RESUMO
PURPOSE: Local recurrence occurs in ~ 19% of sinonasal inverted papilloma (SNIP) surgeries and is strongly associated with incomplete resection. During surgery, it is technically challenging to visualize and resect all SNIP tissue in this anatomically complex area. Proteins that are overexpressed in SNIP, such as vascular endothelial growth factor (VEGF), may serve as a target for fluorescence molecular imaging to guide surgical removal of SNIP. A proof-of-concept study was performed to investigate if the VEGF-targeted near-infrared fluorescent tracer bevacizumab-800CW specifically localizes in SNIP and whether it could be used as a clinical tool to guide SNIP surgery. METHODS: In five patients diagnosed with SNIP, 10 mg of bevacizumab-800CW was intravenously administered 3 days prior to surgery. Fluorescence molecular imaging was performed in vivo during surgery and ex vivo during the processing of the surgical specimen. Fluorescence signals were correlated with final histopathology and VEGF-A immunohistochemistry. We introduced a fluorescence grid analysis to assess the fluorescence signal in individual tissue fragments, due to the nature of the surgical procedure (i.e., piecemeal resection) allowing the detection of small SNIP residues and location of the tracer ex vivo. RESULTS: In all patients, fluorescence signal was detected in vivo during endoscopic SNIP surgery. Using ex vivo fluorescence grid analysis, we were able to correlate bevacizumab-800CW fluorescence of individual tissue fragments with final histopathology. Fluorescence grid analysis showed substantial variability in mean fluorescence intensity (FImean), with SNIP tissue showing a median FImean of 77.54 (IQR 50.47-112.30) compared to 35.99 (IQR 21.48-57.81) in uninvolved tissue (p < 0.0001), although the diagnostic ability was limited with an area under the curve of 0.78. CONCLUSIONS: A fluorescence grid analysis could serve as a valid method to evaluate fluorescence molecular imaging in piecemeal surgeries. As such, although substantial differences were observed in fluorescence intensities, VEGF-A may not be the ideal target for SNIP surgery. TRIAL REGISTRATION: NCT03925285.
Assuntos
Neoplasias de Cabeça e Pescoço , Papiloma Invertido , Bevacizumab/uso terapêutico , Humanos , Imuno-Histoquímica , Imagem Óptica , Papiloma Invertido/diagnóstico por imagem , Papiloma Invertido/metabolismo , Papiloma Invertido/cirurgia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Anastomotic leakage in patients undergoing colorectal surgery is associated with morbidity and mortality. Although multiple risk factors have been identified, the underlying mechanisms are mainly unknown. The aim of this study was to perform a transcriptome analysis of genes underlying the development of anastomotic leakage. METHODS: A set of human samples from the anastomotic site collected during stapled colorectal anastomosis were used in the study. Transcriptomic profiles were generated for patients who developing anastomotic leakage and case-matched controls with normal anastomotic healing to identify genes and biological processes associated with the development of anastomotic leakage. RESULTS: The analysis included 22 patients with and 69 without anastomotic leakage. Differential expression analysis showed that 44 genes had adjusted P < 0.050, consisting of two upregulated and 42 downregulated genes. Co-functionality analysis of the 150 most upregulated and 150 most downregulated genes using the GenetICA framework showed formation of clusters of genes with different enrichment for biological pathways. The enriched pathways for the downregulated genes are involved in immune response, angiogenesis, protein metabolism, and collagen cross-linking. The enriched pathways for upregulated genes are involved in cell division. CONCLUSION: These data indicate that patients who develop anastomotic leakage start the healing process with an error at the level of gene regulation at the time of surgery. Despite normal macroscopic appearance during surgery, the transcriptome data identified several differences in gene expression between patients who developed anastomotic leakage and those who did not. The expressed genes and enriched processes are involved in the different stages of wound healing. These provide therapeutic and diagnostic targets for patients at risk of anastomotic leakage.
Assuntos
Fístula Anastomótica , Perfilação da Expressão Gênica , Transcriptoma , Idoso , Anastomose Cirúrgica , Estudos de Casos e Controles , Colo/cirurgia , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reto/cirurgia , Regulação para CimaRESUMO
Oral cancer patients undergo diagnostic surgeries to detect occult lymph node metastases missed by preoperative structural imaging techniques. Reducing these invasive procedures that are associated with considerable morbidity, requires better preoperative detection. Multispectral optoacoustic tomography (MSOT) is a rapidly evolving imaging technique that may improve preoperative detection of (early-stage) lymph node metastases, enabling the identification of molecular changes that often precede structural changes in tumorigenesis. Here, we characterize the optoacoustic properties of cetuximab-800CW, a tumor-specific fluorescent tracer showing several photophysical properties that benefit optoacoustic signal generation. In this first clinical proof-of-concept study, we explore its use as optoacoustic to differentiate between malignant and benign lymph nodes. We characterize the appearance of malignant lymph nodes and show differences in the distribution of intrinsic chromophores compared to benign lymph nodes. In addition, we suggest several approaches to improve the efficiency of follow-up studies.
RESUMO
Liposomes composed of tetraether lipids originating from the thermoacidophilic archaeon Sulfolobus acidocaldarius were analyzed for their stability and proton permeability from 20 degrees C up to 80 degrees C. At room temperature, these liposomes are considerably more stable and have a much lower proton permeability than liposomes composed of diester lipids originating from the mesophilic bacterium Escherichia coli or the thermophilic bacterium Bacillus stearothermophilus. With increasing temperature, the stability decreased and the proton permeability increased for all liposomes. Liposomes composed from tetraether lipids, however, remain the most stable. These data suggest these liposomes retain the rigidity of the cytoplasmic membrane of S. acidocaldarius needed to endure extreme environmental growth conditions.
Assuntos
Lipídeos/química , Lipossomos/química , Sulfolobales/química , Estabilidade de Medicamentos , Éteres/química , Prótons , TemperaturaRESUMO
Liposomes composed of Escherichia coli phospholipid were coated with polysaccharides bearing hydrophobic palmitoyl anchors. The effect on the stability of liposomes without or with integral membrane proteins was investigated. A high concentration of hydrophobized dextrans protected the liposomes against detergent degradation, decreased the fluidity of the membranes, prevented fusion of the liposomes and enhanced their stability. Proteoliposomes containing beef heart cytochrome-c oxidase and the lactose transport carrier of E. coli were similarly affected by coating with the dextrans. Under these conditions both membrane proteins were still active. Long-term stability of the coated liposomes was obtained only in the absence of the integral membrane proteins.
Assuntos
Dextranos/metabolismo , Proteínas de Escherichia coli , Lipossomos , Proteínas de Transporte de Monossacarídeos , Proteolipídeos/metabolismo , Simportadores , Animais , Cálcio/metabolismo , Sequência de Carboidratos , Cátions Bivalentes , Bovinos , Dextranos/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Escherichia coli/metabolismo , Fluoresceínas , Polarização de Fluorescência , Fluidez de Membrana/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Miocárdio/enzimologiaRESUMO
The prokaryotic tubulin homologue FtsZ polymerizes in vitro in a nucleotide dependent fashion. Here we report that replacement of the strictly conserved Asp212 residue of Escherichia coli FtsZ by a Cys or Asn, but not by a Glu residue results in FtsZ that polymerizes with divalent cations in the absence of added GTP. FtsZ D212C and D212N mutants co-purify with GTP as bound nucleotide, providing an explanation for the unusual phenotype. We conclude that D212 plays a critical role in the coordination of a metal ion and the nucleotide at the interface of two FtsZ monomers.
Assuntos
Ácido Aspártico/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto , Polímeros , Substituição de Aminoácidos , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Proteínas de Bactérias/genética , Cátions Bivalentes , Guanosina Trifosfato/metabolismo , Mutagênese Sítio-DirigidaRESUMO
Archaeal lipids differ considerably from eubacterial and eukaryotic lipids in their structure and physical properties. From the membranes of the extreme thermophilic archaea Sulfolobus acidocaldarius a tetraether lipid fraction was isolated, which can form closed and stable monolayer liposomes in aqueous media. The function of three different primary proton pumps originating from archaeal, bacterial and eukaryotic lipid sources have been studied after reconstitution in these liposomes: bacteriorhodopsin from the archaea Halobacterium halobium; cytochrome-c oxidase from the thermophilic bacterium Bacillus stearothermophilus and cytochrome-c oxidase from beef heart mitochondria. Liposomes composed of tetraether lipids form a competent matrix for all three exogenous proton pumps. Bacteriorhodopsin was inserted inside-out in these liposomes, as normally observed in bilayer-forming lipid. The activities of the two oxidases were inhibited at high tetraether-lipid concentration, probably due to the low fluidity of these membranes. Only bacteriorhodopsin, which originates from diether archaeal lipids is fully functional in the tetraether membranes.
Assuntos
Lipídeos/farmacologia , Proteolipídeos , Bombas de Próton/efeitos dos fármacos , Bombas de Próton/fisiologia , Sulfolobus acidocaldarius/química , Bacteriorodopsinas/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Escherichia coli/química , Geobacillus stearothermophilus/metabolismo , Halobacterium salinarum/metabolismo , Ionóforos/farmacologia , Fluidez de Membrana , Miocárdio/enzimologia , Desacopladores/farmacologiaRESUMO
The homodimeric SecA protein is the peripheral subunit of the preprotein translocase in bacteria. It binds the preprotein and promotes its translocation across the bacterial cytoplasmic membrane by nucleotide modulated coinsertion and deinsertion into the membrane. SecA has two essential nucleotide binding sites (NBS; Mitchell & Oliver, 1993): The high-affinity NBS-I resides in the amino-terminal domain of the protein, and the low-affinity NBS-II is localized at 2/3 of the protein sequence. The nucleotide-bound states of soluble SecA were studied by site directed tryptophan fluorescence spectroscopy, tryptic digestion, differential scanning calorimetry, and dynamic light scattering. A nucleotide-induced conformational change of a carboxy-terminal domain of SecA was revealed by Trp fluorescence spectroscopy. The Trp fluorescence of a single Trp SecA mutant containing Trp775 decreased and increased upon the addition of NBS-I saturating concentrations of ADP or AMP-PNP, respectively. DSC measurements revealed that SecA unfolds as a two domain protein. Binding of ADP to NBS-I increased the interaction between the two domains whereas binding of AMP-PNP did not influence this interaction. When both NBS-I and NBS-II are bound by ADP, SecA seems to have a more compact globular conformation whereas binding of AMP-PNP seems to cause a more extended conformation. It is suggested that the compact ADP-bound conformation resembles the membrane deinserted state of SecA, while the more extended ATP-bound conformation may correspond to the membrane inserted form of SecA.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/isolamento & purificação , Adenilil Imidodifosfato/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Varredura Diferencial de Calorimetria , Escherichia coli/crescimento & desenvolvimento , Modelos Químicos , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Canais de Translocação SEC , Espalhamento de Radiação , Proteínas SecA , Deleção de Sequência , Espectrometria de Fluorescência , Termodinâmica , Tripsina , TriptofanoRESUMO
SecA is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade, and is bound at the membrane surface to the integral membrane domain of the preprotein translocase. Preproteins are thought to be translocated in a stepwise manner by nucleotide-dependent cycles of SecA membrane insertion and de-insertion, or as large polypeptide segments by the protonmotive force (Deltap) in the absence of SecA. To determine the step size of a complete ATP- and SecA-dependent catalytic cycle, translocation intermediates of the preprotein proOmpA were generated at limiting SecA translocation ATPase activity. Distinct intermediates were formed, spaced by intervals of approximately 5 kDa. Inhibition of the SecA ATPase by azide trapped SecA in a membrane-inserted state and shifted the step size to 2-2.5 kDa. The latter corresponds to the translocation elicited by binding of non-hydrolysable ATP analogues to SecA, or by the re-binding of partially translocated polypeptide chains by SecA. Therefore, a complete catalytic cycle of the preprotein translocase permits the stepwise translocation of 5 kDa polypeptide segments by two consecutive events, i.e. approximately 2.5 kDa upon binding of the polypeptide by SecA, and another 2.5 kDa upon binding of ATP to SecA.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras , Precursores de Proteínas/metabolismo , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Azidas/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Catálise , Primers do DNA , Cinética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canais de Translocação SEC , Proteínas SecARESUMO
The molecular chaperone SecB targets preproteins to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA recognizes SecB via its carboxyl-terminal 22 aminoacyl residues, a highly conserved domain that contains 3 cysteines and 1 histidine residue that could potentially be involved in the coordination of a metal ion. Treatment of SecA with a zinc chelator resulted in a loss of the stimulatory effect of SecB on the SecA translocation ATPase activity, while the activity could be restored by the addition of ZnCl2. Interaction of SecB with the SecB binding domain of SecA is disrupted by chelators of divalent cations, and could be restored by the addition of Cu2+ or Zn2+. Atomic absorption and electrospray mass spectrometry revealed the presence of one zinc atom per monomeric carboxyl terminus of SecA. It is concluded that the SecB binding domain of SecA is stabilized by a zinc ion that promotes the functional binding of SecB to SecA.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Zinco/química , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/fisiologia , Sequência de Aminoácidos , Naftalenossulfonato de Anilina/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/fisiologia , Sítios de Ligação/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Cátions Bivalentes , Quelantes/farmacologia , Cisteína/química , Cisteína/metabolismo , Escherichia coli , Etilenodiaminas/farmacologia , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Canais de Translocação SEC , Proteínas SecA , Reagentes de SulfidrilaRESUMO
Membranes of Sulfolobus acidocaldarius, an extreme thermophilic archaebacterium, are composed of unusual bipolar lipids. They consist of macrocyclic tetraethers with two polar heads linked by two hydrophobic C40 phytanyl chains which are thought to be arranged as a monolayer in the cytoplasmic membrane. Fractionation of a total lipid-extract from S. acidocaldarius yielded a lipid fraction which forms closed and stable unilamellar liposomes in aqueous media. Beef heart cytochrome c-oxidase could be functionally reconstituted in these liposomes. In the presence of reduced cytochrome c, a protonmotive force (delta p) across the liposomal membrane was generated of up to -92 mV. Upon fusion of these proteoliposomes with membrane vesicles of Lactococcus lactis, the delta p generated by cytochrome c-oxidase activity was capable to drive uphill transport of leucine. Electron microscopic analysis indicated that the tetraether lipids form a single monolayer liposome. The results demonstrate that tetraether lipids of archaebacteria can form a suitable matrix for the function of exogenous membrane proteins originating from a regular lipid bilayer.
Assuntos
Bicamadas Lipídicas , Lipossomos , Lipídeos de Membrana/metabolismo , Sulfolobus acidocaldarius/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Cromatografia em Camada Fina , Difusão , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Escherichia coli/metabolismo , Técnica de Fratura por Congelamento , Lactococcus lactis/metabolismo , Microscopia Eletrônica , Miocárdio/enzimologia , Oniocompostos/metabolismo , Compostos Organofosforados/metabolismo , Fosfolipídeos/metabolismo , Potássio/metabolismoRESUMO
Bacterial growth at the extremes of temperature has remained a fascinating aspect in the study of membrane function and structure. The stability of the integral membrane proteins of thermophiles make them particularly amenable to study. Respiratory enzymes of thermophiles appear to be functionally similar to the mesophilic enzymes but differ in their thermostability and unusual high turnover rates. Energy coupling at extreme temperatures seems inefficient as suggested by the high maintenance coefficients and the high permeability of the cell membrane to protons. Nevertheless, membranes maintain their structure at these extremes through changes in fatty acid acyl chain composition. Archaebacteria synthesize novel membrane-spanning lipids with unique physical characteristics. Thermophiles have adapted to life at extreme temperatures by using sodium ions rather than protons as coupling ions in solute transport. Genetic and biochemical studies of these systems now reveal fundamental principles of such adaptations. The recent development of reconstitution techniques using membrane-spanning lipids allows a rigorous biochemical characterization of membrane proteins of extreme thermophiles in their natural environment.
Assuntos
Bactérias/metabolismo , Archaea/metabolismo , Transporte Biológico , Transferência de Energia , TemperaturaRESUMO
Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M(r) = 79,000) shares a high degree of similarity with maize branching enzyme I (M(r) = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species.
RESUMO
SecA is the peripheral subunit of the preprotein translocase of Escherichia coli. SecA consists of two independently folding domains, i.e., the N-domain bearing the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors the low-affinity NBS-II. ATP induces SecA insertion into the membrane during preprotein translocation. Domain-specific monoclonal antibodies (mAbs) were developed to analyze the functions of the SecA domains in preprotein translocation. The antigen binding sites of the obtained mAbs were confined to five epitopes. One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the C-domain in a region that has been implicated in membrane insertion. This mAb, either as IgG or as Fab, completely inhibits in vitro proOmpA translocation and SecA translocation ATPase activity. It prevents SecA membrane insertion and, more strikingly, reverses membrane insertion and promotes the release of SecA from the membrane. Surface plasmon resonance measurements demonstrate that the mAb recognizes the ADP- and the AMP-PNP-bound state of SecA either free in solution or bound at the membrane at the SecYEG protein. It is concluded that the mAb actively reverses a conformation essential for membrane insertion of SecA. The other mAbs directed to various epitopes in the N-domain were found to be without effect, although all bind the native SecA. These results demonstrate that the C-domain plays an important role in the SecA membrane insertion, providing further evidence that this process is needed for preprotein translocation.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação de Anticorpos , Proteínas de Escherichia coli , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana Transportadoras , Precursores de Proteínas/antagonistas & inibidores , Adenosina Trifosfatases/imunologia , Adenosina Trifosfatases/metabolismo , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Transporte Biológico/imunologia , Técnicas Biossensoriais , Mapeamento de Epitopos , Escherichia coli/enzimologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Conformação Proteica , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo , Canais de Translocação SEC , Proteínas SecA , SolubilidadeRESUMO
SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide-modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase, the SecY, SecE and SecG polypeptides, results in an increased level of membrane-bound SecA. This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane-associated form of SecA indicates that it exposes a carboxy-terminal domain to the periplasmic face of the membrane.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Expressão Gênica , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Canais de Translocação SEC , Proteínas SecA , Tripsina/metabolismoRESUMO
The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the "so-called" Walker B-motif, hXhhD (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg(2+)-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli secA amber mutant strain, and the E. coli SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg(2+)-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp-215 are involved in the binding of the Mg(2+)-ion when Mg(2+)-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Magnésio/metabolismo , Proteínas de Membrana Transportadoras , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Mutação , Conformação Proteica , Canais de Translocação SEC , Proteínas SecA , Relação Estrutura-AtividadeRESUMO
In Escherichia coli, precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (delta8proOmpA) bearing a non-functional signal sequence. Delta8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prIA4 strain. SecB reduces the translocation of delta8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB-SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.