RESUMO
Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrheal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large-scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas-dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by single guide RNA (sgRNA) to introduce targeted CâT transitions, resulting in internal stop codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter, we were able to generate loss-of-function mutants in both Escherichia coli and Shigella flexneri with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion was in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Furthermore, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella flexneri genes and found the mutant phenotype to be similar to those of the reported gene deletion mutants, with no apparent polar effects at the phenotype level. This method can be used in a 96-well-plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days. IMPORTANCE Loss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches systems biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen-Shigella. This pathogen causes high morbidity and mortality in children, and antibiotic-resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of the utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method has the potential to be programmed to generate multiple mutants in a single, massively parallel reaction. By virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.
Assuntos
Shigella flexneri , Shigella , Criança , Humanos , Shigella flexneri/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , Virulência/genética , Mutagênese , Plasmídeos/genética , Shigella/genética , Escherichia coli/genética , CromossomosRESUMO
Microorganisms are generally sensed by receptors recognizing microbial molecules, which evoke changes in cellular activities and gene expression. Bacterial pathogens induce secretion of the danger signal ATP as an early alert response of intestinal epithelial cells, initiating overt inflammation. However, what triggers ATP secretion during infection is unclear. Here we show that the inherently mechanosensitive plasma membrane channel PIEZO1 acts as a sensor for bacterial entry. PIEZO1 is mechanically activated by invasion-induced membrane ruffles upstream of Ca2+ influx and ATP secretion. Mimicking mechanical stimuli of pathogen uptake with sterile beads equally elicits ATP secretion. Chemical or genetic PIEZO1 inactivation inhibits mechanically induced ATP secretion. Moreover, chemical or mechanical PIEZO1 activation evokes gene expression in immune and barrier pathways. Thus, mechanosensation of invasion-induced plasma membrane distortion initiates immune signaling upon infection, independently of detection of microbial molecules. Hence, PIEZO1-dependent detection of infection is driven by physical signals instead of chemical ligands.
Assuntos
Canais Iônicos , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologiaRESUMO
BACKGROUND: Functionally related genes in bacteria are often organized and transcribed as polycistronic transcriptional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci. RESULTS: Cas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator's sequence, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (ΔatpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non-polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase. CONCLUSIONS: We conclude that Cas12a-mediated mutagenesis is an efficient simple system to generate polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intracellular ATP in the markerless ΔatpIBEFHAGDC mutant.
Assuntos
Sistemas CRISPR-Cas , Escherichia coli , Trifosfato de Adenosina , Escherichia coli/genética , Edição de Genes , Mutagênese , ÓperonRESUMO
Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Proteína BRCA2/metabolismo , Células Cultivadas , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , Proteínas Nucleares/genética , Rad51 Recombinase/metabolismo , Fatores de Transcrição/genética , CoesinasRESUMO
Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.
Assuntos
Antineoplásicos/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Genes Reguladores , Leucemia Mieloide/tratamento farmacológico , Proteínas Nucleares/metabolismo , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular , Relação Dose-Resposta a Droga , Humanos , Leucemia Mieloide/genética , Terapia de Alvo Molecular , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Purinas/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ribossomos/metabolismo , Análise de Sequência de RNA , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Genoma Humano/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , CoesinasRESUMO
The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Transcrição Gênica , Fator de Ligação a CCCTC , Humanos , Proteínas Repressoras/metabolismo , Imagem Individual de Molécula , Fatores de Tempo , CoesinasRESUMO
Cohesion between sister chromatids is established during DNA replication but needs to be maintained to enable proper chromosome-spindle attachments in mitosis or meiosis. Cohesion is mediated by cohesin, but also depends on cohesin acetylation and sororin. Sororin contributes to cohesion by stabilizing cohesin on DNA. Sororin achieves this by inhibiting WAPL, which otherwise releases cohesin from DNA and destroys cohesion. Here we describe mouse models which enable the controlled depletion of sororin by gene deletion or auxin-induced degradation. We show that sororin is essential for embryonic development, cohesion maintenance, and proper chromosome segregation. We further show that the acetyltransferases ESCO1 and ESCO2 are essential for stabilizing cohesin on chromatin, that their only function in this process is to acetylate cohesin's SMC3 subunit, and that DNA replication is also required for stable cohesin-chromatin interactions. Unexpectedly, we find that sororin interacts dynamically with the cohesin complexes it stabilizes. This implies that sororin recruitment to cohesin does not depend on the DNA replication machinery or process itself, but on a property that cohesin acquires during cohesion establishment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Acetiltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Desenvolvimento Embrionário , CamundongosRESUMO
Bacterial type 4 pili (T4P) are long flexible fibers involved in adhesion, DNA uptake, phage transduction, aggregation and a flagella-independent movement called "twitching motility". T4P comprise thousands of copies of the major pilin subunit, which is initially inserted in the plasma membrane, processed and assembled into dynamic helical filaments. T4P are crucial for host colonization and virulence of many Gram-negative bacteria. In enterohemorrhagic Escherichia coli the T4P, called hemorrhagic coli pili (HCP) promote cell adhesion, motility, biofilm formation and signaling. To understand the mechanism of HCP assembly and function, we analyzed the structure of the major subunit prepilin peptidase-dependent protein D (PpdD) (also called HcpA), a 15 kDa pilin with two potential disulfide bonds. Here we present the (1)H, (15)N and (13)C backbone and side chain resonance assignments of the C-terminal globular domain of PpdD as a first step to its structural determination.
Assuntos
Escherichia coli Êntero-Hemorrágica/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Isótopos de Carbono , Hidrogênio , Isótopos de NitrogênioRESUMO
Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.
Assuntos
Cromatina/química , Cromatina/metabolismo , Segregação de Cromossomos , Proteínas/metabolismo , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Cromossomos de Mamíferos/química , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Endopeptidases/metabolismo , Regulação da Expressão Gênica/genética , Genes myc/genética , Interfase , Camundongos , Mitose , Prófase , Proteínas/genética , Separase , CoesinasRESUMO
Type II secretion systems (T2SSs) share common origins and structure with archaeal flagella (archaella) and pili, bacterial competence systems and type IV pili. All of these systems use a conserved ATP-powered machinery to assemble helical fibers that are anchored in the plasma membrane. The T2SSs assemble pseudopili, periplasmic filaments that promote extracellular secretion of folded periplasmic proteins. Comparative analysis of T2SSs and related fiber assembly nanomachines might provide important clues on their functional specificities and dynamics. This review focuses on recent developments in the study of pseudopilus structure and biogenesis, and discusses mechanistic models of pseudopilus function in protein secretion.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Membrana Celular/metabolismo , Fímbrias Bacterianas/metabolismo , Bactérias/genética , Proteínas de Bactérias/genética , Membrana Celular/genética , Fímbrias Bacterianas/genética , Transporte ProteicoRESUMO
In Gram-negative bacteria, type IV pilus assembly (T4PS) and type II secretion (T2SS) systems polymerize inner membrane proteins called major pilins or pseudopilins respectively, into thin filaments. Four minor pilins are required in both systems for efficient fibre assembly. Escherichia coli K-12 has a set of T4PS assembly genes that are silent under standard growth conditions. We studied the heterologous assembly of the E. coli type IV pilin PpdD by the Klebsiella oxytoca T2SS called the Pul system. PpdD pilus assembly in this context depended on the expression of the K. oxytoca minor pseudopilin genes pulHIJK or of the E. coli minor pilin genes ppdAB-ygdB-ppdC. The E. coli minor pilins restored assembly of the major pseudopilin PulG in a pulHIJK mutant, but not the secretion of the T2SS substrate pullulanase. Thus, minor pilins and minor pseudopilins are functionally interchangeable in initiating major pilin assembly, further extending the fundamental similarities between the two systems. The data suggest that, in both systems, minor pilins activate the assembly machinery through a common self-assembly mechanism. When produced together, PulG and PpdD assembled into distinct homopolymers, establishing major pilins as key determinants of pilus elongation and structure.
Assuntos
Sistemas de Secreção Bacterianos/genética , Escherichia coli K12/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Klebsiella oxytoca/enzimologia , Substâncias Macromoleculares/metabolismo , Escherichia coli K12/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Deleção de Genes , Teste de Complementação Genética , Klebsiella oxytoca/genética , Multimerização Proteica , Subunidades ProteicasRESUMO
In Gram-negative bacteria, type II secretion systems (T2SS) assemble inner membrane proteins of the major pseudopilin PulG (GspG) family into periplasmic filaments, which could drive protein secretion in a piston-like manner. Three minor pseudopilins PulI, PulJ and PulK are essential for protein secretion in the Klebsiella oxytoca T2SS, but their molecular function is unknown. Here, we demonstrate that together these proteins prime pseudopilus assembly, without actively controlling its length or secretin channel opening. Using molecular dynamics, bacterial two-hybrid assays, cysteine crosslinking and functional analysis, we show that PulI and PulJ nucleate filament assembly by forming a staggered complex in the plasma membrane. Binding of PulK to this complex results in its partial extraction from the membrane and in a 1-nm shift between their transmembrane segments, equivalent to the major pseudopilin register in the assembled PulG filament. This promotes fully efficient pseudopilus assembly and protein secretion. Therefore, we propose that PulI, PulJ and PulK self-assembly is thermodynamically coupled to the initiation of pseudopilus assembly, possibly setting the assembly machinery in motion.
Assuntos
Fímbrias Bacterianas/fisiologia , Proteínas de Bactérias/metabolismo , Klebsiella oxytoca/fisiologia , Ligação ProteicaRESUMO
Many gram-negative bacteria secrete specific proteins via the type II secretion systems (T2SS). These complex machineries share with the related archaeal flagella and type IV pilus (T4P) biogenesis systems the ability to assemble thin, flexible filaments composed of small, initially inner membrane-localized proteins called "pilins." In the T2SS from Klebsiella oxytoca, periplasmic pseudopili that are essential for pullulanase (PulA) secretion extend beyond the bacterial surface and form pili when the major pilin PulG is overproduced. Here, we describe the detailed, experimentally validated structure of the PulG pilus generated from crystallographic and electron microscopy data by a molecular modeling approach. Two intermolecular salt bridges crucial for function were demonstrated using single and complementary charge inversions. Double-cysteine substitutions in the transmembrane segment of PulG led to position-specific cross-linking of protomers in assembled pili. These biochemical data provided information on residue distances in the filament that were used to derive a refined model of the T2SS pilus at pseudoatomic resolution. PulG is organized as a right-handed helix of subunits, consistent with protomer organization in gonococcal T4P. The conserved character of residues involved in key hydrophobic and electrostatic interactions within the major pseudopilin family supports the general relevance of this model for T2SS pseudopilus structure.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Klebsiella oxytoca/metabolismo , Modelos Moleculares , Substituição de Aminoácidos/genética , Reagentes de Ligações Cruzadas/metabolismo , Cisteína/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredução , Estrutura Secundária de Proteína , Reprodutibilidade dos Testes , Eletricidade EstáticaRESUMO
In mesophiles, triosephosphate isomerase (TIM) is an obligated homodimer. We have previously shown that monomeric folding intermediates are common in the chemical unfolding of TIM, where dissociation provides 75% of the overall conformational stability of the dimer. However, analysis of the crystallographic structure shows that, during unfolding, intermonomeric contacts contribute to only 5% of the overall increase in accessible surface area. In this work several methodologies were used to characterize the thermal dissociation and unfolding of the TIM from Entamoeba histolytica (EhTIM) and a monomeric variant obtained by chemical derivatization (mEhTIM). During EhTIM unfolding, sequential transitions corresponding to dimer dissociation into a compact monomeric intermediate followed by unfolding and further aggregation of the intermediate occurred. In the case of mEhTIM, a single transition, analogous to the second transition of EhTIM, was observed. Calorimetric, spectroscopic, hydrodynamic, and functional evidence shows that dimer dissociation is not restricted to localized interface reorganization. Dissociation represents 55% (DeltaH(Diss) = 146.8 kcal mol(-1)) of the total enthalpy change (DeltaH(Tot) = 266 kcal mol(-1)), indicating that this process is linked to substantial unfolding. We propose that, rather than a rigid body process, subunit assembly is best represented by a fly-casting mechanism. In TIM, catalysis is restricted to the dimer; therefore, the interface can be viewed as the final nucleation motif that directs assembly, folding, and function.
Assuntos
Entamoeba histolytica/enzimologia , Triose-Fosfato Isomerase/química , Animais , Dimerização , Modelos Moleculares , Desnaturação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas , TermodinâmicaRESUMO
In haloarchaea, sensory rhodopsin II (SRII) mediates a photophobic response to avoid photo-oxidative damage in bright light. Upon light activation the receptor undergoes a conformational change that activates a tightly bound transducer molecule (HtrII), which in turn by a chain of homologous reactions transmits the signal to the chemotactic eubacterial two-component system. Here, using single-molecule force spectroscopy, we localize and quantify changes to the intramolecular interactions within SRII of Natronomonas pharaonis (NpSRII) upon NpHtrII binding. Transducer binding affected the interactions at transmembrane alpha helices F and G of NpSRII to which the transducer was in contact. Remarkably, the interactions were distributed asymmetrically and significantly stabilized alpha helix G entirely but alpha helix F only at its extracellular tip. These findings provide unique insights into molecular mechanisms that "prime" the complex for signaling, and guide the receptor toward transmitting light-activated structural changes to its cognate transducer.
Assuntos
Proteínas Arqueais/química , Halorrodopsinas/química , Conformação Proteica , Rodopsinas Sensoriais/química , Análise Espectral/métodos , Halobacteriaceae/química , Modelos Moleculares , Ligação ProteicaRESUMO
Mechanical single-molecule techniques offer exciting possibilities to investigate protein folding and stability in native environments at submolecular resolution. By applying a free-energy reconstruction procedure developed by Hummer and Szabo, which is based on a statistical theorem introduced by Jarzynski, we determined the unfolding free energy of the membrane proteins bacteriorhodopsin (BR), halorhodopsin, and the sodium-proton antiporter NhaA. The calculated energies ranged from 290.5 kcal/mol for BR to 485.5 kcal/mol for NhaA. For the remarkably stable BR, the equilibrium unfolding free energy was independent of pulling rate and temperature ranging between 18 and 42 degrees C. Our experiments also revealed heterogeneous energetic properties in individual transmembrane helices. In halorhodopsin, the stabilization of a short helical segment yielded a characteristic signature in the energy profile. In NhaA, a pronounced peak was observed at a functionally important site in the protein. Since a large variety of single- and multispan membrane proteins can be tackled in mechanical unfolding experiments, our approach provides a basis for systematically elucidating energetic properties of membrane proteins with the resolution of individual secondary-structure elements.
Assuntos
Bacteriorodopsinas/química , Proteínas de Membrana/química , Bacteriorodopsinas/metabolismo , Estabilidade de Medicamentos , Cinética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Desnaturação Proteica , Dobramento de Proteína , TermodinâmicaRESUMO
The biofunctionalization of materials creates interfaces on which proteins, cells, or tissues can fulfill native or desired tasks. Here we report how to control the assembly of type I collagen into well-defined nanoscopic matrices of different patterns. Collagen fibrils in these ultrathin (approximately 3 nm) matrices maintained their native structure as observed in vivo. This opens up the possibility to create programmable biofunctionalized matrices using collagen-binding proteins or proteins fused with collagen-binding domains. Applied to eukaryotic cells, these nanostructured matrices can direct cellular processes such as adhesion, orientation and migration.
Assuntos
Biotecnologia/instrumentação , Colágenos Fibrilares/química , Colágenos Fibrilares/ultraestrutura , Nanoestruturas/química , Animais , Fenômenos Biomecânicos , Soluções Tampão , Adesão Celular , Movimento Celular , Polaridade Celular , Eletrólitos , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Estrutura Quaternária de Proteína , RatosRESUMO
We have characterized early steps of alpha(2)beta(1) integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of alpha(2)beta(1) as a collagen type I receptor, alpha(2)beta(1)-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas alpha(2)beta(1)-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin-collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin-collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of alpha(2)beta(1)-mediated adhesion as weak initial, single-integrin-mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility.
Assuntos
Adesão Celular/fisiologia , Colágeno Tipo I/metabolismo , Integrina alfa2beta1/metabolismo , Actomiosina/metabolismo , Amidas/farmacologia , Animais , Sítios de Ligação , Fenômenos Biomecânicos , Células CHO , Adesão Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacocinética , Adesões Focais/metabolismo , Humanos , Integrina alfa2beta1/genética , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Microscopia de Força Atômica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Quinases Associadas a rhoRESUMO
Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.