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1.
Front Microbiol ; 14: 1291523, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38029211

RESUMO

Genomes of bacteria and archaea contain a much larger fraction of unidirectional (serial) gene pairs than convergent or divergent gene pairs. Many of the unidirectional gene pairs have short overlaps of -4 nt and -1 nt. As shown previously, translation of the genes in overlapping unidirectional gene pairs is tightly coupled. Two alternative models for the fate of the post-termination ribosome predict either that overlaps or very short intergenic distances are essential for translational coupling or that the undissociated post-termination ribosome can scan through long intergenic regions, up to hundreds of nucleotides. We aimed to experimentally resolve the contradiction between the two models by analyzing three native gene pairs from the model archaeon Haloferax volcanii and three native pairs from Escherichia coli. A two reporter gene system was used to quantify the reinitiation frequency, and several stop codons in the upstream gene were introduced to increase the intergenic distances. For all six gene pairs from two species, an extremely strong dependence of the reinitiation efficiency on the intergenic distance was unequivocally demonstrated, such that even short intergenic distances of about 20 nt almost completely abolished translational coupling. Bioinformatic analysis of the intergenic distances in all unidirectional gene pairs in the genomes of H. volcanii and E. coli and in 1,695 prokaryotic species representative of 49 phyla showed that intergenic distances of -4 nt or -1 nt (= short gene overlaps of 4 nt or 1 nt) were by far most common in all these groups of archaea and bacteria. A small set of genes in E. coli, but not in H. volcanii, had intergenic distances of around +10 nt. Our experimental and bioinformatic analyses clearly show that translational coupling requires short gene overlaps, whereas scanning of intergenic regions by the post-termination ribosome occurs rarely, if at all. Short overlaps are enriched among genes that encode subunits of heteromeric complexes, and co-translational complex formation requiring precise subunit stoichiometry likely confers an evolutionary advantage that drove the formation and conservation of overlapping gene pairs during evolution.

2.
Antiviral Res ; 124: 101-9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26546752

RESUMO

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of standard antiviral therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy is based on the exploitation of cell-directed signaling inhibitors. The broad antiinfective drug artesunate (ART) offers additional therapeutic options such as oral bioavailability and low levels of toxic side-effects. Here, novel ART-derived compounds including dimers and trimers were synthesized showing further improvements over the parental drug. Antiviral activity and mechanistic aspects were determined leading to the following statements: (i) ART exerts antiviral activity towards human and animal herpesviruses, (ii) no induction of ART-resistant HCMV mutants occurred in vitro, (iii) chemically modified derivatives of ART showed strongly enhanced anti-HCMV efficacy, (iv) NF-κB reporter constructs, upregulated during HCMV replication, could be partially blocked by ART treatment, (v) ART activity analyzed in stable reporter cell clones indicated an inhibition of stimulated NF-κB but not CREB pathway, (vi) solid-phase immobilized ART was able to bind to NF-κB RelA/p65, and (vii) peptides within NF-κB RelA/p65 represent candidates of ART binding as analyzed by in silico docking and mass spectrometry. These novel findings open new prospects for the future medical use of ART and ART-related drug candidates.


Assuntos
Artemisininas/farmacologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Antivirais/química , Antivirais/farmacologia , Artemisininas/química , Artesunato , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citomegalovirus/genética , Farmacorresistência Viral , Células HEK293 , Herpesviridae/efeitos dos fármacos , Humanos , Mutação , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional , Regulação para Cima
3.
J Biol Chem ; 289(42): 29135-47, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25143388

RESUMO

Cys loop receptors are pentameric arrangements of independent subunits that assemble into functional ion channels. Each subunit shows a domain architecture. Functional ion channels can be reconstituted even from independent, nonfunctional subunit domains, as shown previously for GlyRα1 receptors. Here, we demonstrate that this reconstitution is not restricted to α1 but can be transferred to other members of the Cys loop receptor family. A nonfunctional GlyR subunit, truncated at the intracellular TM3-4 loop by a premature stop codon, can be complemented by co-expression of the missing tail portion of the receptor. Compared with α1 subunits, rescue by domain complementation was less efficient when GlyRα3 or the GABAA/C subunit ρ1 was used. If truncation disrupted an alternative splicing cassette within the intracellular TM3-4 loop of α3 subunits, which also regulates receptor desensitization, functional rescue was not possible. When α3 receptors were restored by complementation using domains with and without the spliced insert, no difference in desensitization was found. In contrast, desensitization properties could even be transferred between α1/α3 receptor chimeras harboring or lacking the α3 splice cassette proving that functional rescue depends on the integrity of the alternative splicing cassette in α3. Thus, an intact α3 splicing cassette in the TM3-4 loop environment is indispensable for functional rescue, and the quality of receptor restoration can be assessed from desensitization properties.


Assuntos
Canais Iônicos/química , Receptores de Glicina/química , Processamento Alternativo , Sequência de Aminoácidos , Biotinilação , Cisteína/química , Teste de Complementação Genética , Glicina/química , Células HEK293 , Humanos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
4.
J Biomol Struct Dyn ; 32(10): 1537-45, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-23968404

RESUMO

Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.


Assuntos
Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/química , Biotecnologia/métodos , Enterotoxinas/biossíntese , Enterotoxinas/química , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/isolamento & purificação , Cromatografia de Afinidade , Enterotoxinas/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Isótopos de Nitrogênio , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
FEBS Lett ; 585(3): 511-6, 2011 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-21219903

RESUMO

Metabotropic glutamate receptors (mGluRs) are regulated by interacting proteins that mostly bind to their intracellular C-termini. Here, we investigated if mGluR6, mGluR7a and mGluR8a C-termini form predefined binding surfaces or if they were rather unstructured. Limited tryptic digest of purified peptides argued against the formation of stable globular folds. Circular dichroism, (1)H NMR and (1)H(15)N HSQC spectra indicated the absence of rigid secondary structure elements. Furthermore, we localized short linear binding motifs in the unstructured receptor domains. Our data provide evidence that protein interactions of the analyzed mGluR C-termini are mediated rather by short linear motifs than by preformed folds.


Assuntos
Domínios e Motivos de Interação entre Proteínas , Receptores de Glutamato Metabotrópico/química , Motivos de Aminoácidos , Animais , Dicroísmo Circular , Biologia Computacional/métodos , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Hidrolisados de Proteína/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Ratos , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
Mutagenesis ; 26(2): 261-8, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20861153

RESUMO

Photosafety testing is of concern for the evaluation of personal care products and pharmaceuticals. Current regulatory guidance state that photosafety should be evaluated for compounds that absorb radiation between 290 and 700 nm with relevant exposure in the skin or eyes. However, oversensitivity and the occurrence of 'pseudo-effects' with current in vitro photo(geno)toxicity assays have become a major problem. Furthermore, at this moment, there are no relevant in vitro assays available to identify the photocarcinogenic potential of compounds, which might result in unnecessary in vivo photocarcinogenicity studies for pharmaceutical ingredients or unnecessary dropouts in the development of ingredients of personal care products. For these reasons, availability of a relevant and highly predictive in vitro model from human origin to identify the photogenotoxic and/or photocarcinogenic potential of compounds is viewed as high priority. In the present study, human skin tissue obtained from surgery was used for developing a photomicronucleus test. Prior to investigations of the photogenotoxic potential of 8-methoxypsoralen, tissue viability (lactate production and lactate dehydrogenase leakage), cell proliferation (Ki-67 expression) and the effect of ultraviolet (UV) exposure on viability (MTT test), proliferation (Ki-67 expression) and p53 expression were determined. Results of the present study indicate that ex vivo human skin seems to be a relevant method for safety evaluation of compounds that reach the skin in combination with UV exposure.


Assuntos
Indústria Farmacêutica/métodos , Pele , Adulto , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Antígeno Ki-67/metabolismo , Masculino , Metoxaleno/toxicidade , Micronúcleos com Defeito Cromossômico , Testes para Micronúcleos , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta
8.
J Biol Chem ; 285(6): 3730-3739, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19959465

RESUMO

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs.


Assuntos
Motivos de Aminoácidos , Núcleo Celular/metabolismo , Receptores de Glicina/metabolismo , Transporte Ativo do Núcleo Celular , Adulto , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Receptores de Glicina/genética , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , alfa Carioferinas/genética , alfa Carioferinas/metabolismo
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