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1.
PLoS One ; 15(1): e0227275, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923209

RESUMO

The aim of this work was to determine current cagA gene EPIYA motifs present in Colombian Helicobacter pylori isolates using a fast and reliable molecular test. DNA from eighty-five Helicobacter pylori-cagA positive strains were analyzed. Strains were obtained from patients diagnosed with functional dyspepsia at Clínica Fundadores in Bogotá. The 3' region of the cagA gene was amplified through conventional Polymerase Chain Reaction (PCR). Obtained amplicons were sequenced using the Sanger method and analyzed with bioinformatics tools. Additionally, a significant Spearman correlation coefficient was determined between the patients' age and the number of EPIYA-C repeats; with p values < 0.05 considered significant. Estimates were obtained using a 95% CI. The 3´ variable region of the cagA gene was amplified and PCR products of the following sizes corresponded to the following EPIYA motifs: 400 bp: EPIYA AB, 500 bp: EPIYA ABC, 600 bp: EPIYA ABCC and 700 bp: ABCCC. A single PCR band was observed for 58 out of 85 Helicobacter pylori isolates, with an EPIYA distribution motif as follows: 7/85 AB (8.2%), 34/85 ABC (40%), 26/85 ABCC (30.6%) and 18/85 ABCCC (21.2%). However, in 27 out of 85 Helicobacter pylori isolates, two or more bands were observed, where the most predominant cagA genotype were ABC-ABCC (26%, 7/27) and ABCC-ABCCC (22.2%, 6/27). A direct proportionality between the number of EPIYA-C repeats and an increase in the patients' age was observed, finding a greater number of EPIYA ABCC and ABCCC repeats in the population over 50 years old. All isolates were of the Western cagA type and 51.8% of them were found to have multiple EPIYA-C repeats. These standardized molecular test allowed to identify the number of EPIYA C motifs based on band size.


Assuntos
Motivos de Aminoácidos/genética , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Testes Diagnósticos de Rotina/normas , Genes Bacterianos/genética , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/genética , Adulto , Idoso , Colômbia/epidemiologia , DNA Bacteriano/genética , Dispepsia/microbiologia , Feminino , Genótipo , Infecções por Helicobacter/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/normas , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA/normas
2.
PLoS One ; 15(1): e0227180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945090

RESUMO

Recent evidence shows that the disruption of constitutive insulated neighbourhoods might lead to oncogene dysregulation. We present here a systematic pan-cancer characterisation of the associations between constitutive boundaries and genome alterations in cancer. Specifically, we investigate the enrichment of somatic mutation, abnormal methylation, and copy number alteration events in the proximity of CTCF bindings overlapping with topological boundaries (junctions) in 26 cancer types. Focusing on CTCF motifs that are both in-boundary (overlapping with junctions) and active (overlapping with peaks of CTCF expression), we find a significant enrichment of somatic mutations in several cancer types. Furthermore, mutated junctions are significantly conserved across cancer types, and we also observe a positive selection of transversions rather than transitions in many cancer types. We also analyzed the mutational signature found on the different classes of CTCF motifs, finding some signatures (such as SBS26) to have a higher weight within in-boundary than off-bounday motifs. Regarding methylation, we find a significant number of over-methylated active in-boundary CTCF motifs in several cancer types; similarly to somatic-mutated junctions, they also have a significant conservation across cancer types. Finally, in several cancer types we observe that copy number alterations tend to overlap with active junctions more often than in matched normal samples. While several articles have recently reported a mutational enrichment at CTCF binding sites for specific cancer types, our analysis is pan-cancer and investigates abnormal methylation and copy number alterations in addition to somatic mutations. Our method is fully replicable and suggests several follow-up tumour-specific analyses.


Assuntos
Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Análise Mutacional de DNA/métodos , Epigênese Genética/genética , Elementos Isolantes/genética , Neoplasias/genética , Mutação Puntual , Motivos de Aminoácidos/genética , Sítios de Ligação/genética , Cromossomos Humanos Par 11/genética , Variações do Número de Cópias de DNA/genética , Metilação de DNA , Éxons/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Humanos , Taxa de Mutação , Regiões Promotoras Genéticas/genética
3.
Phytopathology ; 110(1): 187-193, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31516080

RESUMO

Potyviral helper component protease (HC-Pro), as a major determinant of symptom expression in susceptible plants, is a likely target candidate in the production of attenuated strains for cross-protection. In this study, single or double mutations of Lys (K) to Glu (E) in the Lys-Ile-Thr-Cys motif and Arg (R) to Ile (I) in the Phe-Arg-Asn-Lys motif of the HC-Pro from the severe papaya leaf distortion mosaic virus strain DF (PLDMV-DF) reduced symptom expression and virus accumulation in infected papaya (Carica papaya) plants. The papaya plants infected with the attenuated double mutant of PLDMV-EI presented as symptomless. PLDMV-EI provided effective protection against PLDMV-DF infection in three papaya cultivars and had no effect on plant growth and development. Our result showed that PLDMV-EI is a promising mild strain for the practical use of cross-protection in the field.


Assuntos
Motivos de Aminoácidos , Carica , Peptídeo Hidrolases , Potyvirus , Motivos de Aminoácidos/genética , Carica/virologia , Mutação/genética , Peptídeo Hidrolases/genética , Potyvirus/enzimologia , Potyvirus/genética
4.
Mol Plant Microbe Interact ; 33(1): 78-86, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31486716

RESUMO

Geminiviruses cause severe damage to agriculture worldwide. The replication (Rep) protein is the indispensable viral protein for viral replication. Although various functional domains of Rep protein in Geminivirus spp. have been characterized, the most carboxyl terminus of Rep protein was not available. We have reported the first cassava-infecting geminivirus, Sri Lankan cassava mosaic virus (SLCMV-HN7 strain), in China. In this study, we reported the second Chinese SLCMV strain, SLCMV-Col, and conducted comparative genomic analysis between these two SLCMV strains. The virulence of SLCMV-Col is much stronger than SLCMV-HN7, indicated by the higher virus titer, more severe symptoms, and more extent host defense. We functionally characterized that Rep protein, a 7-amino-acid motif at the most carboxyl terminus, is essential for Rep protein accumulation and virulence of SLCMV. We also provided evidence suggesting that the motif could also enhance triggering of salicylic acid (SA) defense against SLCMV infection in Nicotiana benthamiana. The significance of the balance between virulence and host SA defense responses in expanding invasions of SLCMV is also discussed.


Assuntos
Motivos de Aminoácidos , Begomovirus , Resistência à Doença , Tabaco , Virulência , Motivos de Aminoácidos/genética , Begomovirus/genética , Begomovirus/patogenicidade , China , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/virologia , Tabaco/virologia , Virulência/genética
5.
Plant Mol Biol ; 102(1-2): 123-141, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31776846

RESUMO

KEY MESSAGE: Conserved motif, gene structure, expression and interaction analysis of C2H2-ZFPs in Brassica rapa, and identified types of genes may play essential roles in flower development, and BrZFP38 was proved to function in flower development by affecting pollen formation. Flower development plays a central role in determining the reproduction of higher plants, and Cys2/His2 zinc-finger proteins (C2H2-ZFPs) widely participate in the transcriptional regulation of flower development. C2H2-ZFPs with various structures are the most widespread DNA-binding transcription factors in plants. In this study, conserved protein motif and gene structures were analyzed to investigate systematically the molecular features of Brassica rapa C2H2-ZFP genes. Expression of B. rapa C2H2-ZFPs in multiple tissues showed that more than half of the family members with different types ZFs were expressed in flowers. The specific expression profiles of these C2H2-ZFPs in different B. rapa floral bud stages were further evaluated to identify their potential roles in flower development. Interaction networks were constructed in B. rapa based on the orthology of flower-related C2H2-ZFP genes in Arabidopsis. The putative cis-regulatory elements in the promoter regions of these C2H2-ZFP genes were thoroughly analyzed to elucidate their transcriptional regulation. Results showed that the orthologs of known-function flower-related C2H2-ZFP genes were conserved and differentiated in B. rapa. A C2H2-ZFP was proved to function in B. rapa flower development. Our study provides a systematic investigation of the molecular characteristics and expression profiles of C2H2-ZFPs in B. rapa and promotes further work in function and transcriptional regulation of flower development.


Assuntos
Brassica rapa/genética , Dedos de Zinco CYS2-HIS2/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Brassica rapa/metabolismo , Dedos de Zinco CYS2-HIS2/fisiologia , Flores/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Glucuronidase/metabolismo , Filogenia , Desenvolvimento Vegetal/genética , Desenvolvimento Vegetal/fisiologia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento , Mapas de Interação de Proteínas
6.
Elife ; 82019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31855179

RESUMO

Class A G-protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. Understanding receptor activation mechanism is critical for discovering novel therapeutics since about one-third of all marketed drugs target members of this family. GPCR activation is an allosteric process that couples agonist binding to G-protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue level changes of this movement remain less well understood. Here, we report a framework to quantify conformational changes. By analyzing the conformational changes in 234 structures from 45 class A GPCRs, we discovered a common GPCR activation pathway comprising of 34 residue pairs and 35 residues. The pathway unifies previous findings into a common activation mechanism and strings together the scattered key motifs such as CWxP, DRY, Na+ pocket, NPxxY and PIF, thereby directly linking the bottom of ligand-binding pocket with G-protein coupling region. Site-directed mutagenesis experiments support this proposition and reveal that rational mutations of residues in this pathway can be used to obtain receptors that are constitutively active or inactive. The common activation pathway provides the mechanistic interpretation of constitutively activating, inactivating and disease mutations. As a module responsible for activation, the common pathway allows for decoupling of the evolution of the ligand binding site and G-protein-binding region. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature.


Assuntos
Motivos de Aminoácidos/genética , Ligação Proteica/genética , Conformação Proteica , Receptores Acoplados a Proteínas-G/química , Regulação Alostérica/genética , Sítios de Ligação/genética , Humanos , Ligantes , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/genética
7.
BMB Rep ; 52(12): 700-705, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31722778

RESUMO

The bacterial effector protein RavZ is secreted by the intracellular pathogen Legionella pneumophila and inhibits host autophagy through an irreversible deconjugation of mammalian ATG8 (mATG8) proteins from autophagosome membranes. However, the roles of the LC3 interacting region (LIR) motifs in RavZ function remain unclear. In this study, we show that a membrane-targeting (MT) domain or the LIR motifs of RavZ play major or minor roles in RavZ function. A RavZ mutant that does not bind to mATG8 delipidated all forms of mATG8-phosphatidylethanolamine (PE) as efficiently as did wild-type RavZ. However, a RavZ mutant with a deletion of the MT domain selectively delipidated mATG8-PE less efficiently than did wild-type RavZ. Taken together, our results suggest that the effects of LIR motifs and the MT domain on RavZ activity are complementary and work through independent pathways. [BMB Reports 2019; 52(12): 700-705].


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Legionella pneumophila/genética , Motivos de Aminoácidos/genética , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/antagonistas & inibidores , Proteínas de Bactérias/genética , Células Cultivadas , Fibroblastos , Células HEK293 , Humanos , Legionella pneumophila/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidiletanolaminas/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética
8.
Nat Commun ; 10(1): 4890, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653829

RESUMO

Intrinsically disordered regions (IDRs) are often fast-evolving protein domains of low sequence complexity that can drive phase transitions and are commonly found in many proteins associated with neurodegenerative diseases, including the RNA processing factor TDP43. Yet, how phase separation contributes to the physiological functions of TDP43 in cells remains enigmatic. Here, we combine systematic mutagenesis guided by evolutionary sequence analysis with a live-cell reporter assay of TDP43 phase dynamics to identify regularly-spaced hydrophobic motifs separated by flexible, hydrophilic segments in the IDR as a key determinant of TDP43 phase properties. This heuristic framework allows customization of the material properties of TDP43 condensates to determine effects on splicing function. Remarkably, even a mutant that fails to phase-separate at physiological concentrations can still efficiently mediate the splicing of a quantitative, single-cell splicing reporter and endogenous targets. This suggests that the ability of TDP43 to phase-separate is not essential for its splicing function.


Assuntos
Motivos de Aminoácidos/genética , Proteínas de Ligação a DNA/genética , Interações Hidrofóbicas e Hidrofílicas , Transição de Fase , Processamento de RNA/genética , Processamento Alternativo/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida
9.
Nat Commun ; 10(1): 4502, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582740

RESUMO

The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.


Assuntos
Lisina/metabolismo , Ribonucleoproteínas/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Motivos de Aminoácidos/genética , Biocatálise , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismo
10.
Nat Cell Biol ; 21(10): 1219-1233, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576058

RESUMO

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a ß-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/metabolismo , Motivos de Aminoácidos/genética , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos/genética , Transporte Proteico/fisiologia , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Nexinas de Classificação/genética
11.
J Plant Physiol ; 242: 153018, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31472447

RESUMO

Intracellular vesicular trafficking ensures the exchange of lipids and proteins between the membranous compartments. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) play a central role in membrane fusion and they are key factors for vesicular trafficking in plants, including crops economically important such as tomato (Solanum lycopersicum). Taking advantage of the complete genome sequence available of S. lycopersicum, we identified 63 genes that encode putative SNARE proteins. Then, phylogenetic analysis allowed the classification of SNAREs in five main groups and recognizing their possible functions. A structure analysis of the genes, their syntenic relationships and their location in the chromosomes were also carried out for their characterization. In addition, the expression profiles of SNARE genes in different tissues were investigated using microarray-based analysis. The results indicated that specific SNAREs had a higher induction in leaf, root, flower and mature green fruit. S. lycopersicum is characterized for being a crop sensitive to saline stress unlike its wild relatives, such as Solanum pennellii, Solanum pimpinellifolium, Solanum habrochaites or Solanum chilense, which are tolerant. In this context, we analyzed different microarrays and evaluated and validated the transcript levels through qRT-PCR experiments. The results showed that SlGOS12.2, SlVAMP727 and SlSYP51.2 could have a positive relationship with salt stress and probably an important role in their tolerance. All these data increase our knowledge and can also be utilized to identify potential molecular targets for conferring tolerance to various stresses in tomato.


Assuntos
Lycopersicon esculentum/genética , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Estresse Salino/genética , Motivos de Aminoácidos/genética , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Lycopersicon esculentum/crescimento & desenvolvimento , Fusão de Membrana/genética , Filogenia , Estresse Salino/fisiologia
12.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 9): 599-607, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31475927

RESUMO

Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular-biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7''-phosphotransferase-Ia [APH(7'')-Ia] from Streptomyces hygroscopicus and aminoglycoside 4-phosphotransferase-Ia [APH(4)-Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7'' and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)-Ia was reported as a ternary complex with HygB and 5'-adenylyl-ß,γ-imidodiphosphate (AMP-PNP). To investigate the differences in the substrate-recognition mechanism between APH(7'')-Ia and APH(4)-Ia, the crystal structure of APH(7'')-Ia complexed with HygB is reported. The overall structure of APH(7'')-Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)-Ia, and consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7'')-Ia and APH(4)-Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C-lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7'')-Ia and APH(4)-Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes.


Assuntos
Antibacterianos/química , Higromicina B/química , Canamicina Quinase/química , Streptomyces/enzimologia , Adenilil Imidodifosfato/química , Motivos de Aminoácidos/genética , Aminoglicosídeos/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Escherichia coli/metabolismo , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Fosforilação , Domínios Proteicos , Especificidade por Substrato
13.
BMC Genomics ; 20(1): 691, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477008

RESUMO

BACKGROUND: Mitochondria perform many key roles in their eukaryotic hosts, from integrating signaling pathways through to modulating whole organism phenotypes. The > 1 billion years of nuclear and mitochondrial gene co-evolution has necessitated coordinated expression of gene products from both genomes that maintain mitochondrial, and more generally, eukaryotic cellular function. How mitochondrial DNA (mtDNA) variation modifies host fitness has proved a challenging question but has profound implications for evolutionary and medical genetics. In Drosophila, we have previously shown that recently diverged mtDNA haplotypes within-species can have more impact on organismal phenotypes than older, deeply diverged haplotypes from different species. Here, we tested the effects of mtDNA haplotype variation on gene expression in Drosophila under standardized conditions. Using the Drosophila Genetic Reference Panel (DGRP), we constructed a panel of mitonuclear genotypes that consists of factorial variation in nuclear and mtDNA genomes, with mtDNAs originating in D. melanogaster (2x haplotypes) and D. simulans (2x haplotypes). RESULTS: We show that mtDNA haplotype variation unequivocally alters nuclear gene expression in both females and males, and mitonuclear interactions are pervasive modifying factors for gene expression. There was appreciable overlap between the sexes for mtDNA-sensitive genes, and considerable transcriptional variation attributed to particular mtDNA contrasts. These genes are generally found in low-connectivity gene co-expression networks, occur in gene clusters along chromosomes, are often flanked by non-coding RNA, and are under-represented among housekeeping genes. Finally, we identify the giant (gt) transcription factor motif as a putative regulatory sequence associated with mtDNA-sensitive genes. CONCLUSIONS: There are predictive conditions for nuclear genes that are influenced by mtDNA variation.


Assuntos
Núcleo Celular/genética , Drosophila/genética , Redes Reguladoras de Genes/genética , Genoma Mitocondrial/genética , Motivos de Aminoácidos/genética , Animais , Núcleo Celular/metabolismo , Drosophila/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes/fisiologia , Genes Essenciais/genética , Genes Essenciais/fisiologia , Variação Genética , Genótipo , Haplótipos , Masculino , Família Multigênica , Fenótipo , Mapas de Interação de Proteínas/genética , Mapas de Interação de Proteínas/fisiologia , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , RNA-Seq , Transcriptoma
14.
Nat Commun ; 10(1): 3311, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427571

RESUMO

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.


Assuntos
Acetobacterium/enzimologia , Proteínas de Bactérias/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Metiltransferases/metabolismo , Moorella/enzimologia , Acetobacterium/genética , Motivos de Aminoácidos/genética , Proteínas de Bactérias/genética , Metilação de DNA , DNA Bacteriano/metabolismo , Epigênese Genética , Genoma Bacteriano , Moorella/genética , Análise de Sequência de DNA
15.
J Biol Chem ; 294(40): 14574-14590, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31375564

RESUMO

Human tankyrase-1 (TNKS) is a member of the poly(ADP-ribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose (termed PARylation). The TNKS ankyrin repeat domain mediates interactions with a growing number of structurally and functionally diverse binding partners, linking TNKS activity to multiple critical cell processes, including Wnt signaling, Golgi trafficking, and telomere maintenance. However, some binding partners can engage TNKS without being modified, suggesting that separate parameters influence TNKS interaction and PARylation. Here, we present an analysis of the sequence and structural features governing TNKS interactions with two model binding partners: the PARylated partner telomeric repeat-binding factor 1 (TRF1) and the non-PARylated partner GDP-mannose 4,6-dehydratase (GMD). Using a combination of TNKS-binding assays, PARP activity assays, and analytical ultracentrifugation sedimentation analysis, we found that both the specific sequence of a given TNKS-binding peptide motif and the quaternary structure of individual binding partners play important roles in TNKS interactions. We demonstrate that GMD forms stable 1:1 complexes with the TNKS ankyrin repeat domain; yet, consistent with results from previous studies, we were unable to detect GMD modification. We also report in vitro evidence that TNKS primarily directs PAR modification to glutamate/aspartate residues. Our results suggest that TNKS-binding partners possess unique sequence and structural features that control binding and PARylation. Ultimately, our findings highlight the binding partner:ankyrin repeat domain interface as a viable target for inhibition of TNKS activity.


Assuntos
Hidroliases/química , Complexos Multiproteicos/química , Estrutura Quaternária de Proteína/genética , Tanquirases/química , Proteínas de Ligação a Telômeros/química , Adenosina Difosfato Ribose/química , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Repetição de Anquirina/genética , Ácido Aspártico/genética , Sítios de Ligação/genética , Ácido Glutâmico/genética , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Poli(ADP-Ribose) Polimerases/química , Ligação Proteica/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Tanquirases/genética , Tanquirases/metabolismo , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Via de Sinalização Wnt/genética
16.
Proc Natl Acad Sci U S A ; 116(37): 18528-18536, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31455731

RESUMO

T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis. The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor RORγt. Here, we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt function in Th17 cells. REV-ERBα binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of RORγt-dependent genes including Il17a and Il17f Furthermore, elevated REV-ERBα expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE). These results suggest that modulating REV-ERBα activity may be used to manipulate Th17 cells in autoimmune diseases.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células Th17/imunologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Loci Gênicos , Células HEK293 , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/agonistas , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Pirrolidinas/farmacologia , Pirrolidinas/uso terapêutico , RNA-Seq , Elementos de Resposta/genética , Células Th17/metabolismo , Tiofenos/farmacologia , Tiofenos/uso terapêutico
17.
Methods Mol Biol ; 2033: 95-115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332750

RESUMO

Site-specific incorporation of artificial functionalities into protein targets is an important tool in both basic and applied research and can be a major challenge to protein chemists. Chemical labeling methods often targeting multiple positions within a protein and therefore suffer from lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this contribution we describe the application of the highly specific trypsin variant named trypsiligase for the site-specific modification of virtual any target protein. We present two general routes of modification resulting in either N- or C-terminal functionalized protein products. Both reaction regimes proceed under mild and bioorthogonal conditions in a short period of time which result in homogeneously modified proteins bearing the artificial functionality exclusively at the desired position. We detail protocols for the expression and purification of trypsiligase as well as the construction of peptide or acyl donor ester probes used as substrates for the biocatalyst. In addition, we provide instructions how to perform the ultimate bioconjugation reactions and finally render assistance for the qualitative and quantitative analysis of the reaction course and outcome.


Assuntos
Motivos de Aminoácidos/genética , Ligases/química , Engenharia de Proteínas/métodos , Tripsina/química , Humanos , Ligases/genética , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/genética , Processamento de Proteína Pós-Traducional/genética , Especificidade por Substrato , Tripsina/genética
18.
PLoS One ; 14(7): e0219435, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31291335

RESUMO

Carrier proteins are four-helix bundles that covalently hold metabolites and secondary metabolites, such as fatty acids, polyketides and non-ribosomal peptides. These proteins mediate the production of many pharmaceutically important compounds including antibiotics and anticancer agents. Acyl carrier proteins (ACPs) can be found as part of a multi-domain polypeptide (Type I ACPs), or as part of a multiprotein complex (Type II). Here, the main focus is on ACP2 and ACP3, domains from the type I trans-AT polyketide synthase MmpA, which is a core component of the biosynthetic pathway of the antibiotic mupirocin. During molecular dynamics simulations of their apo, holo and acyl forms ACP2 and ACP3 both form a substrate-binding surface-groove. The substrates bound to this surface-groove have polar groups on their acyl chain exposed and forming hydrogen bonds with the solvent. Bulky hydrophobic residues in the GXDS motif common to all ACPs, and similar residues on helix III, appear to prohibit the formation of a deep tunnel in type I ACPs and type II ACPs from polyketide synthases. In contrast, the equivalent positions in ACPs from type II fatty acid synthases, which do form a deep solvent-excluded substrate-binding tunnel, have the small residue alanine. During simulation, ACP3 with mutations I61A L36A W44L forms a deep tunnel that can fully bury a saturated substrate in the core of the ACP, in contrast to the surface groove of the wild type ACP3. Similarly, in the ACP from E. coli fatty acid synthase, a type II ACP, mutations can change ligand binding from being inside a deep tunnel to being in a surface groove, thus demonstrating how changing a few residues can modify the possibilities for ligand binding.


Assuntos
Proteína de Transporte de Acila/química , Complexos Multiproteicos/química , Peptídeos/química , Policetídeo Sintases/química , Acinetobacter baumannii/química , Acinetobacter baumannii/genética , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Motivos de Aminoácidos/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Vias Biossintéticas/genética , Sequestro de Carbono/genética , Escherichia coli/genética , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Simulação de Dinâmica Molecular , Complexos Multiproteicos/genética , Mupirocina/biossíntese , Mupirocina/metabolismo , Peptídeos/genética , Mutação Puntual/genética , Policetídeo Sintases/genética , Ligação Proteica
19.
PLoS Biol ; 17(6): e3000341, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31233486

RESUMO

Protein kinases catalyse the phosphorylation of target proteins, controlling most cellular processes. The specificity of serine/threonine kinases is partly determined by interactions with a few residues near the phospho-acceptor residue, forming the so-called kinase-substrate motif. Kinases have been extensively duplicated throughout evolution, but little is known about when in time new target motifs have arisen. Here, we show that sequence variation occurring early in the evolution of kinases is dominated by changes in specificity-determining residues. We then analysed kinase specificity models, based on known target sites, observing that specificity has remained mostly unchanged for recent kinase duplications. Finally, analysis of phosphorylation data from a taxonomically broad set of 48 eukaryotic species indicates that most phosphorylation motifs are broadly distributed in eukaryotes but are not present in prokaryotes. Overall, our results suggest that the set of eukaryotes kinase motifs present today was acquired around the time of the eukaryotic last common ancestor and that early expansions of the protein kinase fold rapidly explored the space of possible target motifs.


Assuntos
Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Especificidade por Substrato/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Animais , Evolução Biológica , Domínio Catalítico , Eucariotos/enzimologia , Evolução Molecular , Humanos , Fosforilação , Células Procarióticas/enzimologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Especificidade por Substrato/fisiologia
20.
Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208

RESUMO

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
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