with the TPO allosteric site. It was proposed that conformational change resulting from activator binding to TPO allosteric pocket results from the flexibility of a nearby loop formed by residues Val352-Tyr363. All compounds act as uncompetitive LOX inhibitors. The most effective were tCA and SA, whereas the weakest was FA (IC50 = 0.009 mM and IC50 0.027 mM, respectively). In all cases, an interaction between the inhibitors carboxylic groups and side-chain atoms of Arg102 and Arg139 in an allosteric pocket of LOX was suggested. FA/tCA and FA/SA acted synergistically, whereas tCA/SA demonstrated antagonism. The highest antiradical activity was found in the case of SA (IC50 = 0.22 mM). FA/tCA and tCA/SA acted synergistically, whereas antagonism was found for the SA/FA mixture.


Assuntos
Autoantígenos/metabolismo , Ativadores de Enzimas/farmacologia , Iodeto Peroxidase/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Inibidores de Lipoxigenase/farmacologia , Compostos Fitoquímicos/farmacologia , Proteína-Lisina 6-Oxidase/metabolismo , Autoantígenos/química , Cinamatos/química , Cinamatos/farmacologia , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Relação Dose-Resposta a Droga , Ativadores de Enzimas/química , Ácido Gálico/análogos & derivados , Ácido Gálico/química , Ácido Gálico/farmacologia , Humanos , Concentração Inibidora 50 , Iodeto Peroxidase/química , Proteínas de Ligação ao Ferro/química , Inibidores de Lipoxigenase/química , Modelos Moleculares , Compostos Fitoquímicos/química , Proteína-Lisina 6-Oxidase/química , Relação Estrutura-Atividade
4.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066057

RESUMO

Poly (ADP-ribose) polymerases (PARP) 1-3 are well-known multi-domain enzymes, catalysing the covalent modification of proteins, DNA, and themselves. They attach mono- or poly-ADP-ribose to targets using NAD+ as a substrate. Poly-ADP-ribosylation (PARylation) is central to the important functions of PARP enzymes in the DNA damage response and nucleosome remodelling. Activation of PARP happens through DNA binding via zinc fingers and/or the WGR domain. Modulation of their activity using PARP inhibitors occupying the NAD+ binding site has proven successful in cancer therapies. For decades, studies set out to elucidate their full-length molecular structure and activation mechanism. In the last five years, significant advances have progressed the structural and functional understanding of PARP1-3, such as understanding allosteric activation via inter-domain contacts, how PARP senses damaged DNA in the crowded nucleus, and the complementary role of histone PARylation factor 1 in modulating the active site of PARP. Here, we review these advances together with the versatility of PARP domains involved in DNA binding, the targets and shape of PARylation and the role of PARPs in nucleosome remodelling.


Assuntos
Proteínas de Ciclo Celular/química , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerases/química , Regulação Alostérica/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Humanos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios Proteicos/efeitos dos fármacos
5.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067963

RESUMO

The effects that solid-liquid interfaces exert on the aggregation of proteins and peptides are of high relevance for various fields of basic and applied research, ranging from molecular biology and biomedicine to nanotechnology. While the influence of surface chemistry has received a lot of attention in this context, the role of surface topography has mostly been neglected so far. In this work, therefore, we investigate the aggregation of the type 2 diabetes-associated peptide hormone hIAPP in contact with flat and nanopatterned silicon oxide surfaces. The nanopatterned surfaces are produced by ion beam irradiation, resulting in well-defined anisotropic ripple patterns with heights and periodicities of about 1.5 and 30 nm, respectively. Using time-lapse atomic force microscopy, the morphology of the hIAPP aggregates is characterized quantitatively. Aggregation results in both amorphous aggregates and amyloid fibrils, with the presence of the nanopatterns leading to retarded fibrillization and stronger amorphous aggregation. This is attributed to structural differences in the amorphous aggregates formed at the nanopatterned surface, which result in a lower propensity for nucleating amyloid fibrillization. Our results demonstrate that nanoscale surface topography may modulate peptide and protein aggregation pathways in complex and intricate ways.


Assuntos
Agonistas dos Receptores da Amilina/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Nanoestruturas/química , Agregados Proteicos , Humanos , Modelos Moleculares , Propriedades de Superfície
6.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068879

RESUMO

The redox chemistry of copper(II) is strongly modulated by the coordination to amyloid-ß peptides and by the stability of the resulting complexes. Amino-terminal copper and nickel binding motifs (ATCUN) identified in truncated Aß sequences starting with Phe4 show very high affinity for copper(II) ions. Herein, we study the oxidase activity of [Cu-Aß4-x] and [Cu-Aß1-x] complexes toward dopamine and other catechols. The results show that the CuII-ATCUN site is not redox-inert; the reduction of the metal is induced by coordination of catechol to the metal and occurs through an inner sphere reaction. The generation of a ternary [CuII-Aß-catechol] species determines the efficiency of the oxidation, although the reaction rate is ruled by reoxidation of the CuI complex. In addition to the N-terminal coordination site, the two vicinal histidines, His13 and His14, provide a second Cu-binding motif. Catechol oxidation studies together with structural insight from the mixed dinuclear complexes Ni/Cu-Aß4-x reveal that the His-tandem is able to bind CuII ions independently of the ATCUN site, but the N-terminal metal complexation reduces the conformational mobility of the peptide chain, preventing the binding and oxidative reactivity toward catechol of CuII bound to the secondary site.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Complexos de Coordenação/metabolismo , Cobre/metabolismo , Dopamina/metabolismo , Oxirredutases/metabolismo , Peptídeos beta-Amiloides/química , Complexos de Coordenação/química , Cobre/química , Dopamina/química , Histidina/química , Histidina/metabolismo , Modelos Moleculares , Conformação Molecular , Oxirredução , Oxirredutases/química
7.
Nat Commun ; 12(1): 3266, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075032

RESUMO

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.


Assuntos
Infecções por Flavivirus/terapia , Flavivirus/ultraestrutura , Proteínas do Envelope Viral/ultraestrutura , Vírion/ultraestrutura , Aedes/virologia , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica , Dengue/terapia , Dengue/virologia , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/farmacologia , Desenho de Fármacos , Flavivirus/efeitos dos fármacos , Flavivirus/imunologia , Flavivirus/patogenicidade , Infecções por Flavivirus/virologia , Humanos , Mesocricetus , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Células Vero , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/farmacologia , Vacinas Virais/uso terapêutico , Vírion/efeitos dos fármacos , Vírion/metabolismo
8.
Nat Commun ; 12(1): 3214, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088904

RESUMO

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZCTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that previously reported in bacteria. Phylogenetic analyses of SepF and FtsZ from bacteria and archaea indicate that the two proteins may date back to the Last Universal Common Ancestor (LUCA), and we speculate that the archaeal mode of SepF/FtsZ interaction might reflect an ancestral feature. Our results provide insights into the mechanisms of archaeal cell division and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.


Assuntos
Proteínas Arqueais/metabolismo , Divisão Celular/fisiologia , Methanobrevibacter/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo Celular , Divisão Celular/genética , Sequência Conservada , Cristalografia por Raios X , Evolução Molecular , Methanobrevibacter/genética , Methanobrevibacter/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Filogenia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
9.
Viruses ; 13(6)2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071984

RESUMO

The high sequence identity of the first SARS-CoV-2 samples collected in December 2019 at Wuhan did not foretell the emergence of novel variants in the United Kingdom, North and South America, India, or South Africa that drive the current waves of the pandemic. The viral spike receptor possesses two surface areas of high mutagenic plasticity: the supersite in its N-terminal domain (NTD) that is recognised by all anti-NTD antibodies and its receptor binding domain (RBD) where 17 residues make contact with the human Ace2 protein (angiotensin I converting enzyme 2) and many neutralising antibodies bind. While NTD mutations appear at first glance very diverse, they converge on the structure of the supersite. The mutations within the RBD, on the other hand, hone in on only a small number of key sites (K417, L452, E484, N501) that are allosteric control points enabling spike to escape neutralising antibodies while maintaining or even gaining Ace2-binding activity. The D614G mutation is the hallmark of all variants, as it promotes viral spread by increasing the number of open spike protomers in the homo-trimeric receptor complex. This review discusses the recent spike mutations as well as their evolution.


Assuntos
COVID-19/virologia , Variação Genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Sítios de Ligação/genética , COVID-19/transmissão , Evolução Molecular , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química
10.
Viruses ; 13(6)2021 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072569

RESUMO

The COVID-19 pandemic, which began in Wuhan (Hubei, China), has been ongoing for about a year and a half. An unprecedented number of people around the world have been infected with SARS-CoV-2, the etiological agent of COVID-19. Despite the fact that the mortality rate for COVID-19 is relatively low, the total number of deaths has currently already reached more than three million and continues to increase due to high incidence. Since the beginning of the pandemic, a large number of sequences have been obtained and many genetic variants have been identified. Some of them bear significant mutations that affect biological properties of the virus. These genetic variants, currently Variants of Concern (VoC), include the so-called United Kingdom variant (20I/501Y), the Brazilian variant (20J/501Y.V3), and the South African variant (20H/501Y.V2). We describe here a novel SARS-CoV-2 variant with distinct spike protein mutations, first obtained at the end of January 2021 in northwest Russia. Therefore, it is necessary to pay attention to the dynamics of its spread among patients with COVID-19, as well as to study in detail its biological properties.


Assuntos
SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Análise Mutacional de DNA , DNA Complementar , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Filogenia , Conformação Proteica , Federação Russa , SARS-CoV-2/classificação , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química
11.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068835

RESUMO

Molecular modeling is widely utilized in subjects including but not limited to physics, chemistry, biology, materials science and engineering. Impressive progress has been made in development of theories, algorithms and software packages. To divide and conquer, and to cache intermediate results have been long standing principles in development of algorithms. Not surprisingly, most important methodological advancements in more than half century of molecular modeling are various implementations of these two fundamental principles. In the mainstream classical computational molecular science, tremendous efforts have been invested on two lines of algorithm development. The first is coarse graining, which is to represent multiple basic particles in higher resolution modeling as a single larger and softer particle in lower resolution counterpart, with resulting force fields of partial transferability at the expense of some information loss. The second is enhanced sampling, which realizes "dividing and conquering" and/or "caching" in configurational space with focus either on reaction coordinates and collective variables as in metadynamics and related algorithms, or on the transition matrix and state discretization as in Markov state models. For this line of algorithms, spatial resolution is maintained but results are not transferable. Deep learning has been utilized to realize more efficient and accurate ways of "dividing and conquering" and "caching" along these two lines of algorithmic research. We proposed and demonstrated the local free energy landscape approach, a new framework for classical computational molecular science. This framework is based on a third class of algorithm that facilitates molecular modeling through partially transferable in resolution "caching" of distributions for local clusters of molecular degrees of freedom. Differences, connections and potential interactions among these three algorithmic directions are discussed, with the hope to stimulate development of more elegant, efficient and reliable formulations and algorithms for "dividing and conquering" and "caching" in complex molecular systems.


Assuntos
Modelos Moleculares , Simulação de Dinâmica Molecular , Algoritmos , Termodinâmica
12.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072443

RESUMO

As an important zoonotic pathogen, Streptococcus suis (S. suis) infection has been reported to be a causative agent for variety of diseases in humans and animals, especially Streptococcal toxic shock-like syndrome (STSLS), which is commonly seen in cases of severe S. suis infection. STSLS is often accompanied by excessive production of inflammatory cytokines, which is the main cause of death. This calls for development of new strategies to avert the damage caused by STSLS. In this study, we found for the first time that Baicalein, combined with ampicillin, effectively improved severe S. suis infection. Further experiments demonstrated that baicalein significantly inhibited the hemolytic activity of SLY by directly binding to SLY and destroying its secondary structure. Cell-based assays revealed that Baicalein did not exert toxic effects and conferred protection in S. suis-infected cells. Interestingly, compared with ampicillin alone, Baicalein combined with ampicillin resulted in a higher survival rate in mice severely infected with S. suis. At the same time, we found that baicalein can be combined with meropenem against MRSA. In conclusion, these results indicate that baicalein has a good application prospect.


Assuntos
Antibacterianos/farmacologia , Flavanonas/farmacologia , Infecções Estreptocócicas/microbiologia , Streptococcus suis/efeitos dos fármacos , Animais , Antibacterianos/química , Citocinas/biossíntese , Modelos Animais de Doenças , Farmacorresistência Bacteriana/efeitos dos fármacos , Quimioterapia Combinada , Flavanonas/química , Hemólise/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/patologia , Relação Estrutura-Atividade
13.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072560

RESUMO

The synthesis of new phenothiazine derivatives, analogs of Methylene Blue, is of particular interest in the design of new drugs, as well as in the development of a new generation of agents for photodynamic therapy. In this study, two new derivatives of phenothiazine, i.e., 3,7-bis(4-aminophenylamino)phenothiazin-5-ium chloride dihydrochloride (PTZ1) and 3,7-bis(4-sulfophenylamino)phenothiazin-5-ium chloride (PTZ2), are synthesized for the first time and characterized by NMR, IR spectroscopy, HRMS and elemental analysis. The interaction of the obtained compounds PTZ1 and PTZ2 with salmon sperm DNA is investigated. It is shown by UV-Vis spectroscopy and DFT calculations that substituents in arylamine fragments play a crucial role in dimer formation and interaction with DNA. In the case of PTZ1, two amine groups promote H-aggregate formation and DNA interactions through groove binding and intercalation. In the case of PTZ2, sulfanilic acid fragments prevent any dimer formation and DNA binding due to electrostatic repulsion. DNA interaction mechanisms are studied and confirmed by UV-vis and fluorescence spectroscopy in comparison with Methylene Blue. The obtained results open significant opportunities for the development of new drugs and photodynamic agents.


Assuntos
Aminas/química , Aminas/farmacologia , Azul de Metileno/química , Azul de Metileno/farmacologia , Aminas/síntese química , DNA/química , Dimerização , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Espectroscopia de Ressonância Magnética , Azul de Metileno/síntese química , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Espectrometria de Fluorescência , Relação Estrutura-Atividade
14.
Science ; 372(6546)2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34083463

RESUMO

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRß-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno de Histocompatibilidade H-2D/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Infecções por Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Complexo CD3/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Epitopos de Linfócito T , Feminino , Antígeno de Histocompatibilidade H-2D/química , Antígeno de Histocompatibilidade H-2D/imunologia , Vírus da Influenza A , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transdução de Sinais
15.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070174

RESUMO

This review focuses on the molecular chaperone ClpB that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and its biological function in selected bacterial pathogens, causing a variety of human infectious diseases, including zoonoses. It has been established that ClpB disaggregates and reactivates aggregated cellular proteins. It has been postulated that ClpB's protein disaggregation activity supports the survival of pathogenic bacteria under host-induced stresses (e.g., high temperature and oxidative stress), which allows them to rapidly adapt to the human host and establish infection. Interestingly, ClpB may also perform other functions in pathogenic bacteria, which are required for their virulence. Since ClpB is not found in human cells, this chaperone emerges as an attractive target for novel antimicrobial therapies in combating bacterial infections.


Assuntos
Endopeptidase Clp/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , ATPases Associadas a Diversas Atividades Celulares/fisiologia , Animais , Bactérias/patogenicidade , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/etiologia , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/fisiologia , Zoonoses Bacterianas/etiologia , Endopeptidase Clp/química , Proteínas de Choque Térmico/fisiologia , Humanos , Modelos Moleculares , Conformação Proteica , Virulência/fisiologia
16.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070225

RESUMO

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Peptidoglicano/química , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Quaternária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética
17.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
18.
Nat Commun ; 12(1): 3305, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083522

RESUMO

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química
19.
Science ; 372(6547): 1149-1150, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112680
20.
Nat Commun ; 12(1): 3456, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103492

RESUMO

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.


Assuntos
Proteínas de Transporte/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Dedos de Zinco , Sítios de Ligação , Proteínas de Transporte/química , Sobrevivência Celular , Cristalografia por Raios X , Análise Mutacional de DNA , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Schizosaccharomyces/citologia
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