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1.
Protein Sci ; 32(7): e4704, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37312651

RESUMEN

Pulse EPR measurements provide information on distances and distance distributions in proteins but require the incorporation of pairs of spin labels that are usually attached to engineered cysteine residues. In previous work, we demonstrated that efficient in vivo labeling of the Escherichia coli outer membrane vitamin B12 transporter, BtuB, could only be achieved using strains defective in the periplasmic disulfide bond formation (Dsb) system. Here, we extend these in vivo measurements to FecA, the E. coli ferric citrate transporter. As seen for BtuB, pairs of cysteines cannot be labeled when the protein is present in a standard expression strain. However, incorporating plasmids that permit an arabinose induced expression of FecA into a strain defective in the thiol disulfide oxidoreductase, DsbA, enables efficient spin-labeling and pulse EPR of FecA in cells. A comparison of the measurements made on FecA in cells with measurements made in reconstituted phospholipid bilayers suggests that the cellular environment alters the behavior of the extracellular loops of FecA. In addition to these in situ EPR measurements, the use of a DsbA minus strain for the expression of BtuB improves the EPR signals and pulse EPR data obtained in vitro from BtuB that is labeled, purified, and reconstituted into phospholipid bilayers. The in vitro data also indicate the presence of intermolecular BtuB-BtuB interactions, which had not previously been observed in a reconstituted bilayer system. This result suggests that in vitro EPR measurements on other outer membrane proteins would benefit from protein expression in a DsbA minus strain.


Asunto(s)
Proteínas de Escherichia coli , Proteínas de Transporte de Membrana , Proteínas de Transporte de Membrana/química , Escherichia coli/metabolismo , Disulfuros/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Marcadores de Spin , Chaperonas Moleculares/metabolismo , Receptores de Superficie Celular/química
2.
Protein Sci ; 31(9): e4402, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36040258

RESUMEN

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful tool that monitors protein dynamics in solution. However, the reversible nature of HDX labels has largely limited the application to in vitro systems. Here, we describe a protocol for measuring HDX-MS in living Escherichia coli cells applied to BtuB, a TonB-dependent transporter found in outer membranes (OMs). BtuB is a convenient and biologically interesting system for testing in vivo HDX-MS due to its controllable HDX behavior and large structural rearrangements that occur during the B12 transport cycle. Our previous HDX-MS study in native OMs provided evidence for B12 binding and breaking of a salt bridge termed the Ionic Lock, an event that leads to the unfolding of the amino terminus. Although purified OMs provide a more native-like environment than reconstituted systems, disruption of the cell envelope during lysis perturbs the linkage between BtuB and the TonB complex that drives B12 transport. The in vivo HDX response of BtuB's plug domain (BtuBp) to B12 binding corroborates our previous in vitro findings that B12 alone is sufficient to break the Ionic Lock. In addition, we still find no evidence of B12 binding-induced unfolding in other regions of BtuBp that could enable B12 passage. Our protocol was successful in reporting on the HDX of several endogenous E. coli proteins measured in the same measurement. Our success in performing HDX in live cells opens the possibility for future HDX-MS studies in a native cellular environment. IMPORTANCE: We present a protocol for performing in vivo HDX-MS, focusing on BtuB, a protein whose native membrane environment is believed to be mechanistically important for B12 transport. The in vivo HDX-MS data corroborate the conclusions from our previous in vitro HDX-MS study of the allostery initiated by B12 binding. Our success with BtuB and other proteins opens the possibility for performing additional HDX-MS studies in a native cellular environment.


Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Proteínas de la Membrana Bacteriana Externa/química , Medición de Intercambio de Deuterio , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Proteínas de Transporte de Membrana/química , Vitamina B 12/metabolismo
3.
Biophys J ; 117(8): 1476-1484, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31582182

RESUMEN

Recent advances in the application of electron paramagnetic resonance spectroscopy have demonstrated that it is possible to obtain structural information on bacterial outer membrane (OM) proteins in intact cells from extracellularly labeled cysteines. However, in the Escherichia coli OM B12 transport protein, BtuB, the double labeling of many cysteine pairs is not possible in a wild-type K12-derived E. coli strain. It has also not yet been possible to selectively label single or paired cysteines that face the periplasmic space. Here, we demonstrate that the inability to produce reactive cysteine residues in pairs is a result of the disulfide bond formation system, which functions to oxidize pairs of free-cysteine residues. Mutant strains that are dsbA or dsbB null facilitate labeling pairs of cysteines. Moreover, we demonstrate that the double labeling of sites on the periplasmic-facing surface of BtuB is possible using a dsbA null strain. BtuB is found to exhibit different structures and structural changes in the cell than it does in isolated OMs or reconstituted systems, and the ability to label and perform electron paramagnetic resonance in cells is expected to be applicable to a range of other bacterial OM proteins.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Proteínas de Transporte de Membrana/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Cisteína/metabolismo , Disulfuros/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutación con Pérdida de Función , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteína Disulfuro Isomerasas/genética
4.
Nat Commun ; 10(1): 3904, 2019 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-31467284

RESUMEN

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.


Asunto(s)
Fusión de Membrana/fisiología , Proteínas SNARE/metabolismo , Vesículas Secretoras/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Transporte Biológico , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Membrana Celular/metabolismo , Exocitosis , Humanos , Insulina , Proteínas Munc18/metabolismo , Proteínas del Tejido Nervioso , Células PC12 , Ratas
5.
ACS Chem Biol ; 14(8): 1760-1766, 2019 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-31260252

RESUMEN

The Gram-negative bacterium Francisella tularensis secretes the siderophore rhizoferrin to scavenge necessary iron from the environment. Rhizoferrin, also produced by a variety of fungi and bacteria, comprises two citrate molecules linked by amide bonds to a central putrescine (diaminobutane) moiety. Genetic analysis has determined that rhizoferrin production in F. tularensis requires two enzymes: FslA, a siderophore synthetase of the nonribosomal peptide synthetase-independent siderophore synthetase (NIS) family, and FslC, a pyridoxal-phosphate-dependent decarboxylase. To discern the steps in the biosynthetic pathway, we tested F. tularensis strain LVS and its ΔfslA and ΔfslC mutants for the ability to incorporate potential precursors into rhizoferrin. Unlike putrescine supplementation, supplementation with ornithine greatly enhanced siderophore production by LVS. Radioactivity from L-[U-14C] ornithine, but not from L-[1-14C] ornithine, was efficiently incorporated into rhizoferrin by LVS. Although neither the ΔfslA nor the ΔfslC mutant produced rhizoferrin, a putative siderophore intermediate labeled by both [U-14C] ornithine and [1-14C] ornithine was secreted by the ΔfslC mutant. Rhizoferrin was identified by liquid chromatography and mass spectrometry in LVS culture supernatants, while citryl-ornithine was detected as the siderophore intermediate in the culture supernatant of the ΔfslC mutant. Our findings support a three-step pathway for rhizoferrin production in Francisella; unlike the fungus Rhizopus delemar, where putrescine functions as a primary precursor for rhizoferrin, biosynthesis in Francisella preferentially starts with ornithine as the substrate for FslA-mediated condensation with citrate. Decarboxylation of this citryl ornithine intermediate by FslC is necessary for a second condensation reaction with citrate to produce rhizoferrin.


Asunto(s)
Citratos/metabolismo , Compuestos Férricos/metabolismo , Francisella tularensis/metabolismo , Ornitina/análogos & derivados , Ornitina/metabolismo , Sideróforos/biosíntesis , Proteínas Bacterianas/metabolismo , Radioisótopos de Carbono , Ligasas de Carbono-Nitrógeno/metabolismo , Carboxiliasas/metabolismo , Francisella tularensis/enzimología
6.
Science ; 364(6441): 689-692, 2019 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-31097669

RESUMEN

The ATP-binding cassette subfamily B member 1 (ABCB1) multidrug transporter P-glycoprotein plays a central role in clearance of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. We used double electron electron resonance spectroscopy to uncover the basis of stimulation of P-glycoprotein adenosine 5'-triphosphate (ATP) hydrolysis by multiple substrates and illuminate how substrates and inhibitors differentially affect its transport function. Our results reveal that substrate-induced acceleration of ATP hydrolysis correlates with stabilization of a high-energy, post-ATP hydrolysis state characterized by structurally asymmetric nucleotide-binding sites. By contrast, this state is destabilized in the substrate-free cycle and by high-affinity inhibitors in favor of structurally symmetric nucleotide binding sites. Together with previous data, our findings lead to a general model of substrate and inhibitor coupling to P-glycoprotein.


Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Transporte Biológico , Dibenzocicloheptenos/química , Dibenzocicloheptenos/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Hidrólisis , Modelos Químicos , Estructura Secundaria de Proteína , Quinolinas/química , Quinolinas/farmacología
7.
Semin Cell Dev Biol ; 88: 119-128, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-29432954

RESUMEN

Chemokines are a family of small proteins best known for their ability to orchestrate immune cell trafficking and recruitment to sites of infection. Their role in promoting host defense is multiplied by a number of additional receptor-dependent biological activities, and most, but not all, chemokines have been found to mediate direct antimicrobial effects against a broad range of microorganisms. The molecular mechanism(s) by which antimicrobial chemokines kill bacteria remains unknown; however, recent observations have expanded our fundamental understanding of chemokine-mediated bactericidal activity to reveal increasingly diverse and complex actions. In the current review, we present and consider mechanistic insights of chemokine-mediated antimicrobial activity against bacteria. We also discuss how contemporary advances are reshaping traditional paradigms and opening up new and innovative avenues of research with translational implications. Towards this end, we highlight a developing framework for leveraging chemokine-mediated bactericidal and immunomodulatory effects to advance pioneering therapeutic approaches for treating bacterial infections, including those caused by multidrug-resistant pathogens.


Asunto(s)
Antiinfecciosos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Membrana Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Quimiocinas/farmacología , Animales , Bacterias/química , Bacterias/patogenicidad , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/patología , Membrana Celular/química , Pared Celular/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Humanos , Peptidoglicano/química , Peptidoglicano/metabolismo , Estructura Secundaria de Proteína , Relación Estructura-Actividad
8.
J Mol Biol ; 430(24): 5050-5065, 2018 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-30539761

RESUMEN

The calcium pump of the sarcoplasmic reticulum (SERCA) is an ATP-driven active transporter of Ca2+ ions that functions via an "alternating-access" cycle mechanism. In each cycle, SERCA transports two Ca2+ ions toward the lumen of the sarcoplasmic reticulum and two to three protons to the cytoplasm. How the latter conformational transition is coupled to cytoplasmic release of protons remains poorly understood. The present computational study shows how the mechanism of proton countertransport is coupled to the alternating access gating process in SERCA. Molecular dynamics simulation trajectories are generated starting from a series of configurations taken along the E2 to E1 transition pathway determined by the string method with swarms-of-trajectories. Simulations of different protonation configurations at the binding sites reveal how deprotonation events affect the opening of the cytoplasmic gate. The results show that there is a strong coupling between the chronological order of deprotonation, the entry of water molecules into the TM region, and the opening of the cytoplasmic gate. Deprotonation of E309 and E771 is sequential with E309 being the first to lose the proton. The deprotonation promotes the opening of the cytoplasmic gate but leads to a productive gating transition only if it occurs after the transmembrane domain has reached an intermediate conformation. Deprotonation of E309 and E771 is unproductive when it occurs too early because it causes the re-opening of the luminal gate.


Asunto(s)
Calcio/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Retículo Sarcoplasmático/metabolismo , Sitios de Unión , Citoplasma/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Protones
9.
Mol Pharm ; 15(12): 5585-5590, 2018 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-30351959

RESUMEN

Nanosized extracellular vesicles (EVs) possess the natural machinery needed to enter selectively and transmit complex molecular messages efficiently into targeted cells. The intracellular fate of the vesicular cargos depends on the route of internalization. Therefore, understanding the mechanism of attachment and subsequent intake of these vesicles (before and after exerting any modification) is imperative. Here the extent of communication, the uptake kinetics, and the pathways of endothelial EVs into endothelial cells in the presence of specific pharmacological inhibitors were assessed by imaging flow cytometry. The results showed that the uptake of endothelial EVs into endothelial cells was largely an energy-dependent process using predominantly a receptor-mediated, clathrin-dependent pathway.


Asunto(s)
Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Exosomas/metabolismo , Nanopartículas/metabolismo , Animales , Línea Celular , Clorpromazina/farmacología , Clatrina/metabolismo , Endocitosis/efectos de los fármacos , Células Endoteliales/ultraestructura , Endotelio Vascular/citología , Endotelio Vascular/ultraestructura , Exosomas/ultraestructura , Citometría de Flujo/métodos , Macrólidos/farmacología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Microscopía Fluorescente/métodos , Nistatina/farmacología
10.
Front Microbiol ; 8: 740, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28496437

RESUMEN

The antimicrobial activity of the chemokine CXCL10 against vegetative cells of Bacillus anthracis occurs via both bacterial FtsE/X-dependent and-independent pathways. Previous studies established that the FtsE/X-dependent pathway was mediated through interaction of the N-terminal region(s) of CXCL10 with a functional FtsE/X complex, while the FtsE/X-independent pathway was mediated through the C-terminal α-helix of CXCL10. Both pathways result in cell lysis and death of B. anthracis. In other bacterial species, it has been shown that FtsE/X is involved in cellular elongation though activation of complex-associated peptidoglycan hydrolases. Thus, we hypothesized that the CXCL10-mediated killing of vegetative cells of B. anthracis through the FtsE/X-dependent pathway resulted from the disruption of peptidoglycan processing. Immunofluorescence microscopy studies using fluorescent peptidoglycan probes revealed that incubation of B. anthracis Sterne (parent) strain with CXCL10 or a C-terminal truncated CXCL10 (CTTC) affected peptidoglycan processing and/or incorporation of precursors into the cell wall. B. anthracis ΔftsX or ftsE(K123A/D481N) mutant strains, which lacked a functional FtsE/X complex, exhibited little to no evidence of disruption in peptidoglycan processing by either CXCL10 or CTTC. Additional studies demonstrated that the B. anthracis parent strain exhibited a statistically significant increase in peptidoglycan release in the presence of either CXCL10 or CTTC. While B. anthracis ΔftsX strain showed increased peptidoglycan release in the presence of CXCL10, no increase was observed with CTTC, suggesting that the FtsE/X-independent pathway was responsible for the activity observed with CXCL10. These results indicate that FtsE/X-dependent killing of vegetative cells of B. anthracis results from a loss of cell wall integrity due to disruption of peptidoglycan processing and suggest that FtsE/X may be an important antimicrobial target to study in the search for alternative microbial therapeutics.

11.
Nature ; 543(7647): 738-741, 2017 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-28289287

RESUMEN

ATP binding cassette (ABC) transporters of the exporter class harness the energy of ATP hydrolysis in the nucleotide-binding domains (NBDs) to power the energetically uphill efflux of substrates by a dedicated transmembrane domain (TMD). Although numerous investigations have described the mechanism of ATP hydrolysis and defined the architecture of ABC exporters, a detailed structural dynamic understanding of the transduction of ATP energy to the work of substrate translocation remains elusive. Here we used double electron-electron resonance and molecular dynamics simulations to describe the ATP- and substrate-coupled conformational cycle of the mouse ABC efflux transporter P-glycoprotein (Pgp; also known as ABCB1), which has a central role in the clearance of xenobiotics and in cancer resistance to chemotherapy. Pairs of spin labels were introduced at residues selected to track the putative inward-facing to outward-facing transition. Our findings illuminate how ATP energy is harnessed in the NBDs in a two-stroke cycle and elucidate the consequent conformational motion that reconfigures the TMD, two critical aspects of Pgp transport mechanism. Along with a fully atomistic model of the outward-facing conformation in membranes, the insight into Pgp conformational dynamics harmonizes mechanistic and structural data into a novel perspective on ATP-coupled transport and reveals mechanistic divergence within the efflux class of ABC transporters.


Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/química , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Biocatálisis , Adenosina Trifosfato/metabolismo , Animales , Electrones , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Marcadores de Spin
12.
J Mol Biol ; 429(5): 647-666, 2017 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-28093226

RESUMEN

Ion pumps are integral membrane proteins responsible for transporting ions against concentration gradients across biological membranes. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), a member of the P-type ATPases family, transports two calcium ions per hydrolyzed ATP molecule via an "alternating-access" mechanism. High-resolution crystallographic structures provide invaluable insight on the structural mechanism of the ion pumping process. However, to understand the molecular details of how ATP hydrolysis is coupled to calcium transport, it is necessary to gain knowledge about the conformational transition pathways connecting the crystallographically resolved conformations. Large-scale transitions in SERCA occur at time-scales beyond the current reach of unbiased molecular dynamics simulations. Here, we overcome this challenge by employing the string method, which represents a transition pathway as a chainofstates linking two conformational endpoints. Using a multiscale methodology, we have determined all-atom transition pathways for three main conformational transitions responsible for the alternating-access mechanism. The present pathways provide a clear chronology and ordering of the key events underlying the active transport of calcium ions by SERCA. Important conclusions are that the conformational transition that leads to occlusion with bound ATP and calcium is highly concerted and cooperative, the phosphorylation of Asp351 causes areorganization of the cytoplasmic domains that subsequently drives the opening of the luminal gate, and thereclosing of luminal gate induces a shift in the cytoplasmic domains that subsequently enables the dephosphorylation of Asp351-P. Formation of transient residue-residue contacts along the conformational transitions predicted by the computations provide an experimental route to test the general validity of the computational pathways.


Asunto(s)
ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Biología Computacional , Cristalografía , Cristalografía por Rayos X , Transporte Iónico , Simulación de Dinámica Molecular , Fosforilación , Conformación Proteica
13.
mBio ; 7(3)2016 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-27165799

RESUMEN

UNLABELLED: Bacillus anthracis is killed by the interferon-inducible, ELR(-) CXC chemokine CXCL10. Previous studies showed that disruption of the gene encoding FtsX, a conserved membrane component of the ATP-binding cassette transporter-like complex FtsE/X, resulted in resistance to CXCL10. FtsX exhibits some sequence similarity to the mammalian CXCL10 receptor, CXCR3, suggesting that the CXCL10 N-terminal region that interacts with CXCR3 may also interact with FtsX. A C-terminal truncated CXCL10 was tested to determine if the FtsX-dependent antimicrobial activity is associated with the CXCR3-interacting N terminus. The truncated CXCL10 exhibited antimicrobial activity against the B. anthracis parent strain but not the ΔftsX mutant, which supports a key role for the CXCL10 N terminus. Mutations in FtsE, the conserved ATP-binding protein of the FtsE/X complex, resulted in resistance to both CXCL10 and truncated CXCL10, indicating that both FtsX and FtsE are important. Higher concentrations of CXCL10 overcame the resistance of the ΔftsX mutant to CXCL10, suggesting an FtsX-independent killing mechanism, likely involving its C-terminal α-helix, which resembles a cationic antimicrobial peptide. Membrane depolarization studies revealed that CXCL10 disrupted membranes of the B. anthracis parent strain and the ΔftsX mutant, but only the parent strain underwent depolarization with truncated CXCL10. These findings suggest that CXCL10 is a bifunctional molecule that kills B. anthracis by two mechanisms. FtsE/X-dependent killing is mediated through an N-terminal portion of CXCL10 and is not reliant upon the C-terminal α-helix. The FtsE/X-independent mechanism involves membrane depolarization by CXCL10, likely because of its α-helix. These findings present a new paradigm for understanding mechanisms by which CXCL10 and related chemokines kill bacteria. IMPORTANCE: Chemokines are a class of molecules known for their chemoattractant properties but more recently have been shown to possess antimicrobial activity against a wide range of Gram-positive and Gram-negative bacterial pathogens. The mechanism(s) by which these chemokines kill bacteria is not well understood, but it is generally thought to be due to the conserved amphipathic C-terminal α-helix that resembles cationic antimicrobial peptides in charge and secondary structure. Our present study indicates that the interferon-inducible, ELR(-) chemokine CXCL10 kills the Gram-positive pathogen Bacillus anthracis through multiple molecular mechanisms. One mechanism is mediated by interaction of CXCL10 with the bacterial FtsE/X complex and does not require the presence of the CXCL10 C-terminal α-helix. The second mechanism is FtsE/X receptor independent and kills through membrane disruption due to the C-terminal α-helix. This study represents a new paradigm for understanding how chemokines exert an antimicrobial effect that may prove applicable to other bacterial species.


Asunto(s)
Antibacterianos/farmacología , Bacillus anthracis/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/genética , Quimiocina CXCL10/genética , Quimiocina CXCL10/farmacología , Animales , Bacillus anthracis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Quimiocina CXCL10/química , Quimiocina CXCL10/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Mutación , Unión Proteica , Alineación de Secuencia
14.
Infect Immun ; 84(1): 320-8, 2016 01.
Artículo en Inglés | MEDLINE | ID: mdl-26553462

RESUMEN

Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains of Escherichia coli and report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enables E. coli to resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc, aceE. Resistance to CXCL10 also occurred following deletion of either aceF or lpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both the E. coli parent and aceE deletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine.


Asunto(s)
Antibacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Quimiocina CXCL10/metabolismo , Inmunidad Innata/inmunología , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Sitios de Unión , Dihidrolipoamida Deshidrogenasa/genética , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/genética , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli , Técnicas de Inactivación de Genes , Humanos , Unión Proteica , Piruvato Deshidrogenasa (Lipoamida)/genética
15.
J Biomol NMR ; 61(3-4): 209-26, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25631353

RESUMEN

CLC transporters catalyze the exchange of Cl(-) for H(+) across cellular membranes. To do so, they must couple Cl(-) and H(+) binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state (13)C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H(+)) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H(+)-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl(-)-permeation pathway, to the extracellular solution. The H(+)-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H(+) binding is mechanistically coupled to closing of the intracellular access-pathway for Cl(-).


Asunto(s)
Antiportadores/ultraestructura , Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Antiportadores de Cloruro-Bicarbonato/ultraestructura , Proteínas de Escherichia coli/ultraestructura , Resonancia Magnética Nuclear Biomolecular/métodos , Radioisótopos de Carbono , Cristalografía por Rayos X , Cisteína/química , Escherichia coli/metabolismo , Lisina/química , Metionina/química , Metilación , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína
16.
Int J Nanomedicine ; 9: 4223-30, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25214786

RESUMEN

Exosomes, one subpopulation of nanosize extracellular vesicles derived from multivesicular bodies, ranging from 30 to 150 nm in size, emerged as promising carriers for small interfering ribonucleic acid (siRNA) delivery, as they are capable of transmitting molecular messages between cells through carried small noncoding RNAs, messenger RNAs, deoxyribonucleic acids, and proteins. Endothelial cells are involved in a number of important biological processes, and are a major source of circulating exosomes. In this study, we prepared exosomes from endothelial cells and evaluated their capacity to deliver siRNA into primary endothelial cells. Exosomes were isolated and purified by sequential centrifugation and ultracentrifugation from cultured mouse aortic endothelial cells. Similar to exosome particles from other cell sources, endothelial exosomes are nanometer-size vesicles, examined by both the NanoSight instrument and transmission electron microscopy. Enzyme-linked immunosorbent assay analysis confirmed the expression of two exosome markers: CD9 and CD63. Flow cytometry and fluorescence microscopy studies demonstrated that endothelial exosomes were heterogeneously distributed within cells. In a gene-silencing study with luciferase-expressing endothelial cells, exosomes loaded with siRNA inhibited luciferase expression by more than 40%. In contrast, siRNA alone and control siRNA only suppressed luciferase expression by less than 15%. In conclusion, we demonstrated that endothelial exosomes have the capability to accommodate and deliver short foreign nucleic acids into endothelial cells.


Asunto(s)
Portadores de Fármacos/química , Exosomas , Técnicas de Transferencia de Gen , Nanopartículas/química , ARN Interferente Pequeño/química , Animales , Células Cultivadas , Portadores de Fármacos/metabolismo , Células Endoteliales/citología , Ratones , Ratones Endogámicos C57BL , Nanopartículas/metabolismo , Tamaño de la Partícula , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo
17.
Circ Res ; 111(2): 201-11, 2012 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-22652908

RESUMEN

RATIONALE: Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis. OBJECTIVE: To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation. METHODS AND RESULTS: We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE(-/-)) mice and in C57Bl/6 mice treated with platelet-derived growth factor-BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43(CT)) and produced null phosphorylation Ser>Ala (Cx43(MK4A)/Cx43(CTMK4A)) and phospho-mimetic Ser>Asp (Cx43(MK4D)/Cx43(CTMK4D)) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43(+/+)) and Cx43 null (Cx43(-/-)) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury. CONCLUSIONS: We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.


Asunto(s)
Proliferación Celular , Conexina 43/metabolismo , Ciclina E/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Animales , Animales Recién Nacidos , Aterosclerosis/enzimología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Conexina 43/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación/fisiología , Unión Proteica/fisiología
18.
J Biol Chem ; 287(27): 22771-80, 2012 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-22582396

RESUMEN

In observations of single molecule behavior under V(max) conditions with minimal load, the F(1) sector of the ATP synthase (F-ATPase) rotates through continuous cycles of catalytic dwells (∼0.2 ms) and 120° rotation steps (∼0.6 ms). We previously established that the rate-limiting transition step occurs during the catalytic dwell at the initiation of the 120° rotation. Here, we use the phytopolyphenol, piceatannol, which binds to a pocket formed by contributions from α and ß stator subunits and the carboxyl-terminal region of the rotor γ subunit. Piceatannol did not interfere with the movement through the 120° rotation step, but caused increased duration of the catalytic dwell. The duration time of the intrinsic inhibited state of F(1) also became significantly longer with piceatannol. All of the beads rotated at a lower rate in the presence of saturating piceatannol, indicating that the inhibitor stays bound throughout the rotational catalytic cycle. The Arrhenius plot of the temperature dependence of the reciprocal of the duration of the catalytic dwell (catalytic rate) indicated significantly increased activation energy of the rate-limiting step to trigger the 120° rotation. The activation energy was further increased by combination of piceatannol and substitution of γ subunit Met(23) with Lys, indicating that the inhibitor and the ß/γ interface mutation affect the same transition step, even though they perturb physically separated rotor-stator interactions.


Asunto(s)
Escherichia coli/enzimología , Polifenoles/metabolismo , ATPasas de Translocación de Protón/metabolismo , Estilbenos/metabolismo , Secuencia de Aminoácidos , Antioxidantes/metabolismo , Antioxidantes/farmacología , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Catálisis , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Mutagénesis/fisiología , Polifenoles/química , Polifenoles/farmacología , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genética , Quercetina/metabolismo , Quercetina/farmacología , Estilbenos/farmacología , Temperatura , Termodinámica
19.
Biochim Biophys Acta ; 1817(10): 1711-21, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22459334

RESUMEN

We focus on the rotational catalysis of Escherichia coli F-ATPase (ATP synthase, F(O)F(1)). Using a probe with low viscous drag, we found stochastic fluctuation of the rotation rates, a flat energy pathway, and contribution of an inhibited state to the overall behavior of the enzyme. Mutational analyses revealed the importance of the interactions among ß and γ subunits and the ß subunit catalytic domain. We also discuss the V-ATPase, which has different physiological roles from the F-ATPase, but is structurally and mechanistically similar. We review the rotation, diversity of subunits, and the regulatory mechanism of reversible subunit dissociation/assembly of Saccharomyces cerevisiae and mammalian complexes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , ATPasas de Translocación de Protón/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Dominio Catalítico , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Estructura Terciaria de Proteína , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genética
20.
J Biol Chem ; 285(53): 42058-67, 2010 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-20974856

RESUMEN

ATP hydrolysis-dependent rotation of the F(1) sector of the ATP synthase is a successive cycle of catalytic dwells (∼0.2 ms at 24 °C) and 120° rotation steps (∼0.6 ms) when observed under V(max) conditions using a low viscous drag 60-nm bead attached to the γ subunit (Sekiya, M., Nakamoto, R. K., Al-Shawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401-22410). During the normal course of observation, the γ subunit pauses in a stochastic manner to a catalytically inhibited state that averages ∼1 s in duration. The rotation behavior with adenosine 5'-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 µM) indicates that the rotation is inhibited at the catalytic dwell when the bound ATP undergoes reversible hydrolysis/synthesis. The temperature dependence of rotation shows that F(1) requires ∼2-fold higher activation energy for the transition from the active to the inhibited state compared with that for normal steady-state rotation during the active state. Addition of superstoichiometric ε subunit, the inhibitor of F(1)-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the ε subunit has little effect on the transition between active and inhibited states. Rather, the ε subunit confers lower activation energy of steady-state rotation. These results suggest that the ε subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state.


Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , ATPasas de Translocación de Protón Mitocondriales/metabolismo , ATPasas de Translocación de Protón/química , Adenosina Trifosfato/química , Bioquímica/métodos , Biofisica/métodos , Catálisis , Hidrólisis , Cinética , Magnesio/química , Temperatura , Viscosidad
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