RESUMEN
This review focuses on the molecular chaperone ClpB that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and its biological function in selected bacterial pathogens, causing a variety of human infectious diseases, including zoonoses. It has been established that ClpB disaggregates and reactivates aggregated cellular proteins. It has been postulated that ClpB's protein disaggregation activity supports the survival of pathogenic bacteria under host-induced stresses (e.g., high temperature and oxidative stress), which allows them to rapidly adapt to the human host and establish infection. Interestingly, ClpB may also perform other functions in pathogenic bacteria, which are required for their virulence. Since ClpB is not found in human cells, this chaperone emerges as an attractive target for novel antimicrobial therapies in combating bacterial infections.
Asunto(s)
Endopeptidasa Clp/fisiología , Interacciones Microbiota-Huesped/fisiología , ATPasas Asociadas con Actividades Celulares Diversas/fisiología , Animales , Bacterias/patogenicidad , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/etiología , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/fisiología , Zoonosis Bacterianas/etiología , Endopeptidasa Clp/química , Proteínas de Choque Térmico/fisiología , Humanos , Modelos Moleculares , Conformación Proteica , Virulencia/fisiologíaRESUMEN
Bacterial survival during lethal heat stress relies on the cellular ability to reactivate aggregated proteins. This activity is typically executed by the canonical 70-kDa heat shock protein (Hsp70)-ClpB bichaperone disaggregase, which is most widespread in bacteria. The ClpB disaggregase is a member of the ATPase associated with diverse cellular activities protein family and exhibits an ATP-driven threading activity. Substrate binding and stimulation of ATP hydrolysis depends on the Hsp70 partner, which initiates the disaggregation reaction. Recently elevated heat resistance in gamma-proteobacterial species was shown to be mediated by the ATPase associated with diverse cellular activities protein ClpG as an alternative disaggregase. Pseudomonas aeruginosa ClpG functions autonomously and does not cooperate with Hsp70 for substrate binding, enhanced ATPase activity, and disaggregation. With the underlying molecular basis largely unknown, the fundamental differences in ClpG- and ClpB-dependent disaggregation are reflected by the presence of sequence alterations and additional ClpG-specific domains. By analyzing the effects of mutants lacking ClpG-specific domains and harboring mutations in conserved motifs implicated in ATP hydrolysis and substrate threading, we show that the N-terminal, ClpG-specific N1 domain generally mediates protein aggregate binding as the molecular basis of autonomous disaggregation activity. Peptide substrate binding strongly stimulates ClpG ATPase activity by overriding repression by the N-terminal N1 and N2 domains. High ATPase activity requires two functional nucleotide binding domains and drives substrate threading which ultimately extracts polypeptides from the aggregate. ClpG ATPase and disaggregation activity is thereby directly controlled by substrate availability.
Asunto(s)
Antígenos Bacterianos/metabolismo , Endopeptidasa Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Antígenos Bacterianos/fisiología , Endopeptidasa Clp/fisiología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/fisiología , Agregado de Proteínas , Unión Proteica , Dominios Proteicos/genéticaRESUMEN
ClpXP in Escherichia coli is a proteasome degrading protein substrates. It consists of one hexamer of ATPase (ClpX) and two heptamers of peptidase (ClpP). The ClpX binds ATP and translocates the substrate protein into the ClpP chamber by binding and hydrolysis of ATP. At single molecular level, ClpX harnesses cycles of power stroke (dwell and burst) to unfold the substrates, then releases the ADP and Pi. Based on the construction and function of ClpXP, especially the recent progress on how ClpX unfold protein substrates, in this mini-review, a currently proposed single ClpX molecular model system detected by optical tweezers, and its prospective for the elucidation of the mechanism of force generation of ClpX in its power stroke and the subunit interaction with each other, were discussed in detail.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/fisiología , Endopeptidasa Clp/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/enzimología , Chaperonas Moleculares/fisiología , Imagen Individual de Molécula , ATPasas Asociadas con Actividades Celulares Diversas/química , Investigación Biomédica , Endopeptidasa Clp/química , Proteínas de Escherichia coli/química , Redes y Vías Metabólicas , Mitocondrias/fisiología , Modelos Moleculares , Chaperonas Moleculares/química , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/fisiología , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Caseinolytic peptidase P (CLPP) plays a central role in mitochondrial unfolded protein response (mtUPR) and is required for maintaining protein homeostasis in the mitochondria. Global germline Clpp deletion causes female infertility and accelerated follicular depletion. In the current study, we aimed to characterize the role of CLPP in cumulus cell function, gene expression, and mitochondrial ultrastructure. We found that mitochondria in Clpp-deficient cumulus cells have a smaller aspect ratio (length/width) and have a larger coverage area (mitochondrial area/cytoplasmic area) under electron microscopy. These ultrastructural changes were accompanied with diminished expression of mitochondrial dynamics genes. RNA sequencing analysis revealed a significant change in genes related to cellular metabolism in Clpp-deficient cumulus cells compared to wild type. In addition, apoptosis and phagosome pathways were significantly affected. Immunofluorescence assessment confirmed increased apoptotic activity and decreased cell proliferation in cumulus oophorus complexes (COCs) of Clpp-deficient mice. Our findings demonstrate that deletion of CLPP results in significant structural and functional changes in cumulus cells and suggests that mtUPR is required for cumulus cell function.
Asunto(s)
Apoptosis , Células del Cúmulo/fisiología , Endopeptidasa Clp/fisiología , Mitocondrias/fisiología , Dinámicas Mitocondriales , Estrés Fisiológico , Animales , Células del Cúmulo/metabolismo , Endopeptidasa Clp/genética , Femenino , Expresión Génica , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/ultraestructura , Respuesta de Proteína DesplegadaRESUMEN
ß-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of ß-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of ß-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with ß-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by ß-lactams or by inhibiting an early step in WTA biosynthesis. The finding that ß-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that ß-lactams do not kill S. aureus simply by weakening the cell wall.
Asunto(s)
Proteínas Bacterianas/fisiología , Endopeptidasa Clp/fisiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriólisis/efectos de los fármacos , Bacteriólisis/fisiología , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endopeptidasa Clp/genética , Humanos , Modelos Biológicos , Mutación , Oxacilina/farmacología , Staphylococcus aureus/genética , Ácidos Teicoicos/biosíntesis , Tunicamicina/farmacología , beta-Lactamas/farmacologíaRESUMEN
Both α-Synuclein (αSyn) accumulation and mitochondrial dysfunction have been implicated in the pathology of Parkinson's disease (PD). Although studies suggest that αSyn and its missense mutant, A53T, preferentially accumulate in the mitochondria, the mechanisms by which αSyn and mitochondrial proteins regulate each other to trigger mitochondrial and neuronal toxicity are poorly understood. ATP-dependent Clp protease (ClpP), a mitochondrial matrix protease, plays an important role in regulating mitochondrial protein turnover and bioenergetics activity. Here, we show that the protein level of ClpP is selectively decreased in αSyn-expressing cell culture and neurons derived from iPS cells of PD patient carrying αSyn A53T mutant, and in dopaminergic (DA) neurons of αSyn A53T mice and PD patient postmortem brains. Deficiency in ClpP induces an overload of mitochondrial misfolded/unfolded proteins, suppresses mitochondrial respiratory activity, increases mitochondrial oxidative damage and causes cell death. Overexpression of ClpP reduces αSyn-induced mitochondrial oxidative stress through enhancing the level of Superoxide Dismutase-2 (SOD2), and suppresses the accumulation of αSyn S129 phosphorylation and promotes neuronal morphology in neurons derived from PD patient iPS cells carrying αSyn A53T mutant. Moreover, we find that αSyn WT and A53T mutant interact with ClpP and suppress its peptidase activity. The binding of αSyn to ClpP further promotes a distribution of ClpP from soluble to insoluble cellular fraction in vitro and in vivo, leading to reduced solubility of ClpP. Compensating for the loss of ClpP in the substantia nigra of αSyn A53T mice by viral expression of ClpP suppresses mitochondrial oxidative damage, and reduces αSyn pathology and behavioral deficits of mice. Our findings provide novel insights into the mechanism underlying αSyn-induced neuronal pathology, and they suggest that ClpP might be a useful therapeutic target for PD and other synucleinopathies.
Asunto(s)
Endopeptidasa Clp/fisiología , Mitocondrias/enzimología , Mutación Missense , Proteínas del Tejido Nervioso/fisiología , Enfermedad de Parkinson/genética , alfa-Sinucleína/fisiología , Animales , Respiración de la Célula , Células Cultivadas , Neuronas Dopaminérgicas/metabolismo , Endopeptidasa Clp/antagonistas & inhibidores , Endopeptidasa Clp/deficiencia , Mutación con Ganancia de Función , Genes Reporteros , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/deficiencia , Estrés Oxidativo , Pliegue de Proteína , Mapeo de Interacción de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Especies Reactivas de Oxígeno , Proteínas Recombinantes/metabolismo , Solubilidad , Sustancia Negra/metabolismo , Superóxido Dismutasa/metabolismo , alfa-Sinucleína/genéticaRESUMEN
In prokaryotic cells and eukaryotic organelles, the ClpP protease plays an important role in proteostasis. The disruption of the ClpP function has been shown to influence the infectivity and virulence of a number of bacterial pathogens. More recently, ClpP has been found to be involved in various forms of carcinomas and in Perrault syndrome, which is an inherited condition characterized by hearing loss in males and females and by ovarian abnormalities in females. Hence, targeting ClpP is a potentially viable, attractive option for the treatment of different ailments. Herein, the biochemical and cellular activities of ClpP are discussed along with the mechanisms by which ClpP affects bacterial pathogenesis and various human diseases. In addition, a comprehensive overview is given of the new classes of compounds in development that target ClpP. Many of these compounds are currently primarily aimed at treating bacterial infections. Some of these compounds inhibit ClpP activity, while others activate the protease and lead to its dysregulation. The ClpP activators are remarkable examples of small molecules that inhibit protein-protein interactions but also result in a gain of function.
Asunto(s)
Infecciones Bacterianas/fisiopatología , Endopeptidasa Clp/fisiología , Neoplasias/fisiopatología , Adenosina Trifosfatasas/antagonistas & inhibidores , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Proteínas Bacterianas/antagonistas & inhibidores , Endopeptidasa Clp/antagonistas & inhibidores , Endopeptidasa Clp/química , Inhibidores Enzimáticos/farmacología , Proteínas de Choque Térmico/antagonistas & inhibidores , Humanos , Mitocondrias/fisiología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimologíaRESUMEN
Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA). The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection.
Asunto(s)
Proteínas Bacterianas/efectos de los fármacos , Biopelículas/efectos de los fármacos , Pared Celular/efectos de los fármacos , Endopeptidasa Clp/antagonistas & inhibidores , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Hidrolasas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Transactivadores/efectos de los fármacos , Animales , Autólisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Modelos Animales de Enfermedad , Endopeptidasa Clp/genética , Endopeptidasa Clp/fisiología , Genes Bacterianos/genética , Hidrolasas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/fisiología , Transactivadores/metabolismo , Virulencia/genéticaRESUMEN
The flagellum is a rotary motor that enables bacteria to swim in liquids and swarm over surfaces. Numerous global regulators control flagellar assembly in response to cellular and environmental factors. Previous studies have also shown that flagellar assembly is affected by the growth-rate of the cell. However, a systematic study has not yet been described under controlled growth conditions. Here, we investigated the effect of growth rate on flagellar assembly in Escherichia coli using steady-state chemostat cultures where we could precisely control the cell growth-rate. Our results demonstrate that flagellar abundance correlates with growth rate, where faster growing cells produce more flagella. They also demonstrate that this growth-rate dependent control occurs through the expression of the flagellar master regulator, FlhD4C2. Collectively, our results demonstrate that motility is intimately coupled to the growth-rate of the cell.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Escherichia coli/fisiología , Flagelos/fisiología , Endopeptidasa Clp/fisiología , Proteínas de Escherichia coli/fisiología , MovimientoRESUMEN
The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram-positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl-leucyl-leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP-activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP-dependent protein degradation.
Asunto(s)
Depsipéptidos/uso terapéutico , Endopeptidasa Clp/efectos de los fármacos , Endopeptidasa Clp/metabolismo , Adenosina Trifosfatasas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Depsipéptidos/química , Depsipéptidos/farmacología , Endopeptidasa Clp/fisiología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Péptido Hidrolasas/metabolismo , Proteolisis , Serina Endopeptidasas/metabolismoRESUMEN
The importance of NO(â) to immunity is highlighted by the diversity of pathogens that require NO(â)-defensive systems to establish infections. Proteases have been identified to aid pathogens in surviving macrophage attack, inspiring us to investigate their role during NO(â) stress in Escherichia coli. We discovered that the elimination of ClpP largely impaired NO(â) detoxification by E. coli. Using a quantitative model of NO(â) stress, we employed an ensemble-guided approach to identify the underlying mechanism. Iterations of in silico analyses and corresponding experiments identified the defect to result from deficient transcript levels of hmp, which encodes NO(â) dioxygenase. Interestingly, the defect was not confined to hmp, as ΔclpP imparted widespread perturbations to the expression of NO(â)-responsive genes. This work identified a target for anti-infective therapies based on disabling NO(â) defenses, and demonstrated the utility of model-based approaches for exploring the complex, systems-level stress exerted by NO(â).
Asunto(s)
Dihidropteridina Reductasa/genética , Endopeptidasa Clp/fisiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , Escherichia coli/genética , Hemoproteínas/genética , NADH NADPH Oxidorreductasas/genética , Óxido Nítrico/metabolismo , ARN Mensajero/análisis , Biocatálisis , Escherichia coli/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de ARN , Estrés Fisiológico , Transcripción GenéticaRESUMEN
Intra-plastid proteolysis is essential in plastid biogenesis, differentiation and plastid protein homeostasis (proteostasis). We provide a comprehensive review of the Clp protease system present in all plastid types and we draw lessons from structural and functional information of bacterial Clp systems. The Clp system plays a central role in plastid development and function, through selective removal of miss-folded, aggregated, or otherwise unwanted proteins. The Clp system consists of a tetradecameric proteolytic core with catalytically active ClpP and inactive ClpR subunits, hexameric ATP-dependent chaperones (ClpC,D) and adaptor protein(s) (ClpS1) enhancing delivery of subsets of substrates. Many structural and functional features of the plastid Clp system are now understood though extensive reverse genetics analysis combined with biochemical analysis, as well as large scale quantitative proteomics for loss-of-function mutants of Clp core, chaperone and ClpS1 subunits. Evolutionary diversification of Clp system across non-photosynthetic and photosynthetic prokaryotes and organelles is illustrated. Multiple substrates have been suggested based on their direct interaction with the ClpS1 adaptor or screening of different loss-of-function protease mutants. The main challenge is now to determine degradation signals (degrons) in Clp substrates and substrate delivery mechanisms, as well as functional interactions of Clp with other plastid proteases. This article is part of a Special Issue entitled: Chloroplast Biogenesis.
Asunto(s)
Endopeptidasa Clp/fisiología , Plastidios/metabolismo , Endopeptidasa Clp/química , Estabilidad de Enzimas , Evolución Molecular , Proteolisis , ProteomaRESUMEN
The distribution of essential metal ions over subcellular compartments for use as cofactors requires control of membrane transporters. PAA2/HMA8 is a copper-transporting P1B -type ATPase in the thylakoid membrane, required for the maturation of plastocyanin. When copper is highly available to the plant this transporter is degraded, which implies the action of a protease. In order to identify the proteolytic machinery responsible for PAA2/HMA8 turnover in Arabidopsis, mutant lines defective in five different chloroplast protease systems were analyzed. Plants defective in the chloroplast caseinolytic protease (Clp) system were specifically impaired in PAA2/HMA8 protein turnover on media containing elevated copper concentrations. However, the abundance of a core Clp component was not directly affected by copper. Furthermore, the expression and activity of both cytosolic and chloroplast-localized superoxide dismutases (SODs), which are known to be dependent on copper, were not altered in the clp mutants, indicating that the loss of PAA2/HMA8 turnover in these lines was not caused by a lack of stromal copper. The results suggest that copper excess in the stroma triggers selection of the thylakoid-localized PAA2 transporter for degradation by the Clp protease, but not several other chloroplast proteases, and support a novel role for this proteolytic system in cellular copper homeostasis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Cobre/metabolismo , Endopeptidasa Clp/fisiología , Adenosina Trifosfatasas , Arabidopsis/genética , Proteínas de Transporte de Catión , ATPasas Transportadoras de Cobre , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Homeostasis , Proteínas de Transporte de Membrana/metabolismo , Mutación , Superóxido Dismutasa/metabolismo , Tilacoides/metabolismoRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic mutant, Mtb:ΔRv2745c, is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb as well as to a complemented strain. Together with the fact that the expression of Rv2745c is strongly induced in response to redox stress, these results strongly implicate a role for ClgR in the management of intraphagosomal redox stress. Additionally, we observed that redox stress led to the dysregulation of the expression of the σH/σE regulon in the isogenic mutant, Mtb:ΔRv2745c. Furthermore, induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions that need to be elucidated. Our data, when taken together with that obtained by other groups, indicates that ClgR plays diverse roles in multiple regulatory networks in response to different stress conditions. In addition to redox stress, the expression of Rv2745c correlates with the expression of genes involved in sulfate assimilation as well as in response to hypoxia and reaeration. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in-vivo, regardless of its induction of the Clp proteolytic pathway.
Asunto(s)
Endopeptidasa Clp/fisiología , Mycobacterium tuberculosis , Estrés Oxidativo/genética , Factores de Transcripción/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Diamida/farmacología , Endopeptidasa Clp/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Análisis por Micromatrices , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Organismos Modificados Genéticamente , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Factores de Transcripción/genética , Transcriptoma/efectos de los fármacosRESUMEN
The anaerobic global regulator FNR from Escherichia coli is a [4Fe-4S](2+) cluster-containing dimer that is inactivated by O(2) through disruption of the Fe-S cluster and conversion to the monomeric apoprotein. It was shown that apo-FNR is subject to ClpXP proteolysis, and two recognition sites, amino acids 5-11 and amino acids 249 and 250, are responsible for targeting FNR to the protease. However, how the exposure of these sites is mediated such that only apo-FNR is recognized by the ClpXP protease and is degraded in a regulated manner so that a sufficient and similar FNR level is maintained in both anaerobic and aerobic conditions is unknown. To investigate this, we performed three-alanine scanning on amino acids 2-19 and 236-250 that are in the proximity of the two ClpXP recognition sites, and their functions remain unknown. We found that three-alanine substitution of residues 239-241 (LAQ239-241A(3)) and 242-244 (LAG242-244A(3)) caused reduced FNR protein levels, transcription activities, and growth rates under anaerobic conditions. In vivo degradation assays demonstrated that these mutants were degraded significantly faster than the wild type (WT), and either deletion of clpXP or blocking the ClpXP recognition site of amino acids 249 and 250 stabilizes these proteins. Circular dichroism analysis revealed that introduction of LAQ239-241A(3) caused conformational changes with a significant loss of secondary structures in both WT and an O(2) stable FNR dimer, FNR D154A. We propose that the region of amino acids 239-244 plays a negative role in the proteolysis of FNR by promoting a structural fold that limits the exposure of the proximal ClpXP site to the protease.
Asunto(s)
Regulación hacia Abajo/fisiología , Endopeptidasa Clp/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteínas Hierro-Azufre/antagonistas & inhibidores , Proteínas Hierro-Azufre/química , Fragmentos de Péptidos/química , Secuencia Conservada , Endopeptidasa Clp/fisiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , Proteínas Hierro-Azufre/genética , Fragmentos de Péptidos/fisiología , Proteolisis , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Factores de Transcripción/fisiologíaRESUMEN
Programmed cell death is a gene-directed process involved in the development and homeostasis of multicellular organisms. The most common mode of programmed cell death is apoptosis, which is characterized by a stereotypical set of biochemical and morphological hallmarks. Here we report that Escherichia coli also exhibit characteristic markers of apoptosis-including phosphatidylserine exposure, chromosome condensation, and DNA fragmentation-when faced with cell death-triggering stress, namely bactericidal antibiotic treatment. Notably, we also provide proteomic and genetic evidence for the ability of multifunctional RecA to bind peptide sequences that serve as substrates for eukaryotic caspases, and regulation of this phenotype by the protease, ClpXP, under conditions of cell death. Our findings illustrate that prokaryotic organisms possess mechanisms to dismantle and mark dying cells in response to diverse noxious stimuli and suggest that elaborate, multilayered proteolytic regulation of these features may have evolved in eukaryotes to harness and exploit their deadly potential.
Asunto(s)
Ampicilina/farmacología , Antibacterianos/farmacología , Apoptosis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Gentamicinas/farmacología , Norfloxacino/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Caspasas/metabolismo , Caspasas/fisiología , Cromosomas Bacterianos/efectos de los fármacos , Fragmentación del ADN , Endopeptidasa Clp/fisiología , Escherichia coli/citología , Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , Etiquetado Corte-Fin in Situ , Fosfatidilserinas/análisis , Rec A Recombinasas/metabolismo , Rec A Recombinasas/fisiología , Respuesta SOS en Genética/efectos de los fármacos , Estrés Fisiológico , Especificidad por SustratoRESUMEN
Recently, single-molecule force spectroscopy techniques have provided unprecedented opportunities to apply and to quantify forces that guide protein (un-)folding. A new study provides fascinating insights into the sophisticated mechanism by which an ATP-fueled proteolytic machine generates mechanical forces to unfold and translocate multidomain substrates.
Asunto(s)
Adenosina Trifosfato/metabolismo , Endopeptidasa Clp/fisiología , Complejo de la Endopetidasa Proteasomal/fisiología , Modelos Biológicos , Transporte de Proteínas , Desplegamiento Proteico , Transducción de SeñalRESUMEN
Staphylococcus aureus is a multidrug-resistant pathogen that not only causes a diverse array of human diseases, but also is able to survive in potentially dry and stressful environments, such as the human nose, on skin and on inanimate surfaces such as clothing and surfaces. This study investigated parameters governing desiccation tolerance of S. aureus and identified several components involved in the process. Initially, the role of environmental parameters such as temperature, growth phase, cell density, desiccation time and protectants in desiccation tolerance were determined. This established a robust model of desiccation tolerance in which S. aureus has the ability to survive on dry plastic surfaces for more than 1,097 days. Using a combination of a random screen and defined mutants, clpX, sigB and yjbH were identified as being required for desiccation tolerance. ClpX is a part of the ATP-dependent ClpXP protease, important for protein turnover, and YjbH has a proposed linked function. SigB is an accessory sigma factor with a role in generalized stress resistance. Understanding the molecular mechanisms that govern desiccation tolerance may determine the break points to be exploited to prevent the spread of this dangerous pathogen in hospitals and communities.
Asunto(s)
Staphylococcus aureus/genética , Proteínas Bacterianas/fisiología , Desecación , Endopeptidasa Clp/fisiología , Ambiente , Genes Bacterianos , Prueba de Complementación Genética , Mutación , Factor sigma/fisiologíaRESUMEN
ClpX is a member of Hsp100 (heat-shock protein) family which is conserved among organisms. Hsp100/ Clp implicates in stress resistance, intracellular protein turn-over, DNA replication and regulation of gene expression. Tuberculosis remains one of the major threats to human health. In pathogens, ClpX protease plays an important role in the gene expression regulation, pathogenesis, and resistance of immune stress. The structure, substrates and target genes of ClpX are summarized in this study. The biological function of M. tuberculosis ClpX, such as gene expression regulation, pathogenesis, intracellular survival and persistence, evolution and structural feature, substrates is the focus of this summary.
Asunto(s)
Adenosina Trifosfatasas/química , Endopeptidasa Clp/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Chaperonas Moleculares/química , Conformación Proteica , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/fisiología , Secuencia de Aminoácidos , Northern Blotting , Endopeptidasa Clp/metabolismo , Endopeptidasa Clp/fisiología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/fisiología , Humanos , Hidrolasas/química , Hidrolasas/fisiología , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiología , Datos de Secuencia Molecular , Serina Endopeptidasas/química , Serina Endopeptidasas/fisiologíaRESUMEN
BACKGROUND: Legionella pneumophila, the intracellular bacterial pathogen that causes Legionnaires' disease, exhibit characteristic transmission traits such as elevated stress tolerance, shortened length and virulence during the transition from the replication phase to the transmission phase. ClpP, the catalytic core of the Clp proteolytic complex, is widely involved in many cellular processes via the regulation of intracellular protein quality. RESULTS: In this study, we showed that ClpP was required for optimal growth of L. pneumophila at high temperatures and under several other stress conditions. We also observed that cells devoid of clpP exhibited cell elongation, incomplete cell division and compromised colony formation. Furthermore, we found that the clpP-deleted mutant was more resistant to sodium stress and failed to proliferate in the amoebae host Acanthamoeba castellanii. CONCLUSIONS: The data present in this study illustrate that the ClpP protease homologue plays an important role in the expression of transmission traits and cell division of L. pneumophila, and further suggest a putative role of ClpP in virulence regulation.