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1.
Nat Commun ; 11(1): 4931, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004795

RESUMO

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas de Neoplasias/ultraestrutura , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Motivos de Aminoácidos , Aminoquinolinas/farmacologia , Aminoquinolinas/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mutagênese , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Estudo de Prova de Conceito , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Domínios Proteicos/genética , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
2.
Nat Commun ; 11(1): 4864, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978392

RESUMO

The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal ß-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.


Assuntos
Tolerância a Medicamentos/fisiologia , Fungos/enzimologia , Fungos/metabolismo , Xilosidases/química , Xilosidases/metabolismo , Biocatálise , Domínio Catalítico , Fungos/efeitos dos fármacos , Glicoconjugados/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/química , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese , Especificidade por Substrato , Talaromyces/enzimologia , Talaromyces/genética , Xilosidases/genética
3.
Nat Commun ; 11(1): 4682, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943628

RESUMO

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.


Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Pirazinas/farmacologia , Amidas/farmacocinética , Animais , Antivirais/farmacocinética , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Moleculares , Mutagênese/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Pirazinas/farmacocinética , RNA Replicase/química , RNA Replicase/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Análise de Sequência , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
4.
Nature ; 585(7825): 459-463, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908305

RESUMO

The RNA polymerase II (Pol II) core promoter is the strategic site of convergence of the signals that lead to the initiation of DNA transcription1-5, but the downstream core promoter in humans has been difficult to understand1-3. Here we analyse the human Pol II core promoter and use machine learning to generate predictive models for the downstream core promoter region (DPR) and the TATA box. We developed a method termed HARPE (high-throughput analysis of randomized promoter elements) to create hundreds of thousands of DPR (or TATA box) variants, each with known transcriptional strength. We then analysed the HARPE data by support vector regression (SVR) to provide comprehensive models for the sequence motifs, and found that the SVR-based approach is more effective than a consensus-based method for predicting transcriptional activity. These results show that the DPR is a functionally important core promoter element that is widely used in human promoters. Notably, there appears to be a duality between the DPR and the TATA box, as many promoters contain one or the other element. More broadly, these findings show that functional DNA motifs can be identified by machine learning analysis of a comprehensive set of sequence variants.


Assuntos
Sequência Consenso/genética , Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Máquina de Vetores de Suporte , Transcrição Genética , Sequência de Bases , Células/metabolismo , Simulação por Computador , Conjuntos de Dados como Assunto , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Genéticos , Mutagênese , TATA Box/genética
5.
Science ; 369(6508): 1261-1265, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753553

RESUMO

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90-glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.


Assuntos
Betacoronavirus/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Engenharia de Proteínas , Receptores Virais/genética , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Humanos , Modelos Moleculares , Mutagênese , Mutação , Peptidil Dipeptidase A/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química
6.
Nat Commun ; 11(1): 4046, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792488

RESUMO

2-oxoglutarate (2-OG or α-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide mutagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG dependent dioxygenase function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc)-the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 associates with the OGDHc and maintains catalytic activity of lipoyl domain by preventing the formation of lipoyl adducts, highlighting ABHD11 as a regulator of functional lipoylation and 2-OG metabolism.


Assuntos
Complexo Cetoglutarato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mitocôndrias/metabolismo , Mutagênese/fisiologia , Serina Proteases/metabolismo , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Células HeLa , Humanos , Complexo Cetoglutarato Desidrogenase/genética , Modelos Biológicos , Mutagênese/genética , Serina Proteases/genética
7.
Nat Commun ; 11(1): 4132, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807781

RESUMO

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.


Assuntos
Edição de Genes/métodos , RNA Guia/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Humanos , Mutagênese/genética , Mutagênese/fisiologia , RNA Guia/genética
8.
PLoS One ; 15(7): e0235643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735615

RESUMO

BACKGROUND: Pyrazinamide is an important drug against the latent stage of tuberculosis and is used in both first- and second-line treatment regimens. Pyrazinamide-susceptibility test usually takes a week to have a diagnosis to guide initial therapy, implying a delay in receiving appropriate therapy. The continued increase in multi-drug resistant tuberculosis and the prevalence of pyrazinamide resistance in several countries makes the development of assays for prompt identification of resistance necessary. The main cause of pyrazinamide resistance is the impairment of pyrazinamidase function attributed to mutations in the promoter and/or pncA coding gene. However, not all pncA mutations necessarily affect the pyrazinamidase function. OBJECTIVE: To develop a methodology to predict pyrazinamidase function from detected mutations in the pncA gene. METHODS: We measured the catalytic constant (kcat), KM, enzymatic efficiency, and enzymatic activity of 35 recombinant mutated pyrazinamidase and the wild type (Protein Data Bank ID = 3pl1). From all the 3D modeled structures, we extracted several predictors based on three categories: structural stability (estimated by normal mode analysis and molecular dynamics), physicochemical, and geometrical characteristics. We used a stepwise Akaike's information criterion forward multiple log-linear regression to model each kinetic parameter with each category of predictors. We also developed weighted models combining the three categories of predictive models for each kinetic parameter. We tested the robustness of the predictive ability of each model by 6-fold cross-validation against random models. RESULTS: The stability, physicochemical, and geometrical descriptors explained most of the variability (R2) of the kinetic parameters. Our models are best suited to predict kcat, efficiency, and activity based on the root-mean-square error of prediction of the 6-fold cross-validation. CONCLUSIONS: This study shows a quick approach to predict the pyrazinamidase function only from the pncA sequence when point mutations are present. This can be an important tool to detect pyrazinamide resistance.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Amidoidrolases/química , Amidoidrolases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Cinética , Modelos Lineares , Simulação de Dinâmica Molecular , Mutagênese , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
9.
Chem Biol Interact ; 330: 109219, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32846153

RESUMO

The lack of tissue selectivity of anticancer drugs generates intense collateral and adverse effects of cancer patients, making the incorporation of vitamins or micronutrients into the diet of individuals to reduce side or adverse effects of antineoplastics. The study aimed to evaluate the effects of retinol palmitate (RP) on the toxicogenic damages induced by cyclophosphamide (CPA), doxorubicin (DOX) and its association with the AC protocol (CPA + DOX), in Sarcoma 180 (S-180) tumor cell line, using the micronuclei test with a block of cytokinesis (CBMN); and in non-tumor cells derived from Mus musculus using the comet assay. The results suggest that CPA, DOX and AC protocol induced significant toxicogenic damages (P < 0.05) on the S-180 cells by induction of micronuclei, cytoplasmic bridges, nuclear buds, apoptosis, and cell necrosis, proving their antitumor effects, and significant damage (P < 0.001) to the genetic material of peripheral blood cells of healthy mice, proving the genotoxic potential of these drugs. However, RP modulated the toxicogenic effects of antineoplastic tested both in the CBMN test (P < 0.05), at the concentrations of 1, 10 and 100 IU/mL; as in the comet assay (P < 0.001) at the concentration of 100 IU/kg for the index and frequency of genotoxic damage. The accumulated results suggest that RP reduced the action of antineoplastics in non-tumor cells as well as the cytotoxic, mutagenic, and cell death in neoplastic cells.


Assuntos
Antineoplásicos/toxicidade , Diterpenos/farmacologia , Vitamina A/análogos & derivados , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Ensaio Cometa , Ciclofosfamida/efeitos adversos , Ciclofosfamida/toxicidade , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/efeitos adversos , Doxorrubicina/toxicidade , Interações Medicamentosas , Humanos , Camundongos , Testes para Micronúcleos , Mutagênese/efeitos dos fármacos , Vitamina A/farmacologia
10.
Chem Biol Interact ; 330: 109216, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810488

RESUMO

In our previous study, an antimutagenic compound from spinach (Spinacea oleracea L.), ethoxy-substituted phylloquinone (ESP) was isolated and characterized. The current study deals with elucidation of the possible mechanism of antimutagenicity of ESP against ethyl methanesulfonate (EMS) deploying model systems such as human lymphoblast (TK+/- or TK6) cell line (thymidine kinase gene mutation assay) and Escherichia coli MG1655 (rifampicin resistance assay). Findings of the study ruled out the possibility of direct inactivation of EMS by ESP. DAPI competitive binding assay indicated the DNA minor groove binding activity of ESP. Interestingly, ESP did not display major groove binding or intercalating abilities. Further, proteomics study using 2-D gel electrophoresis in E. coli and subsequent studies involving single gene knockout strains revealed the possible role of tnaA (tryptophanase) and dgcP (diguanylate cyclase) genes in observed antimutagenicity. These genes have been reported to be involved in indole and cyclic-di-GMP biosynthesis, respectively, which eventually lead to cell division inhibition. In case of TK+/- cell line system, ADCY genes (adenylate cyclase), a functional analogue of dgcP gene, were found to be transcriptionally up-regulated. The generation/doubling time were significantly higher in E. coli or TK+/- cells treated with ESP than control cells. The findings indicated inhibition of cell proliferation by ESP through gene regulation as a possible mechanism of antimutagenicity across the biological system. Cell division inhibition actually provides additional time for the repair of damaged DNA leading to antimutagenicity.


Assuntos
Mutagênese/efeitos dos fármacos , Spinacia oleracea/química , Vitamina K 1/química , Vitamina K 1/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Liases/metabolismo , Vitamina K 1/metabolismo
11.
Mol Genet Genomics ; 295(6): 1501-1516, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32767127

RESUMO

Encapsidation by nucleocapsid (N) protein is crucial for viral RNA to serve as a functional template for virus replication. However, the potential region that is vital for RNA encapsidation of Nipah virus (NiV) is still unknown. Thus, this study was aimed to identify these regions using a NiV minireplicon system. A series of broad range internal deletion mutations was generated in the 5' non-translated region (NTR) of the N gene mRNA region of NiV leader promoter via site-directed overlapping PCR-mediated mutagenesis. The mutation effects on synthesis and encapsidation of antigenome RNA, transcription, and RNA binding affinity of N protein were evaluated. The deletions of nucleotides 73-108, 79-108, and 85-108 from NiV leader promoter inhibited the encapsidation of antigenome RNA, while the deletion of nucleotides 103-108 suppressed the synthesis and encapsidation of antigenome RNA, implying that these regions are required for genome replication. Surprisingly, none of the mutations had detrimental effect on viral transcription. Using isothermal titration calorimetry, the binding of NiV N protein to genome or antigenome RNA transcript lacking of nucleotides 73-108 was found to be suppressed. Additionally, in silico analysis on secondary structure of genome RNA further supported the plausible cause of inefficient encapsidation of antigenome RNA by the loss of encapsidation signal in genome template. In conclusion, this study suggests that the nucleotides 73-90 within 5' NTR of the N gene mRNA region in NiV leader promoter contain cis-acting RNA element that is important for efficient encapsidation of antigenome RNA.


Assuntos
Regulação Viral da Expressão Gênica , Vírus Nipah/genética , Regiões Promotoras Genéticas , RNA Viral , Montagem de Vírus , Regiões 5' não Traduzidas , Linhagem Celular , Mutagênese , Proteínas do Nucleocapsídeo/genética , RNA Mensageiro , RNA Viral/fisiologia , Proteínas Recombinantes/genética , Transcrição Genética
12.
PLoS One ; 15(7): e0225351, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735563

RESUMO

Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity, ift57, ift88, and ift172 mutants, which lack cilia, were crossed with the double-transgenic zebrafish strain Tg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with Shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown.


Assuntos
Barreira Hematorretiniana/metabolismo , Cílios/metabolismo , Células Endoteliais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/fisiologia , Células Endoteliais/citologia , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Larva/metabolismo , Mutagênese , Vasos Retinianos/citologia , Transdução de Sinais , Alcaloides de Veratrum/farmacologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(32): 19517-19527, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32727901

RESUMO

Oxidative damage to DNA is a threat to the genomic integrity and coding accuracy of the chromosomes of all living organisms. Guanine is particularly susceptible to oxidation, and 8-oxo-dG (OG), when produced in situ or incorporated by DNA polymerases, is highly mutagenic due to mispairing with adenine. In many bacteria, defense against OG depends on MutT enzymes, which sanitize OG in the nucleotide pool, and the MutM/Y system, which counteracts OG in chromosomal DNA. In Escherichia coli, antibiotic lethality has been linked to oxidative stress and the downstream consequences of OG processing. However, in mycobacteria, the role of these systems in genomic integrity and antibiotic lethality is not understood, in part because mycobacteria encode four MutT enzymes and two MutMs, suggesting substantial redundancy. Here, we definitively probe the role of OG handling systems in mycobacteria. We find that, although MutT4 is the only MutT enzyme required for resistance to oxidative stress, this effect is not due to OG processing. We find that the dominant system that defends against OG-mediated mutagenesis is MutY/MutM1, and this system is dedicated to in situ chromosomal oxidation rather than correcting OG incorporated by accessory polymerases (DinB1/DinB2/DinB3/DnaE2). In addition, we uncover that mycobacteria resist antibiotic lethality through nucleotide sanitization by MutTs, and in the absence of this system, accessory DNA polymerases and MutY/M contribute to antibiotic-induced lethality. These results reveal a complex, multitiered system of OG handling in mycobacteria with roles in oxidative stress resistance, mutagenesis, and antibiotic lethality.


Assuntos
Antibacterianos/metabolismo , Cromossomos Bacterianos/metabolismo , Reparo do DNA/genética , Mycobacterium/genética , Estresse Oxidativo , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA , Tolerância a Medicamentos , Mutagênese , Mutação , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/metabolismo , Oxirredução
14.
Nat Commun ; 11(1): 3790, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728032

RESUMO

Menthol in mints elicits coolness sensation by selectively activating TRPM8 channel. Although structures of TRPM8 were determined in the apo and liganded states, the menthol-bounded state is unresolved. To understand how menthol activates the channel, we docked menthol to the channel and systematically validated our menthol binding models with thermodynamic mutant cycle analysis. We observed that menthol uses its hydroxyl group as a hand to specifically grab with R842, and its isopropyl group as legs to stand on I846 and L843. By imaging with fluorescent unnatural amino acid, we found that menthol binding induces wide-spread conformational rearrangements within the transmembrane domains. By Φ analysis based on single-channel recordings, we observed a temporal sequence of conformational changes in the S6 bundle crossing and the selectivity filter leading to channel activation. Therefore, our study suggested a 'grab and stand' mechanism of menthol binding and how menthol activates TRPM8 at the atomic level.


Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Mentol/farmacologia , Canais de Cátion TRPM/agonistas , Sítios de Ligação/genética , Células HEK293 , Humanos , Mentol/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese , Técnicas de Patch-Clamp , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética
15.
Nat Commun ; 11(1): 3533, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669552

RESUMO

Cyclic dinucleotides (CDNs) are second messengers conserved across all three domains of life. Within eukaryotes they mediate protective roles in innate immunity against malignant, viral, and bacterial disease, and exert pathological effects in autoimmune disorders. Despite their ubiquitous role in diverse biological contexts, CDN detection methods are limited. Here, using structure guided design of the murine STING CDN binding domain, we engineer a Förster resonance energy transfer (FRET) based biosensor deemed BioSTING. Recombinant BioSTING affords real-time detection of CDN synthase activity and inhibition. Expression of BioSTING in live human cells allows quantification of localized bacterial and eukaryotic CDN levels in single cells with low nanomolar sensitivity. These findings establish BioSTING as a powerful kinetic in vitro platform amenable to high throughput screens and as a broadly applicable cellular tool to interrogate the temporal and spatial dynamics of CDN signaling in a variety of infectious, malignant, and autoimmune contexts.


Assuntos
Técnicas Biossensoriais , Proteínas de Membrana/química , Nucleotídeos Cíclicos/análise , Transdução de Sinais , Animais , Bacillus subtilis/química , GMP Cíclico/metabolismo , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Imunidade Inata , Ligantes , Camundongos , Mutagênese , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes
16.
Nat Commun ; 11(1): 3469, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651386

RESUMO

Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon-based mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that differ from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more compromised, a larger proportion has successfully adapted for the activity. Different functional profiles emerge: (i) both strong and weak trade-off between activities are observed; (ii) trade-off is more severe (20- to 35-fold increased kcat/KM in arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in kcat rather than just in KM, suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions.


Assuntos
Mutação INDEL/genética , Evolução Molecular , Humanos , Mutagênese/genética , Mutagênese/fisiologia , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Biologia Sintética/métodos
17.
BMC Bioinformatics ; 21(1): 275, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611389

RESUMO

BACKGROUND: Protein engineering has many applications for industry, such as the development of new drugs, vaccines, treatment therapies, food, and biofuel production. A common way to engineer a protein is to perform mutations in functionally essential residues to optimize their function. However, the discovery of beneficial mutations for proteins is a complex task, with a time-consuming and high cost for experimental validation. Hence, computational approaches have been used to propose new insights for experiments narrowing the search space and reducing the costs. RESULTS: In this study, we developed Proteus (an acronym for Protein Engineering Supporter), a new algorithm for proposing mutation pairs in a target 3D structure. These suggestions are based on contacts observed in other known structures from Protein Data Bank (PDB). Proteus' basic assumption is that if a non-interacting pair of amino acid residues in the target structure is exchanged to an interacting pair, this could enhance protein stability. This trade is only allowed if the main-chain conformation of the residues involved in the contact is conserved. Furthermore, no steric impediment is expected between the proposed mutations and the surrounding protein atoms. To evaluate Proteus, we performed two case studies with proteins of industrial interests. In the first case study, we evaluated if the mutations suggested by Proteus for four protein structures enhance the number of inter-residue contacts. Our results suggest that most mutations proposed by Proteus increase the number of interactions into the protein. In the second case study, we used Proteus to suggest mutations for a lysozyme protein. Then, we compared Proteus' outcomes to mutations with available experimental evidence reported in the ProTherm database. Four mutations, in which our results agree with the experimental data, were found. This could be initial evidence that changes in the side-chain of some residues do not cause disturbances that harm protein structure stability. CONCLUSION: We believe that Proteus could be used combined with other methods to give new insights into the rational development of engineered proteins. Proteus user-friendly web-based tool is available at < http://proteus.dcc.ufmg.br >.


Assuntos
Proteínas/química , Interface Usuário-Computador , Algoritmos , Bases de Dados de Proteínas , Muramidase/química , Muramidase/genética , Muramidase/metabolismo , Mutagênese , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo
18.
J Vis Exp ; (161)2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716393

RESUMO

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The "last-line" antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.


Assuntos
Acinetobacter baumannii/genética , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutagênese
19.
Nat Commun ; 11(1): 3664, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694532

RESUMO

Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Etanol/toxicidade , Taxa de Mutação , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/genética , Mutagênese , Testes de Mutagenicidade , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(29): 17211-17220, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611811

RESUMO

The bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a wide range of cellular functions from biofilm formation to growth and survival. Targeting a second-messenger network is challenging because the system involves a multitude of components with often overlapping functions. Here, we present a strategy to intercept c-di-GMP signaling pathways by directly targeting the second messenger. For this, we developed a c-di-GMP-sequestering peptide (CSP) that was derived from a CheY-like c-di-GMP effector protein. CSP binds c-di-GMP with submicromolar affinity. The elucidation of the CSP⋅c-di-GMP complex structure by NMR identified a linear c-di-GMP-binding motif, in which a self-intercalated c-di-GMP dimer is tightly bound by a network of H bonds and π-stacking interactions involving arginine and aromatic residues. Structure-based mutagenesis yielded a variant with considerably higher, low-nanomolar affinity, which subsequently was shortened to 19 residues with almost uncompromised affinity. We demonstrate that endogenously expressed CSP intercepts c-di-GMP signaling and effectively inhibits biofilm formation in Pseudomonas aeruginosa, the most widely used model for serious biofilm-associated medical implications.


Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Peptídeos/metabolismo , Sistemas do Segundo Mensageiro , Transdução de Sinais , Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli , Modelos Moleculares , Mutagênese , Peptídeos/química , Peptídeos/genética , Mutação Puntual , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/metabolismo
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