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1.
Nat Commun ; 11(1): 4955, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009385

RESUMO

The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960 nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82 Å resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the αß-polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the αß-polypeptides that are hydrogen-bonded to BChl a. The Ca2+-facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of α- and ß-polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside.


Assuntos
Cálcio/metabolismo , Microscopia Crioeletrônica , Complexos de Proteínas Captadores de Luz/ultraestrutura , Peptídeos/metabolismo , Fotossíntese , Sequência de Aminoácidos , Bacterioclorofila A/metabolismo , Sítios de Ligação , Chromatiaceae/metabolismo , Detergentes/química , Dimerização , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Lipídeos/química , Peptídeos/química , Quinonas/química
2.
Sci Signal ; 13(651)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994211

RESUMO

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/química , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Virais/genética , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Fases de Leitura Aberta/genética , Pandemias , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
3.
PLoS One ; 15(9): e0239403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946527

RESUMO

Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.


Assuntos
Betacoronavirus/genética , Primers do DNA/normas , Genoma Viral/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Sequenciamento Completo do Genoma/métodos , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/diagnóstico , Primers do DNA/metabolismo , Dimerização , Amplificação de Genes , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Carga Viral
4.
EBioMedicine ; 59: 102980, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32862101

RESUMO

BACKGROUND: Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease as well as Lou Gehrig's disease, is a progressive neurological disorder selectively affecting motor neurons with no currently known cure. Around 20% of the familial ALS cases arise from dominant mutations in the sod1 gene encoding superoxide dismutase1 (SOD1) enzyme. Aggregation of mutant SOD1 in familial cases and of wild-type SOD1 in at least some sporadic ALS cases is one of the known causes of the disease. Riluzole, approved in 1995 and edaravone in 2017 remain the only drugs with limited therapeutic benefits. METHODS: We have utilised the ebselen template to develop novel compounds that redeem stability of mutant SOD1 dimer and prevent aggregation. Binding modes of compounds have been visualised by crystallography. In vitro neuroprotection and toxicity of lead compounds have been performed in mouse neuronal cells and disease onset delay of ebselen has been demonstrated in transgenic ALS mice model. FINDING: We have developed a number of ebselen-based compounds with improvements in A4V SOD1 stabilisation and in vitro therapeutic effects with significantly better potency than edaravone. Structure-activity relationship of hits has been guided by high resolution structures of ligand-bound A4V SOD1. We also show clear disease onset delay of ebselen in transgenic ALS mice model holding encouraging promise for potential therapeutic compounds. INTERPRETATION: Our finding established the new generation of organo-selenium compounds with better in vitro neuroprotective activity than edaravone. The potential of this class of compounds may offer an alternative therapeutic agent for ALS treatment. The ability of these compounds to target cysteine 111 in SOD may have wider therapeutic applications targeting cysteines of enzymes involved in pathogenic and viral diseases including main protease of SARS-Cov-2 (COVID-19). FUNDING: Project funding was supported by the ALS Association grant (WA1128) and Fostering Joint International Research (19KK0214) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Compostos Organosselênicos/uso terapêutico , Superóxido Dismutase-1/metabolismo , Esclerose Amiotrófica Lateral/mortalidade , Esclerose Amiotrófica Lateral/patologia , Animais , Azóis/química , Azóis/metabolismo , Azóis/uso terapêutico , Betacoronavirus/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Dimerização , Modelos Animais de Doenças , Estabilidade Enzimática , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Compostos Organosselênicos/química , Compostos Organosselênicos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Superóxido Dismutase-1/genética , Taxa de Sobrevida , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo
5.
Comput Biol Med ; 122: 103848, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658735

RESUMO

The recent outbreak of coronavirus disease-19 (COVID-19) continues to drastically affect healthcare throughout the world. To date, no approved treatment regimen or vaccine is available to effectively attenuate or prevent the infection. Therefore, collective and multidisciplinary efforts are needed to identify new therapeutics or to explore effectiveness of existing drugs and drug-like small molecules against SARS-CoV-2 for lead identification and repurposing prospects. This study addresses the identification of small molecules that specifically bind to any of the three essential proteins (RdRp, 3CL-protease and helicase) of SARS-CoV-2. By applying computational approaches we screened a library of 4574 compounds also containing FDA-approved drugs against these viral proteins. Shortlisted hits from initial screening were subjected to iterative docking with the respective proteins. Ranking score on the basis of binding energy, clustering score, shape complementarity and functional significance of the binding pocket was applied to identify the binding compounds. Finally, to minimize chances of false positives, we performed docking of the identified molecules with 100 irrelevant proteins of diverse classes thereby ruling out the non-specific binding. Three FDA-approved drugs showed binding to 3CL-protease either at the catalytic pocket or at an allosteric site related to functionally important dimer formation. A drug-like molecule showed binding to RdRp in its catalytic pocket blocking the key catalytic residues. Two other drug-like molecules showed specific interactions with helicase at a key domain involved in catalysis. This study provides lead drugs or drug-like molecules for further in vitro and clinical investigation for drug repurposing and new drug development prospects.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Domínio Catalítico , Simulação por Computador , Dimerização , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Pandemias , Inibidores de Proteases/química , Quinoxalinas/farmacologia , Rimantadina/farmacologia , Proteínas Virais/química
6.
J Phys Chem Lett ; 11(14): 5661-5667, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32536162

RESUMO

Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible for the global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting the SARS viruses to bypass the host protective response. This work also constitutes a first step in the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus.


Assuntos
Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Quadruplex G/efeitos dos fármacos , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/genética , Betacoronavirus/efeitos dos fármacos , Dimerização , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Pandemias , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
7.
Toxicon ; 184: 48-54, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32473923

RESUMO

Many venomous species, including snakes, bees and scorpions, contain a variety of secreted phospholipases A2 (sPLA2) that contribute to prey digestion and venom toxicity. Based on their primary structures, the different venom sPLA2 have been classified into four groups I, II, III and IX. While the structure of sPLA2 groups II and I has been well characterized, only one crystal structure of group III sPLA2 from bee venom was described. Scorpion venom sPLA2 belong to group III with a particular heterodimeric structure composed of a long enzymatic chain linked by a disulfide bridge to a Short one after the release of five residues (penta-peptide) during the maturation processes. The function of the Short chain is still not clear. Its sequence varies in composition and in length between different scorpion species. Available studies of the structure-function relationships of scorpion venom sPLA2 are limited. Some biological activities of these enzymes such as neurotoxicity, myotoxicity, along with the hemolytic, anticoagulant, anti-tumoral and anti-angiogenic activities have been investigated. In this review, we tentatively summarize the last findings about the biochemical properties, the structure-function relation and the biological activities of scorpion venom phospholipases A2. In addition to expanding the database of structures for these sPLA2 and understanding their properties and functions, this survey is intended to explore the molecular mechanisms and signaling pathways of the described biological activities.


Assuntos
Fosfolipases A2 Secretórias , Venenos de Escorpião , Sequência de Aminoácidos , Animais , Venenos de Abelha , Abelhas , Dimerização , Fosfolipases A2 , Escorpiões , Serpentes
8.
Nat Commun ; 11(1): 2905, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518228

RESUMO

Bacterial nucleoid remodeling dependent on conserved histone-like protein, HU is one of the determining factors in global gene regulation. By imaging of near-native, unlabeled E. coli cells by soft X-ray tomography, we show that HU remodels nucleoids by promoting the formation of a dense condensed core surrounded by less condensed isolated domains. Nucleoid remodeling during cell growth and environmental adaptation correlate with pH and ionic strength controlled molecular switch that regulated HUαα dependent intermolecular DNA bundling. Through crystallographic and solution-based studies we show that these effects mechanistically rely on HUαα promiscuity in forming multiple electrostatically driven multimerization interfaces. Changes in DNA bundling consequently affects gene expression globally, likely by constrained DNA supercoiling. Taken together our findings unveil a critical function of HU-DNA interaction in nucleoid remodeling that may serve as a general microbial mechanism for transcriptional regulation to synchronize genetic responses during the cell cycle and adapt to changing environments.


Assuntos
DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Ciclo Celular , Cromossomos Bacterianos/metabolismo , Cristalografia por Raios X , Dimerização , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Íons , Mutação , Multimerização Proteica , Tomografia por Raios X
9.
PLoS Comput Biol ; 16(6): e1007919, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32497094

RESUMO

Transmembrane helix association is a fundamental step in the folding of helical membrane proteins. The prototypical example of this association is formation of the glycophorin dimer. While its structure and stability have been well-characterized experimentally, the detailed assembly mechanism is harder to obtain. Here, we use all-atom simulations within phospholipid membrane to study glycophorin association. We find that initial association results in the formation of a non-native intermediate, separated by a significant free energy barrier from the dimer with a native binding interface. We have used transition-path sampling to determine the association mechanism. We find that the mechanism of the initial bimolecular association to form the intermediate state can be mediated by many possible contacts, but seems to be particularly favoured by formation of non-native contacts between the C-termini of the two helices. On the other hand, the contacts which are key to determining progression from the intermediate to the native state are those which define the native binding interface, reminiscent of the role played by native contacts in determining folding of globular proteins. As a check on the simulations, we have computed association and dissociation rates from the transition-path sampling. We obtain results in reasonable accord with available experimental data, after correcting for differences in native state stability. Our results yield an atomistic description of the mechanism for a simple prototype of helical membrane protein folding.


Assuntos
Proteínas de Membrana/química , Dimerização , Glicoforinas/química , Simulação de Dinâmica Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína
10.
Life Sci ; 255: 117841, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454156

RESUMO

AIMS: Trefoil factor 3 (TFF3) is a gut mucosal protective molecule that is secreted by intestinal goblet cells. The dimeric structure of TFF3 enables it to function in intestinal mucosal repair and to maintain its own stability. Protein disulfide isomerase a1 (PDIA1) can directly catalyze the formation, isomerization and reduction of disulfide bonds in proteins and may play an important role in the formation of TFF3 dimer. In this study, we focused on the specific molecular mechanism of TFF3 dimerization by PDIA1 and the changes during sepsis. METHODS: We examined the changes of PDIA1 and TFF3 in sepsis rats and cell models and used a variety of experimental techniques to investigate the specific molecular mechanism of PDIA1-catalyzed TFF3 dimerization. KEY FINDINGS: We found that PDIA1 can directly catalyze the dimerization of TFF3. Our MD model proposed that two TFF3 monomers form hydrogen bonds with the region b' of PDIA1 through two stepwise reactions. Furthermore, we propose that the Cys24-Cys27 active site at the region a' of PDIA1 mediates disulfide bond formation between the Cys79 residues of each of the two TFF3 monomers via deprotonation and nucleophilic attack. During sepsis, PDIA1 is downregulated and the excessive release of nitric oxide (NO) promoted PDIA1 nitrosylation. This modification reduced PDIA1 activity, which resulted in the corresponding decrease of TFF3 dimerization and compromised TFF3 dimer function. SIGNIFICANCE: Our study revealed a novel mechanism for the inhibition of intestinal mucosal repair during sepsis and revealed novel targets for the prevention and treatment of sepsis.


Assuntos
Mucosa Intestinal/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Sepse/fisiopatologia , Fator Trefoil-3/metabolismo , Animais , Dimerização , Modelos Animais de Doenças , Regulação para Baixo , Masculino , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Óxido Nítrico/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Isomerases de Dissulfetos de Proteínas/genética , Ratos , Ratos Sprague-Dawley
11.
Nat Commun ; 11(1): 2223, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376862

RESUMO

Stem cells are one of the foundational evolutionary novelties that allowed the independent emergence of multicellularity in the plant and animal lineages. In plants, the homeodomain (HD) transcription factor WUSCHEL (WUS) is essential for the maintenance of stem cells in the shoot apical meristem. WUS has been reported to bind to diverse DNA motifs and to act as transcriptional activator and repressor. However, the mechanisms underlying this remarkable behavior have remained unclear. Here, we quantitatively delineate WUS binding to three divergent DNA motifs and resolve the relevant structural underpinnings. We show that WUS exhibits a strong binding preference for TGAA repeat sequences, while retaining the ability to weakly bind to TAAT elements. This behavior is attributable to the formation of dimers through interactions of specific residues in the HD that stabilize WUS DNA interaction. Our results provide a mechanistic basis for dissecting WUS dependent regulatory networks in plant stem cell control.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Motivos de Nucleotídeos/genética , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , DNA/metabolismo , Dimerização , Proteínas de Homeodomínio/genética , Brotos de Planta/genética , Ligação Proteica , Sequências Repetitivas de Ácido Nucleico/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Life Sci ; 254: 117752, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32387412

RESUMO

AIMS: To design and evaluate novel mono-PEGylated dimeric GLP-1 conjugate with enhanced GLP-1 receptor activation and prolonged anti-diabetes efficacies. MAIN METHODS: All these novel GLP-1 conjugates were produced by using solid-phase synthesis method and further specific cysteine-maleimide modification. In vitro GLP-1R activation assay was performed in CHO cells stably expressing human GLP-1 receptor. The binding affinity for human serum albumin (HSA) in vitro was also conducted using surface plasmon resonance measurement. Subsequently, selected GLP-1 conjugate was subjected to evaluate the acute and chronic efficacies in vivo. KEY FINDINGS: Four novel glucagon-like peptide-1 (GLP-1) conjugates, termed DIG-1 to DIG-4, were designed and prepared with high purity. Moreover, DIG-1(PEG-5 kDa) and DIG-2 (PEG-10 kDa) exerted ~3-fold and ~2-fold higher potencies of GLP-1R activation than native GLP-1, respectively, and both obviously higher than the DIG-3 (PEG-10 kDa) and DIG-4 (PEG-30 kDa). Then DIG-2 exhibited better in vivo glucose-stabilizing and insulinotropic efficacies than DIG-1 by using multiple oral glucose tests (OGTTs) in SD rats. Furthermore, prolonged glucose-lowering ability of DIG-2 exhibited in hypoglycemic duration test and multiple OGTTs in diabetic db/db mice. Pharmacokinetic data of DIG-2 in cynomolgus monkeys revealed a half-life of ~97.2 h and ~120.4 h after a single subcutaneous (s.c.) administration at doses of 100 and 150 nmol/kg, respectively. Chronic treatment of DIG-2 in db/db mice for consecutive 8-week significantly ameliorate the diabetic symptoms including deteriorative % hemoglobin A1C (HbA1C), glucose tolerance and pancreatic function. SIGNIFICANCE: DIG-2, as a novel mono-PEGylated dimeric GLP-1 conjugate, holds enhanced receptor activation and prolonged anti-diabetes efficacies.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hipoglicemiantes/uso terapêutico , Polietilenoglicóis/metabolismo , Animais , Células CHO , Cricetulus , Dimerização , Humanos , Ratos
13.
J Biosci Bioeng ; 130(3): 253-259, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32451246

RESUMO

Ribozymes with modular structures are attractive platforms for the construction of nanoscale RNA objects with biological functions. We designed group I ribozyme dimers as unit ribozyme dimers (Urds), which self-assembled to form their polymeric states and also oligomeric states with defined numbers of Urds. Assembly of Urds yielded catalytic ability of a pair of distinct ribozyme units to cleave two distinct substrates. The morphologies of the assembled ribozyme structures were observed directly by atomic force microscopy (AFM).


Assuntos
Dimerização , Nanoestruturas/química , RNA Catalítico/química , RNA Catalítico/metabolismo , Biocatálise , Conformação de Ácido Nucleico
14.
Nat Commun ; 11(1): 1733, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265441

RESUMO

Dysregulation of extracellular signal-regulated kinases (ERK1/2) is linked to several diseases including heart failure, genetic syndromes and cancer. Inhibition of ERK1/2, however, can cause severe cardiac side-effects, precluding its wide therapeutic application. ERKT188-autophosphorylation was identified to cause pathological cardiac hypertrophy. Here we report that interference with ERK-dimerization, a prerequisite for ERKT188-phosphorylation, minimizes cardiac hypertrophy without inducing cardiac adverse effects: an ERK-dimerization inhibitory peptide (EDI) prevents ERKT188-phosphorylation, nuclear ERK1/2-signaling and cardiomyocyte hypertrophy, protecting from pressure-overload-induced heart failure in mice whilst preserving ERK1/2-activity and cytosolic survival signaling. We also examine this alternative ERK1/2-targeting strategy in cancer: indeed, ERKT188-phosphorylation is strongly upregulated in cancer and EDI efficiently suppresses cancer cell proliferation without causing cardiotoxicity. This powerful cardio-safe strategy of interfering with ERK-dimerization thus combats pathological ERK1/2-signaling in heart and cancer, and may potentially expand therapeutic options for ERK1/2-related diseases, such as heart failure and genetic syndromes.


Assuntos
Cardiotoxicidade , Peptídeos Penetradores de Células/farmacologia , Dimerização , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Técnicas de Cultura de Células , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/toxicidade , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Sistemas de Liberação de Medicamentos , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Medicina Molecular , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
15.
Nat Commun ; 11(1): 1641, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242019

RESUMO

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.


Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/citologia , Corynebacterium glutamicum/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Divisão Celular , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Dimerização , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , Alinhamento de Sequência
16.
Life Sci ; 253: 117651, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32304764

RESUMO

AIMS: To investigate the combination of dimerization and PEGylation to enhance the receptor activation and in vivo stability of Oxyntomodulin (OXM). MAIN METHODS: All LDM peptides were produced by using standard method of solid phase synthesis. The in vitro effects of LDM peptides were assessed by glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GcgR) binding test and Proteolytic stability test. Subsequently, saline, Liraglutide and three doses of LDM-3 treated groups were subjected to the evaluation of aute and long-term efficacy. KEY FINDINGS: Five long-acting OXM conjugates, termed LDM-1 to LDM-5, were designed using cysteine (Cys)-specific modification reaction including the activated PEG, bisMal-NH2, and OXM-Cys, and all prepared with high purity. LDM-3 exhibited greater GLP-1R and GcgR activation and ameliorative serum stability. In addition, LDM-3 was identified with enhanced insulinotropic and glycemic abilities in the gene knockout mice. The prolonged glucose-lowering effects of the LDM-3 were proved by hypoglycemic duration test and multiple oral glucose tolerance tests (OGTTs) in the diet-induced obesity (DIO) mice. Furthermore, the pharmacokinetic tests in Sprague Dawley (SD) rat and cynomolgus monkey exhibited the lifespans of LDM-3 at 90 nmol·kg-1 were 101.5 h and 119.4 h, respectively. Nevertheless, consecutive 8-week administration of LDM-3 improved the cumulative body weight gain, food intake, % HbA1c, glucose tolerance and the pancreatic of the obese mice. SIGNIFICANCE: LDM-3, as a dual GLP-1R and GcgR agonist, holds potential to be developed as a promising therapeutic candidate for both diabetes and obesity.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hipoglicemiantes/química , Oxintomodulina/química , Receptores de Glucagon/metabolismo , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus/metabolismo , Dimerização , Glucagon/metabolismo , Teste de Tolerância a Glucose , Hipoglicemiantes/farmacocinética , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Obesidade/metabolismo , Oxintomodulina/farmacocinética , Polietilenoglicóis/química , Ratos Sprague-Dawley , Técnicas de Síntese em Fase Sólida , Perda de Peso/efeitos dos fármacos
17.
Nucleic Acids Res ; 48(10): 5766-5776, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32313953

RESUMO

Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5'-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5'-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.


Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas p21(ras)/genética , Cristalografia por Raios X , DNA/química , Dimerização , Ligantes , Simulação de Dinâmica Molecular , Mutação , Poli A/química , Água/química
18.
PLoS Biol ; 18(3): e3000649, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32231352

RESUMO

Icosahedral viral capsids must undergo conformational rearrangements to coordinate essential processes during the viral life cycle. Capturing such conformational flexibility has been technically challenging yet could be key for developing rational therapeutic agents to combat infections. Noroviruses are nonenveloped, icosahedral viruses of global importance to human health. They are a common cause of acute gastroenteritis, yet no vaccines or specific antiviral agents are available. Here, we use genetics and cryo-electron microscopy (cryo-EM) to study the high-resolution solution structures of murine norovirus as a model for human viruses. By comparing our 3 structures (at 2.9- to 3.1-Å resolution), we show that whilst there is little change to the shell domain of the capsid, the radiating protruding domains are flexible, adopting distinct states both independently and synchronously. In doing so, the capsids sample a range of conformational space, with implications for maintaining virion stability and infectivity.


Assuntos
Capsídeo/química , Norovirus/química , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Microscopia Crioeletrônica , Dimerização , Temperatura Alta , Camundongos , Modelos Moleculares , Mutação , Norovirus/genética , Norovirus/patogenicidade , Domínios Proteicos , Células RAW 264.7 , Relação Estrutura-Atividade
19.
PLoS One ; 15(4): e0224853, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298262

RESUMO

Diets rich in flavonoids have been related with low obesity rates, which could be related with their potential to inhibit pancreatic lipase, the main enzyme of fat assimilation. Some flavonoids can aggregate in aqueous medium suggesting that the inhibition mechanism could occur on both molecular and colloidal levels. This study investigates the interaction of two flavonoid aggregates, quercetin and epigallocatechin gallate (EGCG), with pancreatic lipase under simplified intestinal conditions. The stability and the morphology of these flavonoid aggregates were studied in four different solutions: Control (water), salt, low lipase concentration and high lipase concentration. Particles were found by optical microscopy in almost all the solutions tested, except EGCG-control. The results show that the precipitation rate decreases for quercetin and increases for EGCG in salt solution and that lipase stabilize quercetin aggregates. In addition, both flavonoids were shown to precipitate together with pancreatic lipase resulting in a sequestering of the enzyme.


Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Mucosa Intestinal/metabolismo , Lipase/metabolismo , Quercetina/farmacologia , Animais , Antioxidantes/metabolismo , Catequina/metabolismo , Catequina/farmacologia , Dimerização , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Lipase/antagonistas & inibidores , Quercetina/metabolismo , Suínos
20.
Nucleic Acids Res ; 48(9): 4839-4857, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32266943

RESUMO

Development of oligodendrocytes and myelin formation in the vertebrate central nervous system is under control of several basic helix-loop-helix transcription factors such as Olig2, Ascl1, Hes5 and the Id proteins. The class I basic helix-loop-helix proteins Tcf3, Tcf4 and Tcf12 represent potential heterodimerization partners and functional modulators for all, but have not been investigated in oligodendrocytes so far. Using mouse mutants, organotypic slice and primary cell cultures we here show that Tcf4 is required in a cell-autonomous manner for proper terminal differentiation and myelination in vivo and ex vivo. Partial compensation is provided by the paralogous Tcf3, but not Tcf12. On the mechanistic level Tcf4 was identified as the preferred heterodimerization partner of the central regulator of oligodendrocyte development Olig2. Both genetic studies in the mouse as well as functional studies on enhancer regions of myelin genes confirmed the relevance of this physical interaction for oligodendrocyte differentiation. Considering that alterations in TCF4 are associated with syndromic and non-syndromic forms of intellectual disability, schizophrenia and autism in humans, our findings point to the possibility of an oligodendroglial contribution to these disorders.


Assuntos
Fator de Transcrição 2 de Oligodendrócitos/genética , Oligodendroglia/citologia , Fator de Transcrição 4/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Dimerização , Feminino , Deleção de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Bainha de Mielina/fisiologia , Oligodendroglia/metabolismo , Ratos Wistar
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