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Debra A Kendall - One of the best experts on this subject based on the ideXlab platform.
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fluorescence spectroscopy of soluble e coli spase i demonstrates conformational changes induced by lipid and Signal Peptide binding
Biophysical Journal, 2012Co-Authors: Meera B Kolayarattil, Sarah M Auclair, Dongmei Yu, Debra A KendallAbstract:The bacterial secretory pathway is responsible for the translocation of exported preproteins, and for the assembly of membrane preproteins. Signal peptidase I (SPase I) is responsible for the cleavage of the Signal Peptide from these preproteins. We have used tryptophan fluorescence spectroscopy of the soluble Escherichia coli SPase I Δ2-75 to study the dynamic conformational changes that occur when the enzyme is in solution and when interacting with lipids and the Signal Peptide. E. coli SPase I Δ2-75 has four tryptophan residues (W261, W284, W300 and W310). We generated single tryptophan mutants, namely, W261, W284, W300 and W310, by mutating the other three Trp residues in each mutant to Phe. Based on fluorescence quenching experiments, W261 and W284 were found to be solvent inaccessible, while W300 and W310 were solvent exposed. However, W300 and W310 were found to become solvent inaccessible in the presence of lipids. This is consistent with their location proximal to the outer leaflet of the periplasmic membrane, where they could interact with membrane phospholipids, thus facilitating Signal Peptide binding. W261, W284 and W300 were found to undergo conformational changes concomitant with Signal Peptide binding. As these residues are located close to the putative substrate binding groove and near residues stabilizing the transition state, they may undergo a restricted structural rearrangement arising from Signal Peptide binding at the enzyme active site. These experiments have helped elucidate how the enzyme positions itself at the membrane and becomes primed to cleave the Signal Peptide of a preprotein during the translocation process.
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Probing the affinity of SecA for Signal Peptide in different environments.
Biochemistry, 2005Co-Authors: Monika Musial-siwek, Sharyn L. Rusch, Debra A KendallAbstract:SecA, the peripheral subunit of the Escherichia coli preprotein translocase, interacts with a number of ligands during export, including Signal Peptides, membrane phospholipids, and nucleotides. Using fluorescence resonance energy transfer (FRET), we studied the interactions of wild-type (WT) and mutant SecAs with IAEDANS-labeled Signal Peptide, and how these interactions are modified in the presence of other transport ligands. We find that residues on the third α-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and Signal Peptide. For SecA in aqueous solution, saturation binding data using FRET analysis fit a single-site binding model and yielded a Kd of 2.4 μM. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low Signal Peptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has two conformational states; our results do not support different oligomeric states of SecA. Using native gel...
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Demonstration of a specific Escherichia coli SecY-Signal Peptide interaction.
Biochemistry, 2004Co-Authors: Ligong Wang, Sharyn L. Rusch, Alexander Miller, Debra A KendallAbstract:Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the Signal Peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic Signal Peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The Signal Peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the Signal Peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The Signal Peptide-SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of Signal Peptide to SecY upon nucleotide hydrolysis at SecA.
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Juxtaposition of Signal-Peptide charge and core region hydrophobicity is critical for functional Signal Peptides.
Archives of Microbiology, 2002Co-Authors: Sharyn L. Rusch, Cynthia L. Mascolo, Maha O. Kebir, Debra A KendallAbstract:In Escherichia coli, exported proteins are synthesized as precursors containing an amino-terminal Signal Peptide which directs transport through the translocase to the proper destination. We have constructed a series of Signal Peptide mutants, incorporating linker sequences of varying lengths between the amino-terminal charge and core region hydrophobicity, to examine the requirement for the juxtaposition of these two structural features in promoting protein transport. In vivo and in vitro analyses indicated that high transport efficiency via Signal Peptides with core regions of marginal hydrophobicity absolutely requires the proximity of sufficient charge.
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Signal Peptide determinants of SecA binding and stimulation of ATPase activity.
Journal of Biological Chemistry, 2000Co-Authors: Ligong Wang, Alexander Miller, Debra A KendallAbstract:Abstract A Signal Peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic Signal Peptides corresponding to the wild type alkaline phosphatase Signal sequence and three sets of model Signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA. Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional Signal sequences but not with transport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the Signal Peptide core region. For Signal Peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the Signal Peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of Signal Peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.
Frank Sargent - One of the best experts on this subject based on the ideXlab platform.
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A regulatory domain controls the transport activity of a twin-arginine Signal Peptide
FEBS Letters, 2013Co-Authors: Lisa Bowman, Tracy Palmer, Frank SargentAbstract:The twin-arginine translocation (Tat) pathway is used by bacteria for the transmembrane transport of folded proteins. Proteins are targeted to the Tat translocase by Signal Peptides that have common tripartite structures consisting of polar n-regions, hydrophobic h-regions, and polar c-regions. In this work, the Signal Peptide of [NiFe] hydrogenase-1 from Escherichia coli has been studied. The hydrogenase-1 Signal Peptide contains an extended n-region that has a conserved primary structure. Genetic and biochemical approaches reveal that the Signal Peptide n-region is essential for hydrogenase assembly and acts as a regulatory domain controlling transport activity of the Signal Peptide.
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Structural diversity in twin-arginine Signal Peptide-binding proteins
Proceedings of the National Academy of Sciences, 2007Co-Authors: Jacques De Maillard, GAIL BUCHANAN, Tracy Palmer, Vijay Lyall, Christian A E M Spronk, Geerten W Vuister, D J Richardson, Frank SargentAbstract:The twin-arginine transport (Tat) system is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by Signal Peptides containing a twin-arginine motif. In Escherichia coli, many Tat substrates bind redox-active cofactors in the cytoplasm before transport. Coordination of cofactor insertion with protein export involves a "Tat proofreading" process in which chaperones bind twin-arginine Signal Peptides, thus preventing premature export. The initial Tat Signal-binding proteins described belonged to the TorD family, which are required for assembly of N- and S-oxide reductases. Here, we report that E. coli NapD is a Tat Signal Peptide-binding chaperone involved in biosynthesis of the Tat-dependent nitrate reductase NapA. NapD binds tightly and specifically to the NapA twin-arginine Signal Peptide and suppresses Signal Peptide translocation activity such that transport via the Tat pathway is retarded. High-resolution, heteronuclear, multidimensional NMR spectroscopy reveals the 3D solution structure of NapD. The chaperone adopts a ferredoxin-type fold, which is completely distinct from the TorD family. Thus, NapD represents a new family of twin-arginine Signal-Peptide-binding proteins.
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Signal Peptide chaperone interactions on the twin arginine protein transport pathway
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kostas Hatzixanthis, D J Richardson, Raymond J. Turner, Thomas A Clarke, Arthur Oubrie, Frank SargentAbstract:The twin-arginine transport (Tat) system is a protein-targeting pathway of prokaryotes and chloroplasts. Most Escherichia coli Tat substrates are complex metalloenzymes that must be correctly folded and assembled before transport, and a preexport chaperone-mediated “proofreading” process is therefore in operation. The paradigm proofreading chaperone is TorD, which coordinates maturation and export of the key respiratory enzyme trimethylamine N-oxide reductase (TorA). It is demonstrated here that purified TorD binds tightly and with exquisite specificity to the TorA twin-arginine Signal Peptide in vitro. It is also reported that the TorD family constitutes a hitherto unexpected class of nucleotide-binding proteins. The affinity of TorD for GTP is enhanced by initial Signal Peptide binding, and it is proposed that GTP governs Signal Peptide binding-and-release cycles during Tat proofreading.
Harris D Bernstein - One of the best experts on this subject based on the ideXlab platform.
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an unusual Signal Peptide extension inhibits the binding of bacterial presecretory proteins to the Signal recognition particle trigger factor and the secyeg complex
Journal of Biological Chemistry, 2006Co-Authors: Janine H Peterson, Rose L Szabady, Harris D BernsteinAbstract:Considerable evidence indicates that the Escherichia coli Signal recognition particle (SRP) selectively targets proteins that contain highly hydrophobic Signal Peptides to the SecYEG complex cotranslationally. Presecretory proteins that contain only moderately hydrophobic Signal Peptides typically interact with trigger factor (TF) and are targeted post-translationally. Here we describe a striking exception to this rule that has emerged from the analysis of an unusual 55-amino acid Signal Peptide associated with the E. coli autotransporter EspP. The EspP Signal Peptide consists of a C-terminal domain that resembles a classical Signal Peptide plus an N-terminal extension that is conserved in other autotransporter Signal Peptides. Although a previous study showed that proteins containing the C-terminal domain of the EspP Signal Peptide are targeted cotranslationally by SRP, we found that proteins containing the full-length Signal Peptide were targeted post-translationally via a novel TF-independent mechanism. Mutation of an invariant asparagine residue in the N-terminal extension, however, restored cotranslational targeting. Remarkably, proteins containing extremely hydrophobic derivatives of the EspP Signal Peptide were also targeted post-translationally. These and other results suggest that the N-terminal extension alters the accessibility of the Signal Peptide to SRP and TF and promotes post-translational export by reducing the efficiency of the interaction between the Signal Peptide and the SecYEG complex. Based on data, we propose that the N-terminal extension mediates an interaction with an unidentified cytoplasmic factor or induces the formation of an unusual Signal Peptide conformation prior to the onset of protein translocation.
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an unusual Signal Peptide facilitates late steps in the biogenesis of a bacterial autotransporter
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Rose L Szabady, Janine H Peterson, Kristen M Skillman, Harris D BernsteinAbstract:Bacterial autotransporters are proteins that use a C-terminal porin-like domain to facilitate the transport of an upstream “passenger domain” across the outer membrane. Although autotransporters are translocated across the inner membrane (IM) via the Sec pathway, some of them contain exceptionally long Signal Peptides distinguished by a unique N-terminal sequence motif. In this study, we used the Escherichia coli O157:H7 autotransporter EspP as a model protein to investigate the function of the unusual Signal Peptides. We found that removal of the N-terminal motif or replacement of the EspP Signal Peptide did not affect translocation of the protein across the IM. Remarkably, modification of the Signal Peptide caused EspP to misfold in the periplasm and blocked transport of the passenger domain across the outer membrane. Further analysis suggested that the EspP Signal Peptide transits slowly through the Sec machinery. Based on these results, we propose that the unusual Signal Peptides not only function as targeting Signals, but also prevent misfolding of the passenger domain in the periplasm by transiently tethering it to the IM.
Bruno Martoglio - One of the best experts on this subject based on the ideXlab platform.
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targeting presenilin type aspartic protease Signal Peptide peptidase with γ secretase inhibitors
Journal of Biological Chemistry, 2003Co-Authors: Andreas Weihofen, Marius K. Lemberg, Elena Friedmann, Heinrich Rueeger, Albert Schmitz, Paolo Paganetti, Giorgio Rovelli, Bruno MartoglioAbstract:Abstract Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the γ-secretase complex that catalyzes intramembrane cleavage of β-amyloid precursor protein, the last step in the generation of amyloidogenic Aβ Peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of γ-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of Aβ Peptides. Recently, we discovered a family of presenilin-type aspartic proteases. The founding member, Signal Peptide peptidase, catalyzes intramembrane cleavage of distinct Signal Peptides in the endoplasmic reticulum membrane of animals. In humans, the protease plays a crucial role in the immune system. Moreover, it is exploited by the hepatitis C virus for the processing of the structural components of the virion and hence is an attractive target for anti-infective intervention. Signal Peptide peptidase and presenilin share identical active site motifs and both catalyze intramembrane proteolysis. These common features let us speculate that γ-secretase inhibitors directed against presenilin may also inhibit Signal Peptide peptidase. Here we demonstrate that some of the most potent known γ-secretase inhibitors efficiently inhibit Signal Peptide peptidase. However, we found compounds that showed higher specificity for one or the other protease. Our findings highlight the possibility of developing selective inhibitors aimed at reducing Aβ generation without affecting other intramembrane-cleaving aspartic proteases.
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identification of Signal Peptide peptidase a presenilin type aspartic protease
Science, 2002Co-Authors: Andreas Weihofen, Marius K. Lemberg, Kathleen Binns, Keith Ashman, Bruno MartoglioAbstract:Signal Peptide peptidase (SPP) catalyzes intramembrane proteolysis of some Signal Peptides after they have been cleaved from a preprotein. In humans, SPP activity is required to generate Signal sequence-derived human lymphocyte antigen-E epitopes that are recognized by the immune system, and to process hepatitis C virus core protein. We have identified human SPP as a polytopic membrane protein with sequence motifs characteristic of the presenilin-type aspartic proteases. SPP and potential eukaryotic homologs may represent another family of aspartic proteases that promote intramembrane proteolysis to release biologically important Peptides.
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Requirements for Signal Peptide peptidase-catalyzed intramembrane proteolysis.
Molecular Cell, 2002Co-Authors: Marius K. Lemberg, Bruno MartoglioAbstract:The presenilin-type aspartic protease Signal Peptide peptidase (SPP) can cleave Signal Peptides within their transmembrane region. SPP is essential for generation of Signal Peptide-derived HLA-E epitopes in humans and is exploited by Hepatitis C virus for processing of the viral polyprotein. Here we analyzed requirements of substrates for intramembrane cleavage by SPP. Comparing Signal Peptides that are substrates with those that are not revealed that helix-breaking residues within the transmembrane region are required for cleavage, and flanking regions can affect processing. Furthermore, Signal Peptides have to be liberated from the precursor protein by cleavage with Signal peptidase in order to become substrates for SPP. We propose that Signal Peptides require flexibility in the lipid bilayer to exhibit an accessible Peptide bond for intramembrane proteolysis.
Takeshi Iwatsubo - One of the best experts on this subject based on the ideXlab platform.
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structural biology of presenilins and Signal Peptide peptidases
Journal of Biological Chemistry, 2013Co-Authors: Taisuke Tomita, Takeshi IwatsuboAbstract:Presenilin and Signal Peptide peptidase are multispanning intramembrane-cleaving proteases with a conserved catalytic GxGD motif. Presenilin comprises the catalytic subunit of γ-secretase, a protease responsible for the generation of amyloid-β Peptides causative of Alzheimer disease. Signal Peptide peptidase proteins are implicated in the regulation of the immune system. Both protease family proteins have been recognized as druggable targets for several human diseases, but their detailed structure still remains unknown. Recently, the x-ray structures of some archaeal GxGD proteases have been determined. We review the recent progress in biochemical and biophysical probing of the structure of these atypical proteases.
