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Sample records for protein translocation complexes

  1. N-terminal lysines are essential for protein translocation via a modified ERAD system in complex plastids.

    PubMed

    Lau, Julia B; Stork, Simone; Moog, Daniel; Sommer, Maik S; Maier, Uwe G

    2015-05-01

    Nuclear-encoded pre-proteins being imported into complex plastids of red algal origin have to cross up to five membranes. Thereby, transport across the second outermost or periplastidal membrane (PPM) is facilitated by SELMA (symbiont-specific ERAD-like machinery), an endoplasmic reticulum-associated degradation (ERAD)-derived machinery. Core components of SELMA are enzymes involved in ubiquitination (E1-E3), a Cdc48 ATPase complex and Derlin proteins. These components are present in all investigated organisms with four membrane-bound complex plastids of red algal origin, suggesting a ubiquitin-dependent translocation process of substrates mechanistically similar to the process of retro-translocation in ERAD. Even if, according to the current model, translocation via SELMA does not end up in the classical poly-ubiquitination, transient mono-/oligo-ubiquitination of pre-proteins might be required for the mechanism of translocation. We investigated the import mechanism of SELMA and were able to show that protein transport across the PPM depends on lysines in the N-terminal but not in the C-terminal part of pre-proteins. These lysines are predicted to be targets of ubiquitination during the translocation process. As proteins lacking the N-terminal lysines get stuck in the PPM, a 'frozen intermediate' of the translocation process could be envisioned and initially characterized.

  2. Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex.

    PubMed Central

    Kassenbrock, C K; Cao, W; Douglas, M G

    1993-01-01

    To search genetically for additional components of the protein translocation apparatus of mitochondria, we have used low fidelity PCR mutagenesis to generate temperature-sensitive mutants in the outer membrane translocation pore component ISP42. A high copy number suppressor of temperature-sensitive isp42 has been isolated and sequenced. This novel gene, denoted ISP6, encodes a 61 amino acid integral membrane protein of the mitochondrial outer membrane, which is oriented with its amino-terminus facing the cytosol. Disruption of the ISP6 gene is without apparent effect in wild type yeast cells, but is lethal in temperature-sensitive isp42 mutants. Immunoprecipitation of the gene product, ISP42p, from mitochondria solubilized under mild conditions reveals a multi-protein complex containing ISP6p and ISP42p. Images PMID:8344244

  3. Protein translocation: what's the problem?

    PubMed

    Corey, Robin A; Allen, William J; Collinson, Ian

    2016-06-15

    We came together in Leeds to commemorate and celebrate the life and achievements of Prof. Stephen Baldwin. For many years we, together with Sheena Radford and Roman Tuma (colleagues also of the University of Leeds), have worked together on the problem of protein translocation through the essential and ubiquitous Sec system. Inspired and helped by Steve we may finally be making progress. My seminar described our latest hypothesis for the molecular mechanism of protein translocation, supported by results collected in Bristol and Leeds on the tractable bacterial secretion process-commonly known as the Sec system; work that will be published elsewhere. Below is a description of the alternative and contested models for protein translocation that we all have been contemplating for many years. This review will consider their pros and cons.

  4. Protein translocation: what's the problem?

    PubMed Central

    Corey, Robin A.; Allen, William J.; Collinson, Ian

    2016-01-01

    We came together in Leeds to commemorate and celebrate the life and achievements of Prof. Stephen Baldwin. For many years we, together with Sheena Radford and Roman Tuma (colleagues also of the University of Leeds), have worked together on the problem of protein translocation through the essential and ubiquitous Sec system. Inspired and helped by Steve we may finally be making progress. My seminar described our latest hypothesis for the molecular mechanism of protein translocation, supported by results collected in Bristol and Leeds on the tractable bacterial secretion process–commonly known as the Sec system; work that will be published elsewhere. Below is a description of the alternative and contested models for protein translocation that we all have been contemplating for many years. This review will consider their pros and cons. PMID:27284038

  5. Protein translocation: what's the problem?

    PubMed

    Corey, Robin A; Allen, William J; Collinson, Ian

    2016-06-15

    We came together in Leeds to commemorate and celebrate the life and achievements of Prof. Stephen Baldwin. For many years we, together with Sheena Radford and Roman Tuma (colleagues also of the University of Leeds), have worked together on the problem of protein translocation through the essential and ubiquitous Sec system. Inspired and helped by Steve we may finally be making progress. My seminar described our latest hypothesis for the molecular mechanism of protein translocation, supported by results collected in Bristol and Leeds on the tractable bacterial secretion process-commonly known as the Sec system; work that will be published elsewhere. Below is a description of the alternative and contested models for protein translocation that we all have been contemplating for many years. This review will consider their pros and cons. PMID:27284038

  6. Characterization of the Translocation-competent Complex between the Helicobacter pylori Oncogenic Protein CagA and the Accessory Protein CagF*

    PubMed Central

    Bonsor, Daniel A.; Weiss, Evelyn; Iosub-Amir, Anat; Reingewertz, Tali H.; Chen, Tiffany W.; Haas, Rainer; Friedler, Assaf; Fischer, Wolfgang; Sundberg, Eric J.

    2013-01-01

    CagA is a virulence factor that Helicobacter pylori inject into gastric epithelial cells through a type IV secretion system where it can cause gastric adenocarcinoma. Translocation is dependent on the presence of secretion signals found in both the N- and C-terminal domains of CagA and an interaction with the accessory protein CagF. However, the molecular basis of this essential protein-protein interaction is not fully understood. Herein we report, using isothermal titration calorimetry, that CagA forms a 1:1 complex with a monomer of CagF with nm affinity. Peptide arrays and isothermal titration calorimetry both show that CagF binds to all five domains of CagA, each with μm affinity. More specifically, a coiled coil domain and a C-terminal helix within CagF contacts domains II-III and domain IV of CagA, respectively. In vivo complementation assays of H. pylori with a double mutant, L36A/I39A, in the coiled coil region of CagF showed a severe weakening of the CagA-CagF interaction to such an extent that it was nearly undetectable. However, it had no apparent effect on CagA translocation. Deletion of the C-terminal helix of CagF also weakened the interaction with CagA but likewise had no effect on translocation. These results indicate that the CagA-CagF interface is distributed broadly across the molecular surfaces of these two proteins to provide maximal protection of the highly labile effector protein CagA. PMID:24072713

  7. Characterization of the translocation-competent complex between the Helicobacter pylori oncogenic protein CagA and the accessory protein CagF.

    PubMed

    Bonsor, Daniel A; Weiss, Evelyn; Iosub-Amir, Anat; Reingewertz, Tali H; Chen, Tiffany W; Haas, Rainer; Friedler, Assaf; Fischer, Wolfgang; Sundberg, Eric J

    2013-11-15

    CagA is a virulence factor that Helicobacter pylori inject into gastric epithelial cells through a type IV secretion system where it can cause gastric adenocarcinoma. Translocation is dependent on the presence of secretion signals found in both the N- and C-terminal domains of CagA and an interaction with the accessory protein CagF. However, the molecular basis of this essential protein-protein interaction is not fully understood. Herein we report, using isothermal titration calorimetry, that CagA forms a 1:1 complex with a monomer of CagF with nM affinity. Peptide arrays and isothermal titration calorimetry both show that CagF binds to all five domains of CagA, each with μM affinity. More specifically, a coiled coil domain and a C-terminal helix within CagF contacts domains II-III and domain IV of CagA, respectively. In vivo complementation assays of H. pylori with a double mutant, L36A/I39A, in the coiled coil region of CagF showed a severe weakening of the CagA-CagF interaction to such an extent that it was nearly undetectable. However, it had no apparent effect on CagA translocation. Deletion of the C-terminal helix of CagF also weakened the interaction with CagA but likewise had no effect on translocation. These results indicate that the CagA-CagF interface is distributed broadly across the molecular surfaces of these two proteins to provide maximal protection of the highly labile effector protein CagA.

  8. Visualization and translocation of ternary Calcineurin-A/Calcineurin-B/Calmodulin-2 protein complexes by dual-color trimolecular fluorescence complementation.

    PubMed

    Offenborn, Jan Niklas; Waadt, Rainer; Kudla, Jörg

    2015-10-01

    Fluorescence complementation (FC) techniques are expedient for analyzing bimolecular protein-protein interactions. Here we aimed to develop a method for visualization of ternary protein complexes using dual-color trimolecular fluorescence complementation (TriFC). Dual-color TriFC combines protein fragments of mCherry and mVenus, in which a scaffold protein is bilaterally fused to C-terminal fragments of both fluorescent proteins and combined with potential interacting proteins fused to an N-terminal fluorescent protein fragment. For efficient visual verification of ternary complex formation, TriFC was combined with a cytoplasm to plasma membrane translocation assay. Modular vector sets were designed which are fully compatible with previously reported bimolecular fluorescence complementation (BiFC) vectors. As a proof-of-principle, the ternary complex formation of the PP2B protein phosphatase Calcineurin-A/Calcineurin-B with Calmodulin-2 was investigated in transiently transformed Nicotiana benthamiana leaf epidermal cells. The results indicate a Calcineurin-B-induced interaction of Calmodulin-2 with Calcineurin-A. TriFC and the translocation of TriFC complexes provide a novel tool to investigate ternary complex formations with the simplicity of a BiFC approach. The robustness of FC applications and the opportunity to quantify fluorescence complementation render this assay suitable for a broad range of interaction analyses.

  9. The archaeal Sec-dependent protein translocation pathway.

    PubMed Central

    Bolhuis, Albert

    2004-01-01

    Over the past three decades, transport of proteins across cellular membranes has been studied extensively in various model systems. One of the major transport routes, the so-called Sec pathway, is conserved in all domains of life. Very little is known about this pathway in the third domain of life, archaea. The core components of the archaeal, bacterial and eucaryal Sec machinery are similar, although the archaeal components appear more closely related to their eucaryal counterparts. Interestingly, the accessory factors of the translocation machinery are similar to bacterial components, which indicates a unique hybrid nature of the archaeal translocase complex. The mechanism of protein translocation in archaea is completely unknown. Based on genomic sequencing data, the most likely system for archaeal protein translocation is similar to the eucaryal co-translational translocation pathway for protein import into the endoplasmic reticulum, in which a protein is pushed across the translocation channel by the ribosome. However, other models can also be envisaged, such as a bacterial-like system in which a protein is translocated post-translationally with the aid of a motor protein analogous to the bacterial ATPase SecA. This review discusses the different models. Furthermore, an overview is given of some of the other components that may be involved in the protein translocation process, such as those required for protein targeting, folding and post-translational modification. PMID:15306407

  10. Toward a structural understanding of co-translational protein translocation.

    PubMed

    Voorhees, Rebecca M; Hegde, Ramanujan S

    2016-08-01

    The translocation of most eukaryotic secreted and integral membrane proteins occurs co-translationally at the endoplasmic reticulum (ER). These nascent polypeptides are recognized on the ribosome by the signal recognition particle (SRP), targeted to the ER, and translocated across or inserted into the membrane by the Sec61 translocation channel. Structural analysis of these co-translational processes has been challenging due to the size, complexity, and flexibility of the targeting and translocation machinery. Recent technological advances in cryo-electron microscopy (cryo-EM) have resulted in increasingly powerful tools to study large, heterogeneous, and low-abundance samples. These advances are being utilized to obtain near-atomic resolution reconstructions of functional translation, targeting, and translocation intermediates, paving the way to a mechanistic understanding of protein biogenesis.

  11. The TIC complex uncovered: The alternative view on the molecular mechanism of protein translocation across the inner envelope membrane of chloroplasts.

    PubMed

    Nakai, Masato

    2015-09-01

    Chloroplasts must import thousands of nuclear-encoded preproteins synthesized in the cytosol through two successive protein translocons at the outer and inner envelope membranes, termed TOC and TIC, respectively, to fulfill their complex physiological roles. The molecular identity of the TIC translocon had long remained controversial; two proteins, namely Tic20 and Tic110, had been proposed to be central to protein translocation across the inner envelope membrane. Tic40 also had long been considered to be another central player in this process. However, recently, a novel 1-megadalton complex consisting of Tic20, Tic56, Tic100, and Tic214 was identified at the chloroplast inner membrane of Arabidopsis and was demonstrated to constitute a general TIC translocon which functions in concert with the well-characterized TOC translocon. On the other hand, direct interaction between this novel TIC transport system and Tic110 or Tic40 was hardly observed. Consequently, the molecular model for protein translocation across the inner envelope membrane of chloroplasts might need to be extensively revised. In this review article, I intend to propose such alternative view regarding the TIC transport system in contradistinction to the classical view. I also would emphasize importance of reevaluation of previous works in terms of with what methods these classical Tic proteins such as Tic110 or Tic40 were picked up as TIC constituents at the very beginning as well as what actual evidence there were to support their direct and specific involvement in chloroplast protein import. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

  12. Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway

    SciTech Connect

    Pohlschroder Mechthild

    2009-02-03

    Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tat pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.

  13. Protein Translocation across the Rough Endoplasmic Reticulum

    PubMed Central

    Mandon, Elisabet C.; Trueman, Steven F.; Gilmore, Reid

    2013-01-01

    The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration. PMID:23251026

  14. A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion

    DOE PAGES

    Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko

    2014-11-06

    Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components thatmore » form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.« less

  15. A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion

    SciTech Connect

    Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko

    2014-11-06

    Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

  16. Ratcheting up protein translocation with anthrax toxin

    PubMed Central

    Feld, Geoffrey K; Brown, Michael J; Krantz, Bryan A

    2012-01-01

    Energy-consuming nanomachines catalyze the directed movement of biopolymers in the cell. They are found both dissolved in the aqueous cytosol as well as embedded in lipid bilayers. Inquiries into the molecular mechanism of nanomachine-catalyzed biopolymer transport have revealed that these machines are equipped with molecular parts, including adjustable clamps, levers, and adaptors, which interact favorably with substrate polypeptides. Biological nanomachines that catalyze protein transport, known as translocases, often require that their substrate proteins unfold before translocation. An unstructured protein chain is likely entropically challenging to bind, push, or pull in a directional manner, especially in a way that produces an unfolding force. A number of ingenious solutions to this problem are now evident in the anthrax toxin system, a model used to study protein translocation. Here we highlight molecular ratchets and current research on anthrax toxin translocation. A picture is emerging of proton-gradient-driven anthrax toxin translocation, and its associated ratchet mechanism likely applies broadly to other systems. We suggest a cyclical thermodynamic order-to-disorder mechanism (akin to a heat-engine cycle) is central to underlying protein translocation: peptide substrates nonspecifically bind to molecular clamps, which possess adjustable affinities; polypeptide substrates compress into helical structures; these clamps undergo proton-gated switching; and the substrate subsequently expands regaining its unfolded state conformational entropy upon translocation. PMID:22374876

  17. Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome

    SciTech Connect

    Kitagawa, Yukiko; Kameoka, Masanori Shoji-Kawata, Sanae; Iwabu, Yukie; Mizuta, Hiroyuki; Tokunaga, Kenzo; Fujino, Masato; Natori, Yukikazu; Yura, Yoshiaki; Ikuta, Kazuyoshi

    2008-03-30

    The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2{alpha}) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2{alpha}. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2{alpha}. Confocal fluorescence microscopy revealed that a subpopulation of AP2{alpha} was not only localized in the cytoplasm but was also partly co-localized with lamin B, importin {beta} and Nup153, implying that AP2{alpha} negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2{alpha} may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle.

  18. What drives the translocation of proteins?

    PubMed Central

    Simon, S M; Peskin, C S; Oster, G F

    1992-01-01

    We propose that protein translocation across membranes is driven by biased random thermal motion. This "Brownian ratchet" mechanism depends on chemical asymmetries between the cis and trans sides of the membrane. Several mechanisms could contribute to rectifying the thermal motion of the protein, such as binding and dissociation of chaperonins to the translocating chain, chain coiling induced by pH and/or ionic gradients, glycosylation, and disulfide bond formation. This helps explain the robustness and promiscuity of these transport systems. Images PMID:1349170

  19. Surface modification of graphene nanopores for protein translocation

    PubMed Central

    Shan, Y. P.; Tiwari, P. B.; Krishnakumar, P.; Vlassiouk, I.; Li, W.Z.; Wang, X.W.; Darici, Y.; Lindsay, S.M.; Wang, H. D.; Smirnov, S.; He, J.

    2014-01-01

    Studies of DNA translocation through graphene nanopores have revealed their potential for DNA sequencing. Here we report a study of protein translocation through chemically modified graphene nanopores. A transmission electron microscope (TEM) was used to cut nanopores with diameters between 5-20 nm in multilayer graphene prepared by chemical vapor deposition (CVD). After oxygen plasma treatment, the dependence of the measured ionic current on salt concentration and pH was consistent with a small surface charge induced by the formation of carboxyl groups. While translocation of gold nanoparticles (10 nm) was readily detected through such treated pores of a larger diameter, translocation of protein ferritin was not observed either for oxygen plasma treated pores, or for pores modified with mercaptohexadecanoic acid. Ferritin translocation events were reliably observed after the pores were modified with the phospholipid-PEG (DPPE-PEG750) amphiphile. The ion current signature of translocation events was complex, suggesting that a series of interactions between the protein and pore occur during the process. PMID:24231385

  20. Effect of the ATPase inhibitor protein IF{sub 1} on H{sup +} translocation in the mitochondrial ATP synthase complex

    SciTech Connect

    Zanotti, Franco; Gnoni, Antonio; Mangiullo, Roberto; Papa, Sergio

    2009-06-19

    The H{sup +} F{sub o}F{sub 1}-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F{sub 1} moiety of the complex protrudes at the inner side of the membrane, the F{sub o} sector spans the membrane reaching the outer side. The IF{sub 1} component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F{sub o}F{sub 1}-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF{sub 1} (residue 42-58) to the F{sub 1}-{alpha}/{beta} subunits. We have analyzed the effect of native purified IF{sub 1} the IF{sub 1}-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K{sup +}] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F{sub o}F{sub 1} complex. The results show that IF{sub 1}, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F{sub 1} to the F{sub o} side and in the opposite direction. Cross-linking of IF{sub 1} to F{sub 1}-{alpha}/{beta} subunits inhibits the ATP-driven H{sup +} translocation but enhances H{sup +} conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F{sub o}F{sub 1} complex.

  1. Synthesis, characterization, and in vitro evaluation of new coordination complexes of platinum(II) and rhenium(I) with a ligand targeting the translocator protein (TSPO).

    PubMed

    Margiotta, Nicola; Denora, Nunzio; Piccinonna, Sara; Laquintana, Valentino; Lasorsa, Francesco Massimo; Franco, Massimo; Natile, Giovanni

    2014-11-21

    The 18 kDa translocator protein (TSPO) is overexpressed in many types of cancers and is also abundant in activated microglial cells occurring in inflammatory neurodegenerative diseases. The TSPO-selective ligand 2-(8-(2-(bis-(pyridin-2-yl-methyl)amino)acetamido)-2-(4-chlorophenyl)H-imidazo[1,2-a]pyridin-3-yl)-N,N-dipropylacetamide (CB256), which fulfills the requirements of a bifunctional chelate approach, has been used to synthesize coordination complexes containing either Pt (1) or Re (3), or both metal ions (2). The new metal complexes showed a cellular uptake markedly greater than that of the precursor metallic compounds and were also able to induce apoptosis in C6 glioma cells. The good cytotoxicity of the free ligand CB256 towards C6, A2780, and A2780cisR tumor cell lines was attenuated after coordination of the dipicolylamine moiety to Pt while coordination of the imidazopyridine residue to Re reduces the affinity towards TSPO. The results of the present investigation are essential for the design of new imidazopyridine bifunctional chelate ligands targeted to TSPO.

  2. The protein translocation machinery of the endoplasmic reticulum.

    PubMed

    Walter, P; Gilmore, R; Müller, M; Blobel, G

    1982-12-24

    The rough endoplasmic reticulum (r.e.r.) has been postulated to possess a single translation-coupled translocation system (in multiple copies) that effects signal sequence-mediated translocation of all secretory and lysosomal proteins and integration of all integral membrane proteins whose port of entry is the rough endoplasmic reticulum (G. Blobel 1980 Proc. natn. Acad. Sci. U.S.A. 77, 1496-1500). Two proteins have been isolated that are components of the r.e.r. translocation system. Their properties and function in protein translocation across and integration into membranes are discussed. PMID:6131460

  3. Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity

    PubMed Central

    Mesén-Ramírez, Paolo; Reinsch, Ferdinand; Blancke Soares, Alexandra; Bergmann, Bärbel; Ullrich, Ann-Katrin; Tenzer, Stefan

    2016-01-01

    Protein export is central for the survival and virulence of intracellular P. falciparum blood stage parasites. To reach the host cell, exported proteins cross the parasite plasma membrane (PPM) and the parasite-enclosing parasitophorous vacuole membrane (PVM), a process that requires unfolding, suggestive of protein translocation. Components of a proposed translocon at the PVM termed PTEX are essential in this phase of export but translocation activity has not been shown for the complex and questions have been raised about its proposed membrane pore component EXP2 for which no functional data is available in P. falciparum. It is also unclear how PTEX mediates trafficking of both, soluble as well as transmembrane proteins. Taking advantage of conditionally foldable domains, we here dissected the translocation events in the parasite periphery, showing that two successive translocation steps are needed for the export of transmembrane proteins, one at the PPM and one at the PVM. Our data provide evidence that, depending on the length of the C-terminus of the exported substrate, these steps occur by transient interaction of the PPM and PVM translocon, similar to the situation for protein transport across the mitochondrial membranes. Remarkably, we obtained constructs of exported proteins that remained arrested in the process of being translocated across the PVM. This clogged the translocation pore, prevented the export of all types of exported proteins and, as a result, inhibited parasite growth. The substrates stuck in translocation were found in a complex with the proposed PTEX membrane pore component EXP2, suggesting a role of this protein in translocation. These data for the first time provide evidence for EXP2 to be part of a translocating entity, suggesting that PTEX has translocation activity and provide a mechanistic framework for the transport of soluble as well as transmembrane proteins from the parasite boundary into the host cell. PMID:27168322

  4. Stepwise nucleosome translocation by RSC remodeling complexes

    PubMed Central

    Harada, Bryan T; Hwang, William L; Deindl, Sebastian; Chatterjee, Nilanjana; Bartholomew, Blaine; Zhuang, Xiaowei

    2016-01-01

    The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1–2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome. DOI: http://dx.doi.org/10.7554/eLife.10051.001 PMID:26895087

  5. Stepwise nucleosome translocation by RSC remodeling complexes.

    PubMed

    Harada, Bryan T; Hwang, William L; Deindl, Sebastian; Chatterjee, Nilanjana; Bartholomew, Blaine; Zhuang, Xiaowei

    2016-02-19

    The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1-2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.

  6. Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein.

    PubMed

    Chungjatupornchai, Wipa; Fa-aroonsawat, Sirirat

    2009-04-01

    The translocation of proteins to cyanobacterial cell envelope is made complex by the presence of a highly differentiated membrane system. To investigate the protein translocation in cyanobacterium Synechococcus PCC 7942 using the truncated ice nucleation protein (InpNC) from Pseudomonas syringae KCTC 1832, the green fluorescent protein (GFP) was fused in frame to the carboxyl-terminus of InpNC. The fluorescence of GFP was found almost entirely as a halo in the outer regions of cells which appeared to correspond to the periplasm as demonstrated by confocal laser scanning microscopy, however, GFP was not displayed on the outermost cell surface. Western blotting analysis revealed that InpNC-GFP fusion protein was partially degraded. The N-terminal domain of InpNC may be susceptible to protease attack; the remaining C-terminal domain conjugated with GFP lost the ability to direct translocation across outer membrane and to act as a surface display motif. The fluorescence intensity of cells with periplasmic GFP was approximately 6-fold lower than that of cells with cytoplasmic GFP. The successful translocation of the active GFP to the periplasm may provide a potential means to study the property of cyanobacterial periplasmic substances in response to environmental changes in a non-invasive manner. PMID:19412603

  7. Crystal Structures of EF-G-Ribosome Complexes Trapped in Intermediate States of Translocation

    SciTech Connect

    Zhou, Jie; Lancaster, Laura; Donohue, John Paul; Noller, Harry F.

    2013-11-12

    Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA–EF-G complexes trapped with β,γ-imidoguanosine 5'-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S ribosomal RNA that intercalate between bases of the mRNA may act as “pawls” of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.

  8. Protein translocation and thylakoid biogenesis in cyanobacteria.

    PubMed

    Frain, Kelly M; Gangl, Doris; Jones, Alexander; Zedler, Julie A Z; Robinson, Colin

    2016-03-01

    Cyanobacteria exhibit a complex form of membrane differentiation that sets them apart from most bacteria. Many processes take place in the plasma membrane, but photosynthetic light capture, electron transport and ATP synthesis take place in an abundant internal thylakoid membrane. This review considers how this system of subcellular compartmentalisation is maintained, and how proteins are directed towards the various subcompartments--specifically the plasma membrane, periplasm, thylakoid membrane and thylakoid lumen. The involvement of Sec-, Tat- and signal recognition particle- (SRP)-dependent protein targeting pathways is discussed, together with the possible involvement of a so-called 'spontaneous' pathway for the insertion of membrane proteins, previously characterised for chloroplast thylakoid membrane proteins. An intriguing aspect of cyanobacterial cell biology is that most contain only a single set of genes encoding Sec, Tat and SRP components, yet the proteomes of the plasma and thylakoid membranes are very different. The implications for protein sorting mechanisms are considered. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux.

  9. Role of binding proteins in the translocation of carcinogen metabolites from cytoplasm into the nucleus

    SciTech Connect

    Srinivasan, K.; Bhargava, M.M.

    1986-05-01

    The role of binding proteins in the transport of azo dye hepatocarcinogen metabolites from the cytoplasm to the nucleus was investigated. When the liver cytosol prepared from rats administered /sup 14/C-3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB) was subjected to Sephadex gel chromatography, four peaks (I-IV) of radioactivity containing proteins and one peak of radioactivity devoid of protein, were obtained. Over 90% of the cytosolic 3'-Me-MAB, a precursor of 3'-Me-MAB-N-sulfate, the ultimate carcinogen metabolite, was recovered in peak II. In an in vitro system, the radioactivity associated with this peak only was translocated into the nucleus. Only 10-12% of the radioactivity associated with this peak could be translocated even in the presence of an excess of nuclei. Addition of the peak II fraction isolated from untreated rats or from rats administered unlabeled 3'-Me-DAB to the translocation system, decreased the translocation of radioactivity into the nucleus. When either /sup 125/I labeled cytosolic proteins or peak II fraction were incubated with nuclei, translocation of two major (14-8K) and several minor proteins to the nucleus was observed. The results suggest that specific protein-metabolite complexes are involved in the translocation of azo dye metabolites from the cytosol to the nucleus.

  10. Structure and Activity of Tryptophan-rich TSPO Translocator Proteins

    PubMed Central

    Guo, Youzhong; Kalathur, Ravi C.; Liu, Qun; Kloss, Brian; Bruni, Renato; Ginter, Christopher; Kloppmann, Edda; Rost, Burkhard; Hendrickson, Wayne A.

    2015-01-01

    TSPO translocator proteins bind steroids and porphyrins, and they are implicated in many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs have tryptophan-rich sequences that are fhighly conserved from bacteria to mammals. We report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7Å resolution, including a complex with the benzodiazepine-like inhibitor PK11195. We also describe BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to a previously undescribed heme derivative. We used structure-inspired mutations to investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress. PMID:25635100

  11. Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation

    PubMed Central

    Vermeire, Kurt; Bell, Thomas W.; Van Puyenbroeck, Victor; Giraut, Anne; Noppen, Sam; Liekens, Sandra; Schols, Dominique; Hartmann, Enno

    2014-01-01

    In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins. PMID:25460167

  12. Light-regulated translocation of signaling proteins in Drosophila photoreceptors

    PubMed Central

    Frechter, Shahar; Minke, Baruch

    2007-01-01

    Illumination of Drosophila photoreceptor cells induces multi-facet responses, which include generation of the photoreceptor potential, screening pigment migration and translocation of signaling proteins which is the focus of recent extensive research. Translocation of three signaling molecules is covered in this review: (1) Light-dependent translocation of arrestin from the cytosol to the signaling membrane, the rhabdomere, determines the lifetime of activated rhodopsin. Arrestin translocates in PIP3 and NINAC myosin III dependent manner, and specific mutations which disrupt the interaction between arrestin and PIP3 or NINAC also impair the light-dependant translocation of arrestin and the termination of the response to light. (2) Activation of Drosophila visual G protein, DGq, causes a massive and reversible, translocation of the α subunit from the signaling membrane to the cytosol, accompanied by activity-dependent architectural changes. Analysis of the translocation and the recovery kinetics of DGqα in wild-type flies and specific visual mutants indicated that DGqα is necessary but not sufficient for the architectural changes. (3) The TRP-like (TRPL) but not TRP channels translocate in a light-dependent manner between the rhabdomere and the cell body. As a physiological consequence of this light-dependent modulation of the TRP/TRPL ratio, the photoreceptors of dark-adapted flies operate at a wider dynamic range, which allows the photoreceptors enriched with TRPL to function better in darkness and dim background illumination. Altogether, signal-dependent movement of signaling proteins plays a major role in the maintenance and function of photoreceptor cells. PMID:16458490

  13. Phosphatidylserine-binding protein lactadherin inhibits protein translocation across the ER membrane.

    PubMed

    Yamamoto, Hitoshi; Kida, Yuichiro; Sakaguchi, Masao

    2013-05-10

    Secretory and membrane proteins are translocated across and inserted into the endoplasmic reticulum membrane via translocon channels. To investigate the effect of the negatively-charged phospholipid phosphatidylserine on the translocation of nascent polypeptide chains through the translocon, we used the phosphatidylserine-binding protein lactadherin C2-domain. Lactadherin inhibited targeting of nascent chain to the translocon by signal sequence and the initiation of translocation. Moreover, lactadherin inhibited the movement of the translocating polypeptide chain regardless of the presence or absence of positively-charged residues. Phosphatidylserine might be critically involved in translocon function, but it is not a major determinant for translocation arrest of positively-charged residues. PMID:23583395

  14. Protein kinase C translocation in human blood platelets

    SciTech Connect

    Wang, Hoauyan; Friedman, E. )

    1990-01-01

    Protein kinase C (PKC) activity and translocation in response to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA), serotonin (5-HT) and thrombin was assessed in human platelets. Stimulation with PMA and 5-HT for 10 minutes or thrombin for 1 minute elicited platelet PKC translocation from cytosol to membrane. The catecholamines, norepinephrine or epinephrine at 10 {mu}M concentrations did not induce redistribution of platelet PKC. Serotonin and the specific 5-HT{sub 2} receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI) but not the 5-HT{sub 1A} or 5-HT{sub 1B} agonists, ({plus minus}) 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) or 5-methoxy-3-3-(1,2,3,6-tetrahydro-4-pyridin) 1H-indole succinate (RU 24969) induced dose-dependent PKC translocations. Serotonin-evoked PKC translocation was blocked by selective 5-HT{sub 2} receptor antagonists, ketanserin and spiroperidol. These results suggest that, in human platelets, PMA, thrombin and 5-HT can elicit PKC translocation from cytosol to membrane. Serotonin-induced PKC translocation in platelets is mediated via 5-HT{sub 2} receptors.

  15. Translocation boost protein-folding efficiency of double-barreled chaperonins.

    PubMed

    Coluzza, Ivan; van der Vies, Saskia M; Frenkel, Daan

    2006-05-15

    Incorrect folding of proteins in living cells may lead to malfunctioning of the cell machinery. To prevent such cellular disasters from happening, all cells contain molecular chaperones that assist nonnative proteins in folding into the correct native structure. One of the most studied chaperone complexes is the GroEL-GroES complex. The GroEL part has a "double-barrel" structure, which consists of two cylindrical chambers joined at the bottom in a symmetrical fashion. The hydrophobic rim of one of the GroEL chambers captures nonnative proteins. The GroES part acts as a lid that temporarily closes the filled chamber during the folding process. Several capture-folding-release cycles are required before the nonnative protein reaches its native state. Here we report molecular simulations that suggest that translocation of the nonnative protein through the equatorial plane of the complex boosts the efficiency of the chaperonin action. If the target protein is correctly folded after translocation, it is released. However, if it is still nonnative, it is likely to remain trapped in the second chamber, which then closes to start a reverse translocation process. This shuttling back and forth continues until the protein is correctly folded. Our model provides a natural explanation for the prevalence of double-barreled chaperonins. Moreover, we argue that internal folding is both more efficient and safer than a scenario where partially refolded proteins escape from the complex before being recaptured.

  16. Protein import into complex plastids: Cellular organization of higher complexity.

    PubMed

    Maier, Uwe G; Zauner, Stefan; Hempel, Franziska

    2015-01-01

    Many protists with high ecological and medical relevance harbor plastids surrounded by four membranes. Thus, nucleus-encoded proteins of these complex plastids have to traverse these barriers. Here we report on the identification of the protein translocators located in two of the plastid surrounding membranes and present recent findings on the mechanisms of protein import into the plastids of diatoms.

  17. Inhibitors of Protein Translocation Across the ER Membrane.

    PubMed

    Kalies, Kai-Uwe; Römisch, Karin

    2015-10-01

    Protein translocation into the endoplasmic reticulum (ER) constitutes the first step of protein secretion. ER protein import is essential in all eukaryotic cells and is particularly critical in fast-growing tumour cells. Thus, the process can serve as target both for potential cancer drugs and for bacterial virulence factors. Inhibitors of protein transport across the ER membrane range from broad-spectrum to highly substrate-specific and can interfere with virtually any stage of this multistep process, and even with transport of endocytosed antigens into the cytosol for cross-presentation. PMID:26122014

  18. Crystal structure of a substrate-engaged SecY protein-translocation channel

    PubMed Central

    Ling, JingJing; Ingram, Jessica; Ploegh, Hidde; Rapoport, Tom A.

    2016-01-01

    Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61α, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid1-10. The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation 11. How a translocating polypeptide inserts into the channel is uncertain, as cryo-EM structures of the active channel have a relatively low resolution (~10Å) or are of insufficient quality 6-8. Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The C-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase. PMID:26950603

  19. Complex translocation in a boy with trichorhinophalangeal syndrome.

    PubMed Central

    Sánchez, L M; Labarta, J D; De Negrotti, T C; Migliorini, A M

    1985-01-01

    We report a boy with a trichorhinophalangeal syndrome (TRP syndrome), severe mental retardation, and transient megacephaly, whose karyotype showed complex, apparently balanced, translocations with breakpoints in bands 3q13, 8p22, 8q13, 11p12, and 11q21. The fact that cases presenting with phenotypes corresponding to the TRP II syndrome and deletions of the long arm of chromosome 8 have been recently reported prompted us to report this case to help in the clarification of the possible relation between 8q chromosomal mutation and the aetiology of TRP syndromes. Images PMID:4045963

  20. Exocyst Sec10 is Involved in Basolateral Protein Translation and Translocation in the Endoplasmic Reticulum

    PubMed Central

    Choi, Soo Young; Fogelgren, Ben; Zuo, Xiaofeng; Huang, Liwei; McKenna, Sarah; Lingappa, Vishwanath R.; Lipschutz, Joshua H.

    2013-01-01

    Background Protein translation and translocation at the rough endoplasmic reticulum (RER) are the first steps in the secretory pathway. The translocon through which newly-made proteins are translocated into or across the RER membrane, consists of three main subunits, Sec61α, β, and γ. Sec61β facilitates translocation, and we and others showed that the highly-conserved eight protein exocyst complex interacts with Sec61β. We also showed that the exocyst was involved in basolateral, and not apical, protein synthesis and delivery. Recently, however, exocyst involvement in apical protein delivery was reported. Furthermore, we showed that the exocyst was necessary for formation of primary cilia, organelles found on the apical surface. Methods GST pulldown was performed on lysate of renal tubule cells to investigate biochemical interactions. Cell-free assays consisting of cell-free extracts from rabbit reticulocytes, pancreatic ER microsomal membranes, transcripts of cDNA from apical and basolateral proteins, ATP/GTP, amino acids, and 35S-methionine for protein detection, were used to investigate the role of the exocyst in synthesis of polarized proteins. P32-orthophosphate and immunoprecipitation with antibody against Sec61β was used to investigate the Sec61β phosphorylation in exocyst Sec10-overexpressing cells. Results Sec10 biochemically interacts with Sec61β using GST pulldown. Using cell-free assays, there is enhanced recruitment to ER membranes following exocyst depletion and basolateral VSVG protein translation, compared to apical HA protein translation. Finally, Sec10 overexpression increases Sec61β phosphorylation. Conclusion These data confirm that the exocyst is preferentially involved in basolateral protein translation and translocation, and may well act through the phosphorylation of Sec61β. PMID:23037926

  1. Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites

    PubMed Central

    1989-01-01

    Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number. PMID:2529262

  2. Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex.

    PubMed

    Tagliazucchi, Mario; Peleg, Orit; Kröger, Martin; Rabin, Yitzhak; Szleifer, Igal

    2013-02-26

    The molecular structure of the yeast nuclear pore complex (NPC) and the translocation of model particles have been studied with a molecular theory that accounts for the geometry of the pore and the sequence and anchoring position of the unfolded domains of the nucleoporin proteins (the FG-Nups), which control selective transport through the pore. The theory explicitly models the electrostatic, hydrophobic, steric, conformational, and acid-base properties of the FG-Nups. The electrostatic potential within the pore, which arises from the specific charge distribution of the FG-Nups, is predicted to be negative close to pore walls and positive along the pore axis. The positive electrostatic potential facilitates the translocation of negatively charged particles, and the free energy barrier for translocation decreases for increasing particle hydrophobicity. These results agree with the experimental observation that transport receptors that form complexes with hydrophilic/neutral or positively charged proteins to transport them through the NPC are both hydrophobic and strongly negatively charged. The molecular theory shows that the effects of electrostatic and hydrophobic interactions on the translocating potential are cooperative and nonequivalent due to the interaction-dependent reorganization of the FG-Nups in the presence of the translocating particle. The combination of electrostatic and hydrophobic interactions can give rise to complex translocation potentials displaying a combination of wells and barriers, in contrast to the simple barrier potential observed for a hydrophilic/neutral translocating particle. This work demonstrates the importance of explicitly considering the amino acid sequence and hydrophobic, electrostatic, and steric interactions in understanding the translocation through the NPC.

  3. Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex.

    PubMed

    Tagliazucchi, Mario; Peleg, Orit; Kröger, Martin; Rabin, Yitzhak; Szleifer, Igal

    2013-02-26

    The molecular structure of the yeast nuclear pore complex (NPC) and the translocation of model particles have been studied with a molecular theory that accounts for the geometry of the pore and the sequence and anchoring position of the unfolded domains of the nucleoporin proteins (the FG-Nups), which control selective transport through the pore. The theory explicitly models the electrostatic, hydrophobic, steric, conformational, and acid-base properties of the FG-Nups. The electrostatic potential within the pore, which arises from the specific charge distribution of the FG-Nups, is predicted to be negative close to pore walls and positive along the pore axis. The positive electrostatic potential facilitates the translocation of negatively charged particles, and the free energy barrier for translocation decreases for increasing particle hydrophobicity. These results agree with the experimental observation that transport receptors that form complexes with hydrophilic/neutral or positively charged proteins to transport them through the NPC are both hydrophobic and strongly negatively charged. The molecular theory shows that the effects of electrostatic and hydrophobic interactions on the translocating potential are cooperative and nonequivalent due to the interaction-dependent reorganization of the FG-Nups in the presence of the translocating particle. The combination of electrostatic and hydrophobic interactions can give rise to complex translocation potentials displaying a combination of wells and barriers, in contrast to the simple barrier potential observed for a hydrophilic/neutral translocating particle. This work demonstrates the importance of explicitly considering the amino acid sequence and hydrophobic, electrostatic, and steric interactions in understanding the translocation through the NPC. PMID:23404701

  4. Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex

    PubMed Central

    Tagliazucchi, Mario; Peleg, Orit; Kröger, Martin; Rabin, Yitzhak; Szleifer, Igal

    2013-01-01

    The molecular structure of the yeast nuclear pore complex (NPC) and the translocation of model particles have been studied with a molecular theory that accounts for the geometry of the pore and the sequence and anchoring position of the unfolded domains of the nucleoporin proteins (the FG-Nups), which control selective transport through the pore. The theory explicitly models the electrostatic, hydrophobic, steric, conformational, and acid-base properties of the FG-Nups. The electrostatic potential within the pore, which arises from the specific charge distribution of the FG-Nups, is predicted to be negative close to pore walls and positive along the pore axis. The positive electrostatic potential facilitates the translocation of negatively charged particles, and the free energy barrier for translocation decreases for increasing particle hydrophobicity. These results agree with the experimental observation that transport receptors that form complexes with hydrophilic/neutral or positively charged proteins to transport them through the NPC are both hydrophobic and strongly negatively charged. The molecular theory shows that the effects of electrostatic and hydrophobic interactions on the translocating potential are cooperative and nonequivalent due to the interaction-dependent reorganization of the FG-Nups in the presence of the translocating particle. The combination of electrostatic and hydrophobic interactions can give rise to complex translocation potentials displaying a combination of wells and barriers, in contrast to the simple barrier potential observed for a hydrophilic/neutral translocating particle. This work demonstrates the importance of explicitly considering the amino acid sequence and hydrophobic, electrostatic, and steric interactions in understanding the translocation through the NPC. PMID:23404701

  5. VirB/D4-dependent protein translocation from Agrobacterium into plant cells.

    PubMed

    Vergunst, A C; Schrammeijer, B; den Dulk-Ras, A; de Vlaam, C M; Regensburg-Tuïnk, T J; Hooykaas, P J

    2000-11-01

    The Agrobacterium VirB/D4 transport system mediates the transfer of a nucleoprotein T complex into plant cells, leading to crown gall disease. In addition, several Virulence proteins must somehow be transported to fulfill a function in planta. Here, we used fusions between Cre recombinase and VirE2 or VirF to directly demonstrate protein translocation into plant cells. Transport of the proteins was monitored by a Cre-mediated in planta recombination event resulting in a selectable phenotype and depended on the VirB/D4 transport system but did not require transferred DNA. PMID:11062129

  6. Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient.

    PubMed

    Krantz, Bryan A; Finkelstein, Alan; Collier, R John

    2006-02-01

    Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (DeltapH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. DeltapH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or phi clamp) drives translocation synergistically under a DeltapH. We propose that a Brownian ratchet mechanism proposed earlier for the phi clamp is cooperatively linked to a protonation-state, DeltapH-driven ratchet acting trans to the phi-clamp site. In a sense, the channel functions as a proton/protein symporter.

  7. Protein Translocation through Tom40: Kinetics of Peptide Release

    PubMed Central

    Mahendran, Kozhinjampara R.; Romero-Ruiz, Mercedes; Schlösinger, Andrea; Winterhalter, Mathias; Nussberger, Stephan

    2012-01-01

    Mitochondrial proteins are almost exclusively imported into mitochondria from the cytosol in an unfolded or partially folded conformation. Regardless of whether they are destined for the outer or inner membrane, the intermembrane space, or the matrix, proteins begin the importation process by crossing the mitochondrial outer membrane via a specialized protein import machinery whose main component is the Tom40 channel. High-resolution ion conductance measurements through the Tom40 channel in the presence of the mitochondrial presequence peptide pF1β revealed the kinetics of peptide binding. Here we show that the rates for association kon and dissociation koff strongly depend on the applied transmembrane voltage. Both kinetic constants increase with an increase in the applied voltage. The increase of koff with voltage provides strong evidence of peptide translocation. This allows us to distinguish quantitatively between substrate blocking and permeation. PMID:22225796

  8. The changing landscape in translocator protein (TSPO) function.

    PubMed

    Selvaraj, Vimal; Stocco, Douglas M

    2015-07-01

    Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is an outer mitochondrial membrane protein. TSPO has been shown to cooperate with steroidogenic acute regulatory protein (StAR) and function in the transport of cholesterol into mitochondria. TSPO has also been considered as a structural component of the mitochondrial permeability transition pore (MPTP). However, recent advances have changed these views of TSPO's functions and have prompted a re-evaluation of established concepts. This review summarizes the history of TSPO, key elements of the debate, and functional experiments that have changed our understanding. Moving forward, we examine how this fundamental change impacts our understanding of TSPO and affects the future of TSPO as a therapeutic and diagnostic target.

  9. Heterologous protein production using the twin arginine translocation pathway

    DOEpatents

    Pohlschroder, Mechtild; Kissinger, Jessica C; Rose, R. Wesley; Brueser, Thomas; Dilks, Kieran

    2008-11-04

    Provided are means for evaluating and identifying putative substrates of the twin arginine translocation (Tat) secretory pathway in Streptomyces and other bacterial species. Also provided, therefore, are simple ways to express, secrete and purify correctly folded heterologous proteins on a large scale using host microorganisms, such as, Streptomyces and the Tat pathway therein. Many of the thus-produced proteins are of significant therapeutic value in the pharmaceutical and biochemical industries, particularly when they can be secreted from the host in fully-folded active form. Accordingly, there are further provided the heterologous proteins produced by the Tat secretion pathway using the foregoing methods, and the computer algorithm used to identify the Tat signal sequence and putative substrates.

  10. Protein Complexes in Bacteria

    PubMed Central

    Caufield, J. Harry; Abreu, Marco; Wimble, Christopher; Uetz, Peter

    2015-01-01

    Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies. PMID:25723151

  11. Periodic forces trigger knot untying during translocation of knotted proteins

    NASA Astrophysics Data System (ADS)

    Szymczak, Piotr

    2016-03-01

    Proteins need to be unfolded when translocated through the pores in mitochondrial and other cellular membranes. Knotted proteins, however, might get stuck during this process, jamming the pore, since the diameter of the pore is smaller than the size of maximally tightened knot. The jamming probability dramatically increases as the magnitude of the driving force exceeds a critical value, Fc. In this numerical study, we show that for deep knots Fc lies below the force range over which molecular import motors operate, which suggest that in these cases the knots will tighten and block the pores. Next, we show how such topological traps might be prevented by using a pulling protocol of a repetitive, on-off character. Such a repetitive pulling is biologically relevant, since the mitochondrial import motor, like other molecular motors transforms chemical energy into directed motions via nucleotide-hydrolysis-mediated conformational changes, which are cyclic in character.

  12. Periodic forces trigger knot untying during translocation of knotted proteins

    PubMed Central

    Szymczak, Piotr

    2016-01-01

    Proteins need to be unfolded when translocated through the pores in mitochondrial and other cellular membranes. Knotted proteins, however, might get stuck during this process, jamming the pore, since the diameter of the pore is smaller than the size of maximally tightened knot. The jamming probability dramatically increases as the magnitude of the driving force exceeds a critical value, Fc. In this numerical study, we show that for deep knots Fc lies below the force range over which molecular import motors operate, which suggest that in these cases the knots will tighten and block the pores. Next, we show how such topological traps might be prevented by using a pulling protocol of a repetitive, on-off character. Such a repetitive pulling is biologically relevant, since the mitochondrial import motor, like other molecular motors transforms chemical energy into directed motions via nucleotide-hydrolysis-mediated conformational changes, which are cyclic in character. PMID:26996878

  13. Minireview: translocator protein (TSPO) and steroidogenesis: a reappraisal.

    PubMed

    Selvaraj, Vimal; Stocco, Douglas M; Tu, Lan N

    2015-04-01

    The 18-kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a transmembrane protein in the outer mitochondrial membrane. TSPO has long been described as being indispensable for mitochondrial cholesterol import that is essential for steroid hormone production. In contrast to this initial proposition, recent experiments reexamining TSPO function have demonstrated that it is not involved in steroidogenesis. This fundamental change has forced a reexamination of the functional interpretations made for TSPO that broadly impacts both basic and clinical research across multiple fields. In this minireview, we recapitulate the key studies from 25 years of TSPO research and concurrently examine their limitations that perhaps led towards the incorrect association of TSPO and steroid hormone production. Although this shift in understanding raises new questions regarding the molecular function of TSPO, these recent developments are poised to have a significant positive impact for research progress in steroid endocrinology.

  14. Evolving understanding of translocator protein 18 kDa (TSPO).

    PubMed

    Li, Fei; Liu, Jian; Garavito, R Michael; Ferguson-Miller, Shelagh

    2015-09-01

    The translocator protein 18 kDa (TSPO) has been the focus of intense research by the biomedical community and the pharmaceutical industry because of its apparent involvement in many disease-related processes. These include steroidogenesis, apoptosis, inflammation, neurological disease and cancer, resulting in the use of TSPO as a biomarker and its potential as a drug target. Despite more than 30 years of study, the precise function of TSPO remains elusive. A recent breakthrough in determining the high-resolution crystal structures of bacterial homologs of mitochondrial TSPO provides new insight into the structural and functional properties at a molecular level and new opportunities for investigating the significance of this ancient and highly conserved protein family. The availability of atomic level structural information from different species also provides a platform for structure-based drug development. Here we briefly review current knowledge regarding TSPO and the implications of the new structures with respect to hypotheses and controversies in the field.

  15. Periodic forces trigger knot untying during translocation of knotted proteins.

    PubMed

    Szymczak, Piotr

    2016-01-01

    Proteins need to be unfolded when translocated through the pores in mitochondrial and other cellular membranes. Knotted proteins, however, might get stuck during this process, jamming the pore, since the diameter of the pore is smaller than the size of maximally tightened knot. The jamming probability dramatically increases as the magnitude of the driving force exceeds a critical value, Fc. In this numerical study, we show that for deep knots Fc lies below the force range over which molecular import motors operate, which suggest that in these cases the knots will tighten and block the pores. Next, we show how such topological traps might be prevented by using a pulling protocol of a repetitive, on-off character. Such a repetitive pulling is biologically relevant, since the mitochondrial import motor, like other molecular motors transforms chemical energy into directed motions via nucleotide-hydrolysis-mediated conformational changes, which are cyclic in character. PMID:26996878

  16. Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

    PubMed Central

    Hugueney, P; Bouvier, F; Badillo, A; d'Harlingue, A; Kuntz, M; Camara, B

    1995-01-01

    Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation. Images Fig. 1 Fig. 3 Fig. 4 PMID:7777561

  17. Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

    PubMed Central

    Feldheim, D; Rothblatt, J; Schekman, R

    1992-01-01

    SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex. Images PMID:1620130

  18. The prediction of novel multiple lipid-binding regions in protein translocation motor proteins: a possible general feature.

    PubMed

    Keller, Rob C A

    2011-03-01

    Protein translocation is an important cellular process. SecA is an essential protein component in the Sec system, as it contains the molecular motor that facilitates protein translocation. In this study, a bioinformatics approach was applied in the search for possible lipid-binding helix regions in protein translocation motor proteins. Novel lipid-binding regions in Escherichia coli SecA were identified. Remarkably, multiple lipid-binding sites were also identified in other motor proteins such as BiP, which is involved in ER protein translocation. The prokaryotic signal recognition particle receptor FtsY, though not a motor protein, is in many ways related to SecA, and was therefore included in this study. The results demonstrate a possible general feature for motor proteins involved in protein translocation. PMID:20957445

  19. Translocator Protein 2 Is Involved in Cholesterol Redistribution during Erythropoiesis*

    PubMed Central

    Fan, Jinjiang; Rone, Malena B.; Papadopoulos, Vassilios

    2009-01-01

    Translocator protein (TSPO) is an 18-kDa cholesterol- and drug-binding protein conserved from bacteria to humans. While surveying for Tspo-like genes, we identified its paralogous gene, Tspo2, encoding an evolutionarily conserved family of proteins that arose by gene duplications before the divergence of avians and mammals. Comparative analysis of Tspo1 and Tspo2 functions suggested that Tspo2 has become subfunctionalized, typical of duplicated genes, characterized by the loss of diagnostic drug ligand-binding but retention of cholesterol-binding properties, hematopoietic tissue- and erythroid cell-specific distribution, and subcellular endoplasmic reticulum and nuclear membrane localization. Expression of Tspo2 in erythroblasts is strongly correlated with the down-regulation of the enzymes involved in cholesterol biosynthesis. Overexpression of TSPO2 in erythroid cells resulted in the redistribution of intracellular free cholesterol, an essential step in nucleus expulsion during erythrocyte maturation. Taken together, these data identify the TSPO2 family of proteins as mediators of cholesterol redistribution-dependent erythroblast maturation during mammalian erythropoiesis. PMID:19729679

  20. Mitochondrial translocator protein (TSPO): From physiology to cardioprotection.

    PubMed

    Morin, Didier; Musman, Julien; Pons, Sandrine; Berdeaux, Alain; Ghaleh, Bijan

    2016-04-01

    The mitochondrial translocator protein (TSPO) is a high affinity cholesterol binding protein which is primarily located in the outer mitochondrial membrane where it has been shown to interact with proteins implicated in mitochondrial permeability transition pore (mPTP) formation. TSPO is found in different species and is expressed at high levels in tissues that synthesize steroids but is also present in other peripheral tissues especially in the heart. TSPO has been involved in the import of cholesterol into mitochondria, a key step in steroidogenesis. This constitutes the main established function of the protein which was recently challenged by genetic studies. TSPO has also been associated directly or indirectly with a wide range of cellular functions such as apoptosis, cell proliferation, differentiation, regulation of mitochondrial function or porphyrin transport. In the heart the role of TSPO remains undefined but a growing body of evidence suggests that TSPO plays a critical role in regulating physiological cardiac function and that TSPO ligands may represent interesting drugs to protect the heart under pathological conditions. This article briefly reviews current knowledge regarding TSPO and discusses its role in the cardiovascular system under physiological and pathologic conditions. More particularly, it provides evidence that TSPO can represent an alternative strategy to develop new pharmacological agents to protect the myocardium against ischemia-reperfusion injury.

  1. Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

    PubMed

    Ruano-Gallego, David; Álvarez, Beatriz; Fernández, Luis Ángel

    2015-09-18

    Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these "molecular syringes" for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells. PMID:26017572

  2. Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

    PubMed

    Ruano-Gallego, David; Álvarez, Beatriz; Fernández, Luis Ángel

    2015-09-18

    Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these "molecular syringes" for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells.

  3. Autocrine Signaling Underlies Fast Repetitive Plasma Membrane Translocation of Conventional and Novel Protein Kinase C Isoforms in β Cells.

    PubMed

    Wuttke, Anne; Yu, Qian; Tengholm, Anders

    2016-07-15

    PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca(2+)- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In β cells, exocytosis of insulin granules evokes brief (<10 s) local DAG elevations ("spiking") at the plasma membrane because of autocrine activation of P2Y1 purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein-tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MIN6 cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCδ, PKCϵ, and PKCη. The conventional PKCα, PKCβI, and PKCβII isoforms showed a more complex pattern with both rapid and slow translocation. K(+) depolarization-induced PKCϵ translocation entirely mirrored DAG spiking, whereas PKCβI translocation showed a sustained component, reflecting the subplasma membrane Ca(2+) concentration ([Ca(2+)]pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca(2+)]pm elevation or sustained PKCβI translocation. The muscarinic agonist carbachol induced pronounced transient PKCβI translocation and sustained recruitment of PKCϵ. When rise of [Ca(2+)]pm was prevented, the carbachol-induced DAG and PKCϵ responses were somewhat reduced, but PKCβI translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the β cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of β cell function.

  4. Autocrine Signaling Underlies Fast Repetitive Plasma Membrane Translocation of Conventional and Novel Protein Kinase C Isoforms in β Cells*

    PubMed Central

    Wuttke, Anne; Yu, Qian; Tengholm, Anders

    2016-01-01

    PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca2+- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In β cells, exocytosis of insulin granules evokes brief (<10 s) local DAG elevations (“spiking”) at the plasma membrane because of autocrine activation of P2Y1 purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein-tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MIN6 cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCδ, PKCϵ, and PKCη. The conventional PKCα, PKCβI, and PKCβII isoforms showed a more complex pattern with both rapid and slow translocation. K+ depolarization-induced PKCϵ translocation entirely mirrored DAG spiking, whereas PKCβI translocation showed a sustained component, reflecting the subplasma membrane Ca2+ concentration ([Ca2+]pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca2+]pm elevation or sustained PKCβI translocation. The muscarinic agonist carbachol induced pronounced transient PKCβI translocation and sustained recruitment of PKCϵ. When rise of [Ca2+]pm was prevented, the carbachol-induced DAG and PKCϵ responses were somewhat reduced, but PKCβI translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the β cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of β cell function. PMID:27226533

  5. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  6. Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells

    PubMed Central

    Sakalis, Philippe A; van Heusden, G Paul H; Hooykaas, Paul J J

    2014-01-01

    Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20–25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli. PMID:24376037

  7. Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells.

    PubMed

    Sakalis, Philippe A; van Heusden, G Paul H; Hooykaas, Paul J J

    2014-02-01

    Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20-25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli. PMID:24376037

  8. YspC: a unique translocator exhibits structural alteration in the complex form with chaperone SycB.

    PubMed

    Basu, Abhishek; Chatterjee, Rakesh; Datta, Saumen

    2012-08-01

    YspC is an annotated translocator of Yersinia secretion apparatus-Yersinia secretion protein type three secretion system of Yersinia enterocolitica, it forms an 1:1 complex with its cognate chaperone SycB. Unlike other translocators, YspC is highly soluble inspite of having a transmembrane region. Size exclusion chromatography shows that YspC exists predominantly in a monomeric form. Multiple sequence alignment and ConSurf (a web based bioinformatic tool) analysis confirm its significant deviation from the closest class of minor translocators. YspC also possesses a tertiary structure signal seen from near UV CD, further confirming its unique nature amongst the groups of translocators. Far UV CD depicts that YspC is predominantly an α-helical protein; however, its secondary structure alters in the YspC-SycB complex. Thermal denaturation curve predicts a cooperative melting behaviour for YspC which is altered in the YspC-SycB complex. Furthermore, trypsinolysis data confirms a different digestion pattern for YspC in isolation, when compared to the complex form with SycB. From the Forsters resonance energy transfer analysis, it can be predicted that the two tetratricopeptide repeat regions of SycB are masked while it forms a complex with YspC and this is further confirmed by the interaction studies of YspC with two truncated forms of SycB. YspC interacted with ∆SycB₁₋₁₁₄ and ∆SycB₃₆₋₁₁₄ (possessing only the two TPR regions). However, the complexes formed between YspC and truncated forms of SycB have altered physiological states.

  9. Detergent disruption of bacterial inner membranes and recovery of protein translocation activity

    SciTech Connect

    Cunningham, K.; Wickner, W.T. )

    1989-11-01

    Isolation of the integral membrane components of protein translocation requires methods for fractionation and functional reconstitution. The authors treated inner-membrane vesicles of Escherichia coli with mixtures of octyl {beta}-D-glucoside, phospholipids, and an integral membrane carrier protein under conditions that extract most of the membrane proteins into micellar solution. Upon dialysis, proteoliposomes were reconstituted that supported translocation of radiochemically pure ({sup 35}S)pro-OmpA (the precursor of outer membrane protein A). Translocation into these proteoliposomes required ATP hydrolysis and membrane proteins, indicating that the reaction is that of the inner membrane. The suspension of membranes in detergent was separated into supernatant and pellet fractions by ultracentrifugation. After reconstitution, translocation activity was observed in both fractions, but processing by leader peptidase of translocated pro-OmpA to OmpA was not detectable in the reconstituted pellet fraction. Processing activity was restored by addition of pure leader peptidase as long as this enzyme was added before detergent removal, indicating that the translocation activity is not associated with detergent-resistant membrane vesicles. These results show that protein translocation activity can be recovered from detergent-disrupted membrane vesicles, providing a first step towards the goal of isolating the solubilized components.

  10. A role for a eukaryotic GrpE-related protein, Mge1p, in protein translocation.

    PubMed Central

    Laloraya, S; Gambill, B D; Craig, E A

    1994-01-01

    The 70-kDa heat shock proteins (hsp70s) function as molecular chaperones in a wide variety of cellular processes through cycles of binding and release from substrate proteins coupled to cycles of ATP hydrolysis. In the prokaryote Escherichia coli, the hsp70 DnaK functions with two other proteins, DnaJ and GrpE, which modulate the activity of DnaK. While numerous hsp70s and DnaJ-related proteins have been identified in eukaryotes, to our knowledge no GrpE-related proteins have been reported. We report the isolation and characterization of a eukaryotic grpE-related gene, MGE1. MGE1, an essential nuclear gene of the yeast Saccharomyces cerevisiae, encodes a soluble protein of the mitochondrial matrix. Cells with reduced expression of Mge1p accumulate the precursor form of a mitochondrial protein. Since mitochondrial hsp70 is required for translocation of precursors of mitochondrial proteins from the cytosol into the matrix of mitochondria, these data suggest that Mge1p acts in concert with mitochondrial hsp70 in protein translocation. Images PMID:8022808

  11. Identification of a mitochondrial ATP synthase-adenine nucleotide translocator complex in Leishmania.

    PubMed

    Detke, Siegfried; Elsabrouty, Rania

    2008-01-01

    The ATP synthasome is a macromolecular complex consisting of ATP synthase, adenine nucleotide translocator and phosphate carrier. To determine if this complex is evolutionary old or young, we searched for its presence in Leishmania, a mitochondria containing protozoan which evolved from the main eukaryote line soon after eukaryotes split from prokaryotes. Sucrose gradient centrifugation showed that the distribution of ANT among the fractions coincided with the distribution of ATP synthase. In addition, ATP synthase co-precipitated with FLAG tagged and wild type adenine nucleotide translocator isolated with anti FLAG and anti adenine nucleotide translocator antibodies, respectively. These data indicate that the adenine nucleotide translocator interacted with the ATP synthase to form a stable structure referred to as the ATP synthasome. The presence of the ATP synthasome in Leishmania, an organism branching off the main line of eukaryotes early in the development of eukaryotes, as well as in higher eukaryotes suggests that the ATP synthasome is a phylogenetically ancient structure. PMID:17920025

  12. Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein*

    PubMed Central

    Wolters, Manuel; Boyle, Erin C.; Lardong, Kerstin; Trülzsch, Konrad; Steffen, Anika; Rottner, Klemens; Ruckdeschel, Klaus; Aepfelbacher, Martin

    2013-01-01

    Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac. PMID:23803609

  13. Prediction of lipid-binding regions in cytoplasmic and extracellular loops of membrane proteins as exemplified by protein translocation membrane proteins.

    PubMed

    Keller, Rob C A

    2013-01-01

    The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation. PMID:22961045

  14. Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein

    PubMed Central

    Ylösmäki, Leena; Fagerlund, Riku; Kuisma, Inka; Julkunen, Ilkka; Saksela, Kalle

    2016-01-01

    The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif. PMID:27092521

  15. Translocator protein mediates the anxiolytic and antidepressant effects of midazolam.

    PubMed

    Qiu, Zhi-Kun; Li, Ming-Sheng; He, Jia-Li; Liu, Xu; Zhang, Guan-Hua; Lai, Sha; Ma, Jian-Chun; Zeng, Jia; Li, Yan; Wu, Hong-Wei; Chen, Yong; Shen, Yong-Gang; Chen, Ji-Sheng

    2015-12-01

    The translocator protein (18 kDa) (TSPO) plays an important role in stress-related disorders, such as anxiety, depression and post-traumatic stress disorder (PTSD), caused by neurosteroids (e.g. allopregnanolone). The present study sought to evaluate the significance of TSPO in anxiolytic and antidepressant effects induced by midazolam. The animals were administrated midazolam (0.25, 0.5 and 1 mg/kg, i.p.) and subjected to behavioral tests, including Vogel-type conflict test, elevated plus-maze test, forced swimming test. Midazolam produced anxiolytic- and antidepressant-like effects Vogel-type conflict test (1 mg/kg, i.p.), elevated plus-maze test (0.5 and 1 mg/kg, i.p.), and forced swimming test (0.5 and 1 mg/kg, i.p.). These effects of Midazolam were totally blocked by the TSPO antagonist PK11195 (3 mg/kg, i.p.). To evaluate the role of allopregnanolone in the anxiolytic- and antidepressant-like effects of midazolam, the animals were decapitated at the end of the behavioral tests. The allopregnanolone levels of the prefrontal cortex and hippocampus were measured by enzyme-linked immunosorbent assay (ELISA). The allopregnanolone level of the prefrontal cortex and hippocampus was increased by midazolam (0.5, 1 mg/kg, i.p.) and the increase was reversed by PK11195 (3 mg/kg, i.p.). Overall, the results indicated that the anxiolytic- and antidepressant-like effects of midazolam were mediated by TSPO, via stimulation of allopregnanolone biosynthesis. PMID:26455280

  16. Quantifying the role of chaperones in protein translocation by computational modeling.

    PubMed

    Assenza, Salvatore; De Los Rios, Paolo; Barducci, Alessandro

    2015-01-01

    The molecular chaperone Hsp70 plays a central role in the import of cytoplasmic proteins into organelles, driving their translocation by binding them from the organellar interior. Starting from the experimentally-determined structure of the E. coli Hsp70, we computed, by means of molecular simulations, the effective free-energy profile for substrate translocation upon chaperone binding. We then used the resulting free energy to quantitatively characterize the kinetics of the import process, whose comparison with unassisted translocation highlights the essential role played by Hsp70 in importing cytoplasmic proteins.

  17. Protein sorting in complex plastids.

    PubMed

    Sheiner, Lilach; Striepen, Boris

    2013-02-01

    Taming a cyanobacterium in a pivitol event of endosymbiosis brought photosynthesis to eukaryotes, and gave rise to the plastids found in glaucophytes, red and green algae, and the descendants of the latter, the plants. Ultrastructural as well as molecular research over the last two decades has demonstrated that plastids have enjoyed surprising lateral mobility across the tree of life. Numerous independent secondary and tertiary endosymbiosis have led to a spread of plastids into a variety of, up to that point, non-photosynthetic lineages. Happily eating and subsequently domesticating one another protists conquered a wide variety of ecological niches. The elaborate evolution of secondary, or complex, plastids is reflected in the numerous membranes that bound them (three or four compared to the two membranes of the primary plastids). Gene transfer to the host nucleus is a hallmark of endosymbiosis and provides centralized cellular control. Here we review how these proteins find their way back into the stroma of the organelle and describe the advances in the understanding of the molecular mechanisms that allow protein translocation across four membranes. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  18. Apratoxin Kills Cells by Direct Blockade of the Sec61 Protein Translocation Channel.

    PubMed

    Paatero, Anja O; Kellosalo, Juho; Dunyak, Bryan M; Almaliti, Jehad; Gestwicki, Jason E; Gerwick, William H; Taunton, Jack; Paavilainen, Ville O

    2016-05-19

    Apratoxin A is a cytotoxic natural product that prevents the biogenesis of secretory and membrane proteins. Biochemically, apratoxin A inhibits cotranslational translocation into the ER, but its cellular target and mechanism of action have remained controversial. Here, we demonstrate that apratoxin A prevents protein translocation by directly targeting Sec61α, the central subunit of the protein translocation channel. Mutagenesis and competitive photo-crosslinking studies indicate that apratoxin A binds to the Sec61 lateral gate in a manner that differs from cotransin, a substrate-selective Sec61 inhibitor. In contrast to cotransin, apratoxin A does not exhibit a substrate-selective inhibitory mechanism, but blocks ER translocation of all tested Sec61 clients with similar potency. Our results suggest that multiple structurally unrelated natural products have evolved to target overlapping but non-identical binding sites on Sec61, thereby producing distinct biological outcomes.

  19. A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation

    PubMed Central

    Thoma, Lina; Dobrowinski, Hyazinth; Finger, Constanze; Guezguez, Jamil; Linke, Dirk; Sepulveda, Edgardo

    2015-01-01

    ABSTRACT Conjugative DNA transfer in mycelial Streptomyces is a unique process involving the transfer of a double-stranded plasmid from the donor into the recipient and the subsequent spreading of the transferred plasmid within the recipient mycelium. This process is associated with growth retardation of the recipient and manifested by the formation of circular inhibition zones, named pocks. To characterize the unique Streptomyces DNA transfer machinery, we replaced each gene of the conjugative 12.1-kbp Streptomyces venezuelae plasmid pSVH1, with the exception of the rep gene required for plasmid replication, with a hexanucleotide sequence. Only deletion of traB, encoding the FtsK-like DNA translocase, affected efficiency of the transfer dramatically and abolished pock formation. Deletion of spdB3, spd79, or spdB2 had a minor effect on transfer but prevented pock formation and intramycelial plasmid spreading. Biochemical characterization of the encoded proteins revealed that the GntR-type regulator TraR recognizes a specific sequence upstream of spdB3, while Orf108, SpdB2, and TraR bind to peptidoglycan. SpdB2 promoted spheroplast formation by T7 lysozyme and formed pores in artificial membranes. Bacterial two-hybrid analyses and chemical cross-linking revealed that most of the pSVH1-encoded proteins interacted with each other, suggesting a multiprotein DNA translocation complex of TraB and Spd proteins which directs intramycelial plasmid spreading. PMID:26015502

  20. Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells

    PubMed Central

    2015-01-01

    Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these “molecular syringes” for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells. PMID:26017572

  1. Multifunctional roles for the protein translocation machinery in RNA anchoring to the endoplasmic reticulum.

    PubMed

    Jagannathan, Sujatha; Hsu, Jack C-C; Reid, David W; Chen, Qiang; Thompson, Will J; Moseley, Arthur M; Nicchitta, Christopher V

    2014-09-12

    Signal sequence-encoding mRNAs undergo translation-dependent localization to the endoplasmic reticulum (ER) and at the ER are anchored via translation on Sec61-bound ribosomes. Recent investigations into the composition and membrane association characteristics of ER-associated mRNAs have, however, revealed both ribosome-dependent (indirect) and ribosome-independent (direct) modes of mRNA association with the ER. These findings raise important questions regarding our understanding of how mRNAs are selected, localized, and anchored to the ER. Using semi-intact tissue culture cells, we performed a polysome solubilization screen and identified conditions that distinguish polysomes engaged in the translation of distinct cohorts of mRNAs. To gain insight into the molecular basis of direct mRNA anchoring to the ER, we performed RNA-protein UV photocross-linking studies in rough microsomes and demonstrate that numerous ER integral membrane proteins display RNA binding activity. Quantitative proteomic analyses of HeLa cytosolic and ER-bound polysome fractions identified translocon components as selective polysome-interacting proteins. Notably, the Sec61 complex was highly enriched in polysomes engaged in the translation of endomembrane organelle proteins, whereas translocon accessory proteins, such as ribophorin I, were present in all subpopulations of ER-associated polysomes. Analyses of the protein composition of oligo(dT)-selected UV photocross-linked ER protein-RNA adducts identified Sec61α,β and ribophorin I as ER-poly(A) mRNA-binding proteins, suggesting unexpected roles for the protein translocation and modification machinery in mRNA anchoring to the ER. In summary, we propose that multiple mechanisms of mRNA and ribosome association with ER operate to enable an mRNA transcriptome-wide function for the ER in protein synthesis.

  2. Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein.

    PubMed

    Yang, Sung-Tae; Lim, Sung In; Kiessling, Volker; Kwon, Inchan; Tamm, Lukas K

    2016-01-01

    Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein's folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca(2+)-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches. PMID:27605302

  3. Nucleoporin translocated promoter region (Tpr) associates with dynein complex, preventing chromosome lagging formation during mitosis.

    PubMed

    Nakano, Hiroshi; Funasaka, Tatsuyoshi; Hashizume, Chieko; Wong, Richard W

    2010-04-01

    Gain or loss of whole chromosomes is often observed in cancer cells and is thought to be due to aberrant chromosome segregation during mitosis. Proper chromosome segregation depends on a faithful interaction between spindle microtubules and kinetochores. Several components of the nuclear pore complex/nucleoporins play critical roles in orchestrating the rapid remodeling events that occur during mitosis. Our recent studies revealed that the nucleoporin, Rae1, plays critical roles in maintaining spindle bipolarity. Here, we show association of another nucleoporin, termed Tpr (translocated promoter region), with the molecular motors dynein and dynactin, which both orchestrate with the spindle checkpoints Mad1 and Mad2 during cell division. Overexpression of Tpr enhanced multinucleated cell formation. RNA interference-mediated knockdown of Tpr caused a severe lagging chromosome phenotype and disrupted spindle checkpoint proteins expression and localization. Next, we performed a series of rescue and dominant negative experiments to confirm that Tpr orchestrates proper chromosome segregation through interaction with dynein light chain. Our data indicate that Tpr functions as a spatial and temporal regulator of spindle checkpoints, ensuring the efficient recruitment of checkpoint proteins to the molecular motor dynein to promote proper anaphase formation.

  4. Entropic pulling: how Hsp70 chaperones translocate proteins through membrane pores

    NASA Astrophysics Data System (ADS)

    de Los Rios, Paolo; Ben-Zvi, Anat; Slutsky, Olga; Azem, Abdussalam; Goloubinoff, Pierre

    2006-03-01

    Hsp70s are highly conserved ATPase molecular chaperones mediating the translocation of proteins across membranes and the active unfolding and disassembly of stress-induced protein aggregates. Here, we introduce a mechanism named entropic pulling, based on entropy loss due to excluded volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable to drive the translocation of polypeptides into mitochondria. Entropic pulling represents a possible solution to the long-standing debate between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a unifying mechanism for the different Hsp70 functions.

  5. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

    PubMed Central

    Chen, Xin; Li, Wenjuan; Ren, Junming; Huang, Deli; He, Wan-ting; Song, Yunlong; Yang, Chao; Li, Wanyun; Zheng, Xinru; Chen, Pengda; Han, Jiahuai

    2014-01-01

    Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture. PMID:24366341

  6. The Candida albicans Kar2 protein is essential and functions during the translocation of proteins into the endoplasmic reticulum

    PubMed Central

    Janke, Megan R.; Lund, Kyle; Morrison, Emily P.; Paulson, Benjamin A.

    2012-01-01

    Since the secretory pathway is essential for Candida albicans to transition from a commensal organism to a pathogen, an understanding of how this pathway functions may be beneficial for identifying novel drug targets to prevent candidiasis. We have cloned the C. albicans KAR2 gene, which performs many roles during the translocation of proteins into the endoplasmic reticulum (ER) during the first committed step of the secretory pathway in many eukaryotes. Our results show that C. albicans KAR2 is essential, and that the encoded protein rescues a temperature-sensitive growth defect found in a Saccharomyces cerevisiae strain harboring a mutant form of the Kar2 protein. Additionally, S. cerevisiae containing CaKAR2 as the sole copy of this essential gene are viable, and ER microsomes prepared from this strain exhibit wild-type levels of post-translational translocation during in vitro translocation assays. Finally, ER microsomes isolated from a C. albicans strain expressing reduced amounts of KAR2 mRNA are defective for in vitro translocation of a secreted substrate protein, establishing a new method to study ER translocation in this organism. Together, these results suggest that C. albicans Kar2p functions during the translocation of proteins into the ER during the first step of the secretory pathway. PMID:20886215

  7. Analysis of polypeptide movement in the SecY channel during SecA-mediated protein translocation.

    PubMed

    Erlandson, Karl J; Or, Eran; Osborne, Andrew R; Rapoport, Tom A

    2008-06-01

    In bacteria most secretory proteins are transported across the plasma membrane by the interplay of the ATPase SecA with the translocation channel formed by the SecY complex; SecA uses cycles of ATP hydrolysis to "push" consecutive segments of a polypeptide substrate through the channel. Here we have addressed the mechanism of this process by following the fate of stalled translocation intermediates. These were generated by using a polypeptide substrate containing a bulky disulfide-bonded loop, thus preventing the final residues from passing through the channel. Protease protection experiments showed that the intermediates were stable in the presence of ATP and could complete translocation once the block was removed. The translocation intermediate was also stable when SecA associated with ATPgammaS, a poorly hydrolyzable ATP analog, or ADP plus AlF(4), which mimics the transition state during ATP hydrolysis. In contrast, when SecA was in its ADP-bound state, the translocating polypeptide moved back into the cytosol, as indicated by the disappearance of the protected fragment. Backsliding was not significantly altered by deletion of the plug domain, a short helix in the center of the SecY channel, but it was slowed down when changes were introduced into the pore ring, the constriction of the hourglass-shaped channel. In all cases, backsliding was significantly slower than forward translocation. Together, these data suggest that SecA binds the polypeptide chain in its ATP state and releases it in the ADP state. The channel itself does not bind the polypeptide chain but provides "friction" that minimizes backsliding when ADP-bound SecA resets to "grab" the next segment of the substrate. PMID:18359943

  8. Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein

    PubMed Central

    Yang, Sung-Tae; Lim, Sung In; Kiessling, Volker; Kwon, Inchan; Tamm, Lukas K.

    2016-01-01

    Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein’s folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca2+-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches. PMID:27605302

  9. A Hands-On Approach to Teaching Protein Translation & Translocation into the ER

    ERIC Educational Resources Information Center

    LaBonte, Michelle L.

    2013-01-01

    The process of protein translation and translocation into the endoplasmic reticulum (ER) can often be challenging for introductory college biology students to visualize. To help them understand how proteins become oriented in the ER membrane, I developed a hands-on activity in which students use Play-Doh to simulate the process of protein…

  10. SecA Alone Can Promote Protein Translocation and Ion Channel Activity

    PubMed Central

    Hsieh, Ying-hsin; Zhang, Hao; Lin, Bor-ruei; Cui, Ningren; Na, Bing; Yang, Hsiuchin; Jiang, Chun; Sui, Sen-fang; Tai, Phang C.

    2011-01-01

    SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1–2 μm. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity. PMID:22033925

  11. Detection of a complex translocation using fluorescent in situ hybridization (FISH)

    SciTech Connect

    Rosen, B.A.; Abuelo, D.N.; Mark, H.F.

    1994-09-01

    The use of fluorescent in situ hybridization (FISH) allowed the detection of a complex 3-way translocation in a patient with multiple congenital malformations and mental retardation. The patient was a 10-year-old girl with mental retardation, seizures, repaired cleft palate, esotropia, epicanthal folds, broad nasal bridge, upward slanting palpebral fissures, single transverse palmar crease, brachydactyly, hypoplastic nails, ectrodactyly between the third and fourth right toes, and hypoplasia of the left third toe. Chromosome analysis performed at birth was reported as normal. We performed high resolution banding analysis which revealed an apparently balanced translocation between chromosomes 2 and 9. However, because of her multiple abnormalities, further studies were ordered. Fluorescent in situ hybridization (FISH) using chromosome painting probes revealed a karyotype of 46,XX,t(2;8;9) (2pter{yields}q31::8q21.2{yields}8qter; 8pter{yields}q21.2::2q31{yields}q34::9q34{yields}qter; 9pter{yields}q34::2q34{yields}qter). The 3-way translocation appears to be de novo, as neither parent is a translocation carrier. This case illustrates the importance of using FISH to further investigate cases of apparently balanced translocations in the presence of phenotypic abnormalities and/or mental retardation.

  12. Protein co-translocational unfolding depends on the direction of pulling

    NASA Astrophysics Data System (ADS)

    Rodriguez-Larrea, David; Bayley, Hagan

    2014-09-01

    Protein unfolding and translocation through pores occurs during trafficking between organelles, protein degradation and bacterial toxin delivery. In vivo, co-translocational unfolding can be affected by the end of the polypeptide that is threaded into the pore first. Recently, we have shown that co-translocational unfolding can be followed in a model system at the single-molecule level, thereby unravelling molecular steps and their kinetics. Here, we show that the unfolding kinetics of the model substrate thioredoxin, when pulled through an α-haemolysin pore, differ markedly depending on whether the process is initiated from the C terminus or the N terminus. Further, when thioredoxin is pulled from the N terminus, the unfolding pathway bifurcates: some molecules finish unfolding quickly, while others finish ~100 times slower. Our findings have important implications for the understanding of biological unfolding mechanisms and in the application of nanopore technology for the detection of proteins and their modifications.

  13. Impaired translocation and activation of mitochondrial Akt1 mitigated mitochondrial oxidative phosphorylation Complex V activity in diabetic myocardium.

    PubMed

    Yang, Jia-Ying; Deng, Wu; Chen, Yumay; Fan, Weiwei; Baldwin, Kenneth M; Jope, Richard S; Wallace, Douglas C; Wang, Ping H

    2013-06-01

    Insulin can translocate Akt to mitochondria in cardiac muscle. The goals of this study were to define sub-mitochondrial localization of the translocated Akt, to dissect the effects of insulin on Akt isoform translocation, and to determine the direct effect of mitochondrial Akt activation on Complex V activity in normal and diabetic myocardium. The translocated Akt sequentially localized to the mitochondrial intermembrane space, inner membrane, and matrix. To confirm Akt translocation, in vitro import assay showed rapid entry of Akt into mitochondria. Akt isoforms were differentially regulated by insulin stimulation, only Akt1 translocated into mitochondria. In the insulin-resistant Type 2 diabetes model, Akt1 translocation was blunted. Mitochondrial activation of Akt1 increased Complex V activity by 24% in normal myocardium in vivo and restored Complex V activity in diabetic myocardium. Basal mitochondrial Complex V activity was lower by 22% in the Akt1(-/-) myocardium. Insulin-stimulated Complex V activity was not impaired in the Akt1(-/-) myocardium, due to compensatory translocation of Akt2 to mitochondria. Akt1 is the primary isoform that relayed insulin signaling to mitochondria and modulated mitochondrial Complex V activity. Activation of mitochondrial Akt1 enhanced ATP production and increased phosphocreatine in cardiac muscle cells. Dysregulation of this signal pathway might impair mitochondrial bioenergetics in diabetic myocardium.

  14. The mechanism of coupling between electron transfer and proton translocation in respiratory complex I.

    PubMed

    Sazanov, Leonid A

    2014-08-01

    NADH-ubiquinone oxidoreductase (complex I) is the first and largest enzyme in the respiratory chain of mitochondria and many bacteria. It couples the transfer of two electrons between NADH and ubiquinone to the translocation of four protons across the membrane. Complex I is an L-shaped assembly formed by the hydrophilic (peripheral) arm, containing all the redox centres performing electron transfer and the membrane arm, containing proton-translocating machinery. Mitochondrial complex I consists of 44 subunits of about 1 MDa in total, whilst the prokaryotic enzyme is simpler and generally consists of 14 conserved "core" subunits. Recently we have determined the first atomic structure of the entire complex I, using the enzyme from Thermus thermophilus (536 kDa, 16 subunits, 9 Fe-S clusters, 64 TM helices). Structure suggests a unique coupling mechanism, with redox energy of electron transfer driving proton translocation via long-range (up to ~200 Å) conformational changes. It resembles a steam engine, with coupling elements (akin to coupling rods) linking parts of this molecular machine. PMID:24943718

  15. The mechanism of coupling between electron transfer and proton translocation in respiratory complex I.

    PubMed

    Sazanov, Leonid A

    2014-08-01

    NADH-ubiquinone oxidoreductase (complex I) is the first and largest enzyme in the respiratory chain of mitochondria and many bacteria. It couples the transfer of two electrons between NADH and ubiquinone to the translocation of four protons across the membrane. Complex I is an L-shaped assembly formed by the hydrophilic (peripheral) arm, containing all the redox centres performing electron transfer and the membrane arm, containing proton-translocating machinery. Mitochondrial complex I consists of 44 subunits of about 1 MDa in total, whilst the prokaryotic enzyme is simpler and generally consists of 14 conserved "core" subunits. Recently we have determined the first atomic structure of the entire complex I, using the enzyme from Thermus thermophilus (536 kDa, 16 subunits, 9 Fe-S clusters, 64 TM helices). Structure suggests a unique coupling mechanism, with redox energy of electron transfer driving proton translocation via long-range (up to ~200 Å) conformational changes. It resembles a steam engine, with coupling elements (akin to coupling rods) linking parts of this molecular machine.

  16. Stochastic but highly coordinated protein unfolding and translocation by the CIpXP proteolytic machine

    PubMed Central

    Cordova, Juan Carlos; Olivares, Adrian O.; Shin, Yongdae; Stinson, Benjamin M.; Calmat, Stephane; Schmitz, Karl R.; Aubin-Tam, Marie-Eve; Baker, Tania A.; Lang, Matthew J.; Sauer, Robert T.

    2014-01-01

    CIpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known if these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows CIpXP unfolding to be directly visualized and reveals translocation steps of ~1–4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear. By studying single-molecule degradation using different multi-domain substrates and CIpXP variants, we answer many of these questions and provide evidence for stochastic unfolding and translocation. We also present a mechanochemical model that accounts for single-molecule, biochemical, and structural results, for our observation of enzymatic memory in translocation stepping, for the kinetics of translocation steps of different sizes, and for probabilistic but highly coordinated subunit activity within the CIpX ring. PMID:25083874

  17. All-Atom Molecular Dynamics Simulation of Protein Translocation through an α-Hemolysin Nanopore.

    PubMed

    Di Marino, Daniele; Bonome, Emma Letizia; Tramontano, Anna; Chinappi, Mauro

    2015-08-01

    Nanopore sensing is attracting the attention of a large and varied scientific community. One of the main issues in nanopore sensing is how to associate the measured current signals to specific features of the molecule under investigation. This is particularly relevant when the translocating molecule is a protein and the pore is sufficiently narrow to necessarily involve unfolding of the translocating protein. Recent experimental results characterized the cotranslocational unfolding of Thioredoxin (Trx) passing through an α-hemolisin pore, providing evidence for the existence of a multistep process. In this study we report the results of all-atom molecular dynamics simulations of the same system. Our data indicate that Trx translocation involves two main barriers. The first one is an unfolding barrier associated with a translocation intermediate where the N-terminal region of Trx is stuck at the pore entrance in a conformation that strongly resembles the native one. After the abrupt unfolding of the N-terminal region, the Trx enters the α-hemolisin vestibule. During this stage, the constriction is occupied not only by the translocating residue but also by a hairpin-like structure forming a tangle in the constriction. The second barrier is associated with the disentangling of this region.

  18. Manipulation of Protein Translocation through Nanopores by Flow Field Control and Application to Nanopore Sensors.

    PubMed

    Hsu, Wei-Lun; Daiguji, Hirofumi

    2016-09-20

    The control of biomolecule translocation through nanopores is important in nanopore protein detection. Improvement in current nanopore molecule control is desired to enhance capture rates, extend translocation times, and ensure the effective detection of various proteins in the same solutions. We present a method that simultaneously resolves these issues through the use of a gate-modulated conical nanopore coupled with solutions of varying salt concentration. Simulation results show that the presence of an induced reverse electroosmotic flow (IREOF) results in inlet flows from the two ends of the nanopore centerline entering into the nanopore in opposite directions, which simultaneously elevates the capture rate and immobilizes the protein in the nanopore, thus enabling steady current blockage measurements for a range of proteins. In addition, it is shown that proteins with different size/charge ratios can be trapped by a gate modulation intensified flow field at a similar location in the nanopore in the same solution conditions.

  19. Translocator Protein 18 kDa (TSPO): An Old Protein with New Functions?

    PubMed

    Li, Fei; Liu, Jian; Liu, Nan; Kuhn, Leslie A; Garavito, R Michael; Ferguson-Miller, Shelagh

    2016-05-24

    Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer's and Parkinson's disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO. An ortholog in the photosynthetic bacterium Rhodobacter was independently discovered as the tryptophan-rich sensory protein (TspO) and found to play a role in the response to changes in oxygen and light conditions that regulate photosynthesis and respiration. As part of this highly conserved protein family found in all three kingdoms, the rat TSPO is able to rescue the knockout phenotype in Rhodobacter, indicating functional as well as structural conservation. Recently, a major breakthrough in the field was achieved: the determination of atomic-resolution structures of TSPO from different species by several independent groups. This now allows us to reexamine the function of TSPO with a molecular perspective. In this review, we focus on recently determined structures of TSPO and their implications for potential functions of this ubiquitous multifaceted protein. We suggest that TSPO is an ancient bacterial receptor/stress sensor that has developed additional interactions, partners, and roles in its mitochondrial outer membrane environment in eukaryotes.

  20. Dynamic translocation of ligand-complexed DNA through solid-state nanopores with optical tweezers

    NASA Astrophysics Data System (ADS)

    Sischka, Andy; Spiering, Andre; Khaksar, Maryam; Laxa, Miriam; König, Janine; Dietz, Karl-Josef; Anselmetti, Dario

    2010-11-01

    We investigated the threading and controlled translocation of individual lambda-DNA (λ-DNA) molecules through solid-state nanopores with piconewton force sensitivity, millisecond time resolution and picoampere ionic current sensitivity with a set-up combining quantitative 3D optical tweezers (OT) with electrophysiology. With our virtually interference-free OT set-up the binding of RecA and single peroxiredoxin protein molecules to λ-DNA was quantitatively investigated during dynamic translocation experiments where effective forces and respective ionic currents of the threaded DNA molecule through the nanopore were measured during inward and outward sliding. Membrane voltage-dependent experiments of reversible single protein/DNA translocation scans yield hysteresis-free, asymmetric single-molecule fingerprints in the measured force and conductance signals that can be attributed to the interplay of optical trap and electrostatic nanopore potentials. These experiments allow an exact localization of the bound protein along the DNA strand and open fascinating applications for label-free detection of DNA-binding ligands, where structural and positional binding phenomena can be investigated at a single-molecule level.

  1. Dynamic translocation of ligand-complexed DNA through solid-state nanopores with optical tweezers.

    PubMed

    Sischka, Andy; Spiering, Andre; Khaksar, Maryam; Laxa, Miriam; König, Janine; Dietz, Karl-Josef; Anselmetti, Dario

    2010-11-17

    We investigated the threading and controlled translocation of individual lambda-DNA (λ-DNA) molecules through solid-state nanopores with piconewton force sensitivity, millisecond time resolution and picoampere ionic current sensitivity with a set-up combining quantitative 3D optical tweezers (OT) with electrophysiology. With our virtually interference-free OT set-up the binding of RecA and single peroxiredoxin protein molecules to λ-DNA was quantitatively investigated during dynamic translocation experiments where effective forces and respective ionic currents of the threaded DNA molecule through the nanopore were measured during inward and outward sliding. Membrane voltage-dependent experiments of reversible single protein/DNA translocation scans yield hysteresis-free, asymmetric single-molecule fingerprints in the measured force and conductance signals that can be attributed to the interplay of optical trap and electrostatic nanopore potentials. These experiments allow an exact localization of the bound protein along the DNA strand and open fascinating applications for label-free detection of DNA-binding ligands, where structural and positional binding phenomena can be investigated at a single-molecule level.

  2. Translocation of mitochondrial inner-membrane proteins: conformation matters.

    PubMed

    de Marcos-Lousa, Carine; Sideris, Dionisia P; Tokatlidis, Kostas

    2006-05-01

    Most of the mitochondrial inner-membrane proteins are generated without a presequence and their targeting depends on inadequately defined internal segments. Despite the numerous components of the import machinery identified by proteomics, the properties of hydrophobic import substrates remain poorly understood. Recent studies support several principles for these membrane proteins: first, they become organized into partially assembled forms within the translocon; second, they present noncontiguous targeting signals; and third, they induce conformational changes in translocase subunits, thereby mediating "assembly on demand" of the import machinery. It is possible that the energy needed for these proteins to pass across the outer membrane, to travel through the intermembrane space and to target the inner-membrane surface is provided by conformational changes involving import components that seem to have natively unfolded structures. Such structural malleability might render some of the translocase subunits more adept at driving the protein import process.

  3. Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei

    PubMed Central

    Sinclair, Sara H. G.; Garcia-Garcia, Jose C.; Dumler, J. Stephen

    2015-01-01

    Obligate intracellular bacteria have an arsenal of proteins that alter host cells to establish and maintain a hospitable environment for replication. Anaplasma phagocytophilum secrets Ankyrin A (AnkA), via a type IV secretion system, which translocates to the nucleus of its host cell, human neutrophils. A. phagocytophilum-infected neutrophils have dramatically altered phenotypes in part explained by AnkA-induced transcriptional alterations. However, it is unlikely that AnkA is the sole effector to account for infection-induced transcriptional changes. We developed a simple method combining bioinformatics and iTRAQ protein profiling to identify potential bacterial-derived nuclear-translocated proteins that could impact transcriptional programming in host cells. This approach identified 50 A. phagocytophilum candidate genes or proteins. The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells. We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455. Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin. Additionally, application of this approach to other intracellular bacteria such as Mycobacterium tuberculosis, Chlamydia trachomatis and other intracellular bacteria identified multiple candidate genes to be investigated. PMID:25705208

  4. Stoichiometry of proton translocation by respiratory complex I and its mechanistic implications

    PubMed Central

    Wikström, Mårten; Hummer, Gerhard

    2012-01-01

    Complex I (NADH-ubiquinone oxidoreductase) in the respiratory chain of mitochondria and several bacteria functions as a redox-driven proton pump that contributes to the generation of the protonmotive force across the inner mitochondrial or bacterial membrane and thus to the aerobic synthesis of ATP. The stoichiometry of proton translocation is thought to be 4 H+ per NADH oxidized (2 e-). Here we show that a H+/2 e- ratio of 3 appears more likely on the basis of the recently determined H+/ATP ratio of the mitochondrial F1Fo-ATP synthase of animal mitochondria and of a set of carefully determined ATP/2 e- ratios for different segments of the mitochondrial respiratory chain. This lower H+/2 e- ratio of 3 is independently supported by thermodynamic analyses of experiments with both mitochondria and submitochondrial particles. A reduced H+/2 e- stoichiometry of 3 has important mechanistic implications for this proton pump. In a rough mechanistic model, we suggest a concerted proton translocation mechanism in the three homologous and tightly packed antiporter-like subunits L, M, and N of the proton-translocating membrane domain of complex I. PMID:22392981

  5. Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of proteins associated with the translocation channel.

    PubMed

    Black, Virginia H; Sanjay, Archana; van Leyen, Klaus; Lauring, Brett; Kreibich, Gert

    2005-10-01

    Steroid-secreting cells are characterized by abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. Yet they have relatively little morphologically identifiable rough endoplasmic reticulum, presumably required for synthesis and maintenance of the smooth membranes. In this study, we demonstrate that adrenal smooth microsomal subfractions enriched in smooth endoplasmic reticulum membranes contain high levels of translocation apparatus and oligosaccharyltransferase complex proteins, previously thought confined to rough endoplasmic reticulum. We further demonstrate that these smooth microsomal subfractions are capable of effecting cotranslational translocation, signal peptide cleavage, and N-glycosylation of newly synthesized polypeptides. This shifts the paradigm for distinction between smooth and rough endoplasmic reticulum. Confocal microscopy revealed the proteins to be distributed throughout the abundant tubular endoplasmic reticulum in these cells, which is predominantly smooth surfaced. We hypothesize that the broadly distributed translocon and oligosaccharyltransferase proteins participate in local synthesis and/or quality control of membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally regulated manner.

  6. G-protein coupling and nuclear translocation of the human abscisic acid receptor LANCL2

    PubMed Central

    Fresia, Chiara; Vigliarolo, Tiziana; Guida, Lucrezia; Booz, Valeria; Bruzzone, Santina; Sturla, Laura; Di Bona, Melody; Pesce, Mattia; Usai, Cesare; De Flora, Antonio; Zocchi, Elena

    2016-01-01

    Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the α subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation. PMID:27222287

  7. Real-time imaging of dynamic translocation of fluorescent proteins at synapses in living neurons.

    NASA Astrophysics Data System (ADS)

    De Koninck, Paul

    2003-02-01

    To understand the biology of living cells, such as the neurons in our brain, we focus on the molecular signaling interactions that proteins perform intracellularly. We have been studying the behavior of an enzyme, termed 'CaMKII', inside living neurons maintained in tissue culture. This enzyme plays a critical role in the control of synaptic transmission. Such role may involve the dynamic translocation of the enzyme at synaptic sites upon specific stimuli. To study this translocation, we use a cellular imaging technique that allows us to monitor the movement and targeting of CaMKII tagged by genetic engineering with a green fluorescent protein (GFP). We find that the enzyme translocates within seconds to synapses upon synaptic activation by neurotransmitter application. Our approach has lead to several key findings on the regulation of CaMKII translocation to the synapse and on its potential role in synaptic plasticity. However, several new advances in photonics and image analysis, which we will implement in our laboratory, will greatly help pushing the limits of our resolution of such type molecular event in living cells.

  8. Translocator Protein-Mediated Stabilization of Mitochondrial Architecture during Inflammation Stress in Colonic Cells

    PubMed Central

    Issop, Leeyah; Ostuni, Mariano A.; Lee, Sunghoon; Laforge, Mireille; Péranzi, Gabriel; Rustin, Pierre; Benoist, Jean-François; Estaquier, Jérome; Papadopoulos, Vassilios; Lacapère, Jean-Jacques

    2016-01-01

    Chronic inflammation of the gastrointestinal tract increasing the risk of cancer has been described to be linked to the high expression of the mitochondrial translocator protein (18 kDa; TSPO). Accordingly, TSPO drug ligands have been shown to regulate cytokine production and to improve tissue reconstruction. We used HT-29 human colon carcinoma cells to evaluate the role of TSPO and its drug ligands in tumor necrosis factor (TNF)-induced inflammation. TNF-induced interleukin (IL)-8 expression, coupled to reactive oxygen species (ROS) production, was followed by TSPO overexpression. TNF also destabilized mitochondrial ultrastructure, inducing cell death by apoptosis. Treatment with the TSPO drug ligand PK 11195 maintained the mitochondrial ultrastructure, reducing IL-8 and ROS production and cell death. TSPO silencing and overexpression studies demonstrated that the presence of TSPO is essential to control IL-8 and ROS production, so as to maintain mitochondrial ultrastructure and to prevent cell death. Taken together, our data indicate that inflammation results in the disruption of mitochondrial complexes containing TSPO, leading to cell death and epithelia disruption. Significance: This work implicates TSPO in the maintenance of mitochondrial membrane integrity and in the control of mitochondrial ROS production, ultimately favoring tissue regeneration. PMID:27054921

  9. Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

    PubMed

    Unterweger, Daniel; Kostiuk, Benjamin; Ötjengerdes, Rina; Wilton, Ashley; Diaz-Satizabal, Laura; Pukatzki, Stefan

    2015-08-13

    Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.

  10. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    SciTech Connect

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  11. Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation.

    PubMed

    Vermeire, Kurt; Allan, Susanne; Provinciael, Becky; Hartmann, Enno; Kalies, Kai-Uwe

    2015-09-01

    Here, we demonstrate that pancreatic microsomal membranes from pigs, sheep, or cattle destined for human consumption can be used as a valuable and ethically correct alternative to dog microsomes for cell-free protein translocation. By adding adequate ribonuclease (RNase) inhibitors to the membrane fraction, successful in vitro co-translational translocation of wild-type and chimeric pre-prolactin into the lumen of rough microsomes was obtained. In addition, the human type I integral membrane proteins CD4 and VCAM-1 were efficiently glycosylated in RNase-treated microsomes. Thus, RNase-neutralized pancreatic membrane fractions from pig, cow, or sheep are a cheap, easily accessible, and fulfilling alternative to canine microsomes. PMID:26050631

  12. Genic Heterozygosity and Variation in Permanent Translocation Heterozygotes of the OENOTHERA BIENNIS Complex

    PubMed Central

    Levy, Morris; Levin, Donald A.

    1975-01-01

    Genic heterozygosity and variation were studied in the permanent translocation heterozygotes Oenothera biennis I, Oe. biennis II, Oe. biennis III, Oe. strigosa, Oe. parviflora I, Oe. parviflora II, and in the related bivalent formers Oe. argillicola and Oe. hookeri. From variation at 20 enzyme loci, we find that translocation heterozygosity for the entire chromosome complex is accompanied by only moderate levels of genic heterozygosity: 2.8% in Oe. strigosa, 9.5% in Oe. biennis and 14.9% in Oe. parviflora. Inbred garden strains of Oe. argillicola exhibited 8% heterozygosity; neither garden nor wild strains of Oe. hookeri displayed heterozygosity and only a single allozyme genotype was found. The mean number of alleles per locus is only 1.30 in Oe. strigosa, 1.40 in Oe. biennis, and 1.55 in Oe. parviflora, compared to 1.40 in Oe. argillicola. Clearly, the ability to accumulate and/or retain heterozygosity and variability has not been accompanied by extraordinary levels of either. Clinal variation is evident at some loci in each ring-former. A given translocation complex may vary geographically in its allozymic constitution. From gene frequencies, Oe. biennis I, II, and III, Oe. strigosa and Oe. hookeri are judged to be very closely related, whereas Oe. argillicola seems quite remote; Oe. parviflora is intermediate to the two phylads. Gene frequencies also suggest that Oe. argillicola diverged from the Euoenothera progenitor about 1,000,000 years ago, whereas most of the remaining evolution in the complex has occurred within the last 150,000 years. PMID:17248680

  13. Translocator protein (TSPO) role in aging and Alzheimer's disease.

    PubMed

    Repalli, Jayanthi

    2014-01-01

    Cellular damage and deregulated apoptotic cell death lead to functional impairment, and a main consequence of these events is aging. Cellular damage is initiated by different stress/risk factors such as oxidative stress, inflammation, and heavy metals. These stress/risk factors affect the cellular homeostasis by altering methylation status of several aging and Alzheimer's disease associated genes; these effects can be manifested immediately after exposure to stress and at later stages of life. However, when cellular damage exceeds certain threshold levels apoptosis is initiated. This review discusses the stress factors involved in cellular damage and the role and potential of TSPO-mediated cell death in aging as well as in Alzheimer's disease, which is also characterized by extensive cell death. Mitochondrial-mediated apoptotic death through the release of cytochrome c is regulated by TSPO, and increased expression of this protein is observed in both elderly people and in patients with Alzheimer's disease. TSPO forms and mediates opening of the mitochondrial membrane pore, mPTP and oxidizes cardiolipin, and these events lead to the leakage of apoptotic death mediators, such as cytochrome c, resulting in cell death. However, TSPO has many proposed functions and can also increase steroid synthesis, which leads to inhibition of inflammation and inhibition of the release of apoptotic factors, thereby decreasing cell damage and promoting cell survival. Thus, TSPO mediates apoptosis and decreases the cell damage, which in turn dictates the process of aging as well as the functionality of organs such as the brain. TSPO modulation with ligands in the Alzheimer's disease mouse model showed improvement in behavioral symptoms, and studies in Drosophila species showed increased cell survival and prolonged lifespan in flies after TSPO inhibition. These data suggest that since effects/signs of stress can manifest at any time, prevention through change in lifestyle and TSPO

  14. Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes.

    PubMed

    Simons, Michelle; Szczelkun, Mark D

    2011-09-01

    The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5'-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can 'turnover' in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase-nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed.

  15. Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation

    NASA Astrophysics Data System (ADS)

    Mereuta, Loredana; Roy, Mahua; Asandei, Alina; Lee, Jong Kook; Park, Yoonkyung; Andricioaei, Ioan; Luchian, Tudor

    2014-01-01

    The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.

  16. Single color FRET based measurements of conformational changes of proteins resulting from translocation inside cells.

    PubMed

    Gahl, Robert F; Tekle, Ephrem; Tjandra, Nico

    2014-03-15

    Translocation of proteins to different parts of the cell is necessary for many cellular mechanisms as a means for regulation and a variety of other functions. Identifying how these proteins undergo conformational changes or interact with various partners during these events is critical to understanding how these mechanisms are executed. A protocol is presented that identifies conformational changes in a protein that occur during translocation while overcoming challenges in extracting distance information in very different environments of a living cell. Only two samples are required to be prepared and are observed with one optical setup. Live-cell FRET imaging has been applied to identify conformational changes between two native cysteines in Bax, a member of the Bcl-2 family of proteins that regulates apoptosis. Bax exists in the cytosol and translocates to the mitochondria outer membrane upon apoptosis induction. The distance, r, between the two native cysteines in the cytosolic structure of Bax necessitates the use of a FRET donor-accepter pair with R0~r as the most sensitive probe for identifying structural changes at these positions. Alexa Fluor 546 and Dabcyl, a dark acceptor, were used as FRET pairs - resulting in single color intensity variations of Alexa-546 as a measure of FRET efficiency. An internal reference, conjugated to Bax, was employed to normalize changes in fluorescence intensity of Alexa Fluor 546 due to inherent inhomogeneities in the living cell. This correction allowed the true FRET effects to be measured with increased precision during translocation. Normalization of intensities to the internal reference identified a FRET efficiency of 0.45±0.14 in the cytosol and 0.11±0.20 in the mitochondria. The procedure for the conjugation of the internal reference and FRET probes as well as the data analysis is presented.

  17. Structure and dynamics of the mammalian ribosomal pre-translocation complex

    PubMed Central

    Budkevich, Tatyana; Giesebrecht, Jan; Altman, Roger B.; Munro, James B.; Mielke, Thorsten; Nierhaus, Knud H.; Blanchard, Scott C.; Spahn, Christian M.T.

    2011-01-01

    Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pre-translocation complex reveal spontaneous, large-scale conformational changes including an inter-subunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favoured within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor, cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action. PMID:22017870

  18. BCL2 protein expression in follicular lymphomas with t(14;18) chromosomal translocations.

    PubMed

    Masir, Noraidah; Campbell, Lisa J; Goff, Lindsey K; Jones, Margaret; Marafioti, Teresa; Cordell, Jacqueline; Clear, Andrew J; Lister, T Andrew; Mason, David Y; Lee, Abigail M

    2009-03-01

    The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein overexpression in most follicular lymphomas. However the expression of BCL2 is not always homogeneous and may demonstrate a variable degree of heterogeneity. This study analysed BCL2 protein expression pattern in 33 cases of t(14;18)-positive follicular lymphomas using antibodies against two different epitopes (i.e. the widely used antibody BCL2/124 and an alternative antibody E17). 16/33 (49%) cases demonstrated strong BCL2 expression. In 10/33 (30%) cases, BCL2 expression was heterogeneous and in some of these, its loss appeared to be correlated with cell proliferation, as indicated by Ki67 expression. Double immunofluorescence labelling confirmed an inverse BCL2/Ki67 relationship, where in 24/28 (86%) cases cellular expression of BCL2 and Ki67 was mutually exclusive. In addition, seven BCL2 'pseudo-negative' cases were identified in which immunostaining was negative with antibody BCL2/124, but positive with antibody E17. Genomic DNA sequencing of these 'pseudo-negative' cases demonstrated eleven mutations in four cases and nine of these were missense mutations. It can be concluded that in follicular lymphomas, despite carrying the t(14;18) translocations, BCL2 protein expression may be heterogeneous and loss of BCL2 could be related to cell proliferation. Secondly, mutations in translocated BCL2 genes appear to be common and may cause BCL2 pseudo-negative immunostaining. PMID:19120369

  19. a Computational Approach to Explore Protein Translocation Through Type III Secretion Apparatus

    NASA Astrophysics Data System (ADS)

    Rathinavelan, Thenmalarchelvi; Im, Wonpil

    2010-01-01

    Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus (TTSA) that is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. Interestingly, the electronegative channel interior creates an energy barrier for MxiH to enter the channel, while the same may facilitate the ejection of the effectors into host cells. Structurally-known basal regions and ATPase underneath the basal region have also such electronegative interior, while effector proteins have considerable electronegative patches on their surfaces. Based on these observations, we propose a repulsive electrostatic mechanism for protein translocation through the TTSA. This mechanism is supported by the suggestion that an ATPase is required for protein translocation through these nanomachines, which may provide the energy to overcome the initial electrostatic energy barrier. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported.

  20. Complexation of arsenite with phytochelatins reduces arsenite efflux and translocation from roots to shoots in Arabidopsis.

    PubMed

    Liu, Wen-Ju; Wood, B Alan; Raab, Andrea; McGrath, Steve P; Zhao, Fang-Jie; Feldmann, Jörg

    2010-04-01

    Complexation of arsenite [As(III)] with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray ionization-mass spectrometry coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis (Arabidopsis thaliana) exposed to arsenate [As(V)]. As(V) was efficiently reduced to As(III) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC4, As(III)-PC3, and As(III)-(PC2)2. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to As(V) than the wild type. In cad1-3 roots, only 8% of As was complexed with GSH as As(III)-(GS)3 and no As(III)-PCs were detected, while in cad2-1 roots, As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 to 12 times higher shoot-to-root As concentration ratio than the wild type. Roots also effluxed a substantial proportion of the As(V) taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to l-buthionine sulfoximine or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops. PMID:20130102

  1. Complexation of arsenite with phytochelatins reduces arsenite efflux and translocation from roots to shoots in Arabidopsis.

    PubMed

    Liu, Wen-Ju; Wood, B Alan; Raab, Andrea; McGrath, Steve P; Zhao, Fang-Jie; Feldmann, Jörg

    2010-04-01

    Complexation of arsenite [As(III)] with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray ionization-mass spectrometry coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis (Arabidopsis thaliana) exposed to arsenate [As(V)]. As(V) was efficiently reduced to As(III) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC4, As(III)-PC3, and As(III)-(PC2)2. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to As(V) than the wild type. In cad1-3 roots, only 8% of As was complexed with GSH as As(III)-(GS)3 and no As(III)-PCs were detected, while in cad2-1 roots, As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 to 12 times higher shoot-to-root As concentration ratio than the wild type. Roots also effluxed a substantial proportion of the As(V) taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to l-buthionine sulfoximine or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops.

  2. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  3. Phenethyl alcohol disorders phospholipid acyl chains and promotes translocation of the mitochondrial precursor protein apocytochrome c across a lipid bilayer.

    PubMed

    Jordi, W; Nibbeling, R; de Kruijff, B

    1990-02-12

    The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.

  4. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System

    PubMed Central

    Dubytska, Lidiya P.; Rogge, Matthew L.

    2016-01-01

    ABSTRACT Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000

  5. In search of Brucella abortus Type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

    PubMed Central

    Marchesini, María Inés; Herrmann, Claudia K.; Salcedo, Suzana P.; Gorvel, Jean-Pierre; Comerci, Diego J.

    2011-01-01

    SUMMARY Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells. PMID:21707904

  6. The Novel Dipeptide Translocator Protein Ligand, Referred to As GD-23, Exerts Anxiolytic and Nootropic Activities.

    PubMed

    Povarnina, P Yu; Yarkov, S A; Gudasheva, T A; Yarkova, M A; Seredenin, S B

    2015-01-01

    The translocator protein (TSPO) promotes the translocation of cholesterol to the inner mitochondrial membrane and mediates steroid formation. In this study, we first report on a biological evaluation of the dipeptide GD-23 (N-carbobenzoxy-L tryptophanyl-L isoleucine amide), a structural analogue of Alpidem, the principal TSPO ligand. We show that GD-23 in a dose range of 0.05 to 0.5 mg/kg (i.p.) exhibits anxiolytic activity in the elevated plus maze test and nootropic activity in the object recognition test in scopolamine-induced amnesia in rodents. It was shown that GD-23 did not affect spontaneous locomotor activity, holding promise as a nonsedative anxiolytic agent. The anxiolytic and nootropic activities of GD-23 were abrogated by the TSPO specific ligand PK11195, which thus suggests a role for TSPO in mediating the pharmacological activity of GD-23. PMID:26483966

  7. Subcellular Dynamics of Multifunctional Protein Regulation: Mechanisms of GAPDH Intracellular Translocation

    PubMed Central

    Sirover, Michael A.

    2012-01-01

    Multidimensional proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) exhibit distinct activities unrelated to their originally identified functions. Apart from glycolysis, GAPDH participates in iron metabolism, membrane trafficking, histone biosynthesis, the maintenance of DNA integrity and receptor mediated cell signaling. Further, multifunctional proteins exhibit distinct changes in their subcellular localization reflecting their new activities. As such, GAPDH is not only a cytosolic protein but is localized in the membrane, the nucleus, polysomes, the ER and the Golgi. In addition, although the initial subcellular localizations of multifunctional proteins may be of significance, dynamic changes in intracellular distribution may occur as a consequence of those new activities. As such, regulatory mechanisms may exist through which cells control multifunctional protein expression as a function of their subcellular localization. The temporal sequence through which subcellular translocation and the acquisition of new GAPDH functions is considered as well as post-translational modification as a basis for its intracellular transport. PMID:22388977

  8. The Philadelphia (Ph) chromosome in leukemia. III. Complex Ph translocation plus inversion in chronic myelocytic leukemia.

    PubMed

    Morgan, R; Stebbins, R D; Hecht, F; Sandberg, A A

    1985-01-01

    Remarkable chromosome abnormalities were observed in bone marrow cells from a woman with chronic myelocytic leukemia and atypical tuberculosis due to Mycobacterium avium-intracellulare infection. Four chromosome breaks occurred at bands 1p13, 1q32, 11p15, and 22q11. These breaks resulted in a complex Philadelphia (Ph) translocation between chromosomes #1, #11, and #22 and in an inversion of chromosome #1. Oncogenes on these chromosomes include N-ras and c-sk on chromosome #1, c-H-ras on chromosome #11, and c-sis on chromosome #22. Complex chromosome rearrangements may facilitate multiple oncogene changes, thereby permitting several steps in cancer development to occur simultaneously.

  9. The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions.

    PubMed

    Hartland, E L; Daniell, S J; Delahay, R M; Neves, B C; Wallis, T; Shaw, R K; Hale, C; Knutton, S; Frankel, G

    2000-03-01

    Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for 'attaching and effacing' (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.

  10. Complex/variant translocations in chronic myelogenous leukemia (CML): genesis and prognosis with 4 new cases.

    PubMed

    Mendiola, Christina; Ortega, Veronica; Tonk, Vijay S; Coviello, Jean M; Velagaleti, Gopalrao

    2014-08-01

    In 5-10% of cases with CML, variant or complex translocations (CT) are seen that may result in atypical fluorescence in situ hybridization signal patterns. Dual color, dual fusion fluorescence in situ hybridization (D-FISH) patterns are instrumental in identifying the genesis of these CT, but their prognostic implications remain controversial. The most common mechanism is a two-step process in which a standard two-way translocation (9;22) is followed by subsequent rearrangements involving other chromosomes. The second common mechanism is the one-step process wherein breakage occurs simultaneously on different chromosomes leading to CT. The typical D-FISH pattern seen with the one-step mechanism is 1F2G2R, while the pattern for the two-step mechanism can be variable (2F1G1R, 1F1G1R, 1F1G2R, 1F2G1R, etc.). We have studied 4 cases of CT using metaphase FISH with triple color, dual fusion ASS1, ABL1 and BCR probes to understand the genesis of these CT. All the patients were treated with imatinib, but only patients 3 and 4 showed remission. Our results indicate that the CT in cases 1, 3 and 4 arose from a one-step mechanism and case 2 from a multi-step mechanism. Response to imatinib varied from full remission to no response. Long term follow-up is necessary to evaluate the prognostic implications of these CT.

  11. Expression and translocation of the chlamydial major outer membrane protein in Escherichia coli.

    PubMed

    Dascher, C; Roll, D; Bavoil, P M

    1993-12-01

    The entire gene encoding the major outer membrane protein (MOMP) from Chlamydia psittaci strain GPIC has been cloned and expressed in Escherichia coli. A tightly regulated T7 promoter is used to control expression of the protein in Escherichia coli. Upon induction of expression, the precursor (pre-MOMP) is synthesized in the cell. This is followed by the appearance of a lower molecular weight protein that comigrates with mature MOMP from chlamydial elementary bodies by both one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. When E. coli cells expressing MOMP are converted to spheroplasts and subjected to protease treatment, MOMP is quantitatively degraded while cytoplasmic pre-MOMP is protected from degradation. Whole cells subjected to the same protease treatment show no degradation of MOMP. Furthermore, MOMP is not detected in surface-labeling experiments using several MOMP-specific antibodies. These data indicate that pre-MOMP is translocated to the periplasmic space and processed but is not surface exposed in E. coli. Expression of MOMP in this system causes a significant reduction in cell viability. In addition, coexpression in E. coli of MOMP or a MOMP-PhoA fusion with various chaperone proteins does not alter the level of MOMP translocation.

  12. The role of protein kinase C alpha translocation in radiation-induced bystander effect.

    PubMed

    Fang, Zihui; Xu, An; Wu, Lijun; Hei, Tom K; Hong, Mei

    2016-01-01

    Ionizing radiation is a well known human carcinogen. Evidence accumulated over the past decade suggested that extranuclear/extracellular targets and events may also play a critical role in modulating biological responses to ionizing radiation. However, the underlying mechanism(s) of radiation-induced bystander effect is still unclear. In the current study, AL cells were irradiated with alpha particles and responses of bystander cells were investigated. We found out that in bystander AL cells, protein kinase C alpha (PKCα) translocated from cytosol to membrane fraction. Pre-treatment of cells with PKC translocation inhibitor chelerythrine chloride suppressed the induced extracellular signal-regulated kinases (ERK) activity and the increased cyclooxygenase 2 (COX-2) expression as well as the mutagenic effect in bystander cells. Furthermore, tumor necrosis factor alpha (TNFα) was elevated in directly irradiated but not bystander cells; while TNFα receptor 1 (TNFR1) increased in the membrane fraction of bystander cells. Further analysis revealed that PKC activation caused accelerated internalization and recycling of TNFR1. Our data suggested that PKCα translocation may occur as an early event in radiation-induced bystander responses and mediate TNFα-induced signaling pathways that lead to the activation of ERK and up-regulation of COX-2. PMID:27165942

  13. Familial complex autosomal translocations involving chromosomes 7, 8, and 9 exhibiting male and female transmission with segregation and recombination.

    PubMed Central

    Walker, S; Howard, P J; Hunter, D

    1985-01-01

    A family showing a complex translocation between chromosomes 7, 8, and 9 with breakpoints at 7q21, 7q33, 8p23, and 9p23 is described. The proband had been referred because of repeated spontaneous abortions. This is only the second family to be ascertained in this way. Twenty-three other cases of complex translocations are briefly reviewed, eight of which were de novo in origin and 15 familial. All but one of the familial cases showed maternal transmission only. The present family shows both maternal and paternal transmission and is thought to be the first exhibiting recombination from a male carrier. The origin and transmission of the complex translocation is followed through three generations. Images PMID:3841161

  14. Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane

    SciTech Connect

    Devirgiliis, Chiara; Gaetani, Sancia; Apreda, Marianna; Bellovino, Diana . E-mail: bellovino@inran.it

    2005-07-01

    Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH{sub 2}-terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process.

  15. Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus.

    PubMed

    Agarwal, Shivani; Yadav, Sunita Kumari; Dixit, Aparna

    2011-05-01

    Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proliferation of normal cells under pathophysiological conditions. In order to evaluate its potential as a pharmaceutical target, the protein must be produced and purified in sufficiently high yields. In the present study, we report the high level expression of Tpr protein of R. norvegicus employing heterologous host, Escherichia coli, to permit its structural characterization in great detail. We here demonstrate that the Tpr protein was expressed in soluble form and approximately 90 mg/L of the purified protein at the shake flask level could be achieved to near homogeneity using single step-metal chelate affinity chromatography. The amino acid sequence of the protein was confirmed by mass spectroscopic analysis. The highly unstable and disordered Tpr protein was imparted structural and functional stability by the addition of glycerol and it has been shown that the natively unfolded Tpr protein retains DNA binding ability under these conditions only. Thus, the present study emphasizes the significance of an efficient prokaryotic system, which results in a high level soluble expression of a DNA binding protein of eukaryotic origin. Thus, the present strategy employed for purification of the R. norvegicus Tpr protein bypasses the need for the tedious expression strategies associated with the eukaryotic expression systems.

  16. Full cytogenetic characterization of a new neuroblastoma cell line with a complex 17q translocation.

    PubMed

    Panarello, C; Morerio, C; Russo, I; Pasquali, F; Rapella, A; Corrias, M V; Morando, A; Rosanda, C

    2000-01-15

    Recent studies have shown that structural abnormalities of chromosome 17 resulting in gain of material are the most frequent genetic changes in neuroblastoma. We have established a new neuroblastoma cell line from a patient whose disease had evolved from stage 4s to 4, without evidence of deletion of the short arm of chromosome 1 and MYCN amplification, which are considered the most typical genetic indicators of aggressive disease. The cytogenetic study allowed a full characterization of the chromosome changes, and revealed a complex translocation of chromosome 17 leading to a derivative marker which may be described as follows: der(11)t(11;17)(p15;q12)t(11;17) (q22;q12). This resulted in a gain of part of the long arms of chromosome 17, which was recently associated with poor prognosis.

  17. Proton transfer is rate-limiting for translocation of precursor proteins by the Escherichia coli translocase.

    PubMed Central

    Driessen, A J; Wickner, W

    1991-01-01

    The protonmotive force stimulates translocation in vivo, in crude in vitro reactions, and in a purified, reconstituted reaction. Translocation activity is a function of the pH at the inner face of the membrane. Both the transmembrane pH gradient and the transmembrane electrical potential stimulate translocation. A late-stage translocation intermediate of the proOmpA preprotein completes its translocation in the absence of ATP when a protonmotive force is imposed. This completion of translocation is retarded by a factor of greater than 3 in deuterium oxide relative to water, demonstrating that translocation involves proton-transfer reactions in rate-limiting steps. Images PMID:1826054

  18. Signal-on Protein Detection via Dye Translocation between Aptamer and Quantum Dot.

    PubMed

    Lao, Yeh-Hsing; Chi, Chun-Wei; Friedrich, Sarah M; Peck, Konan; Wang, Tza-Huei; Leong, Kam W; Chen, Lin-Chi

    2016-05-18

    A unique interaction between the cyanine dye and negatively charged quantum dot is used to construct a signal-on biaptameric quantum dot (QD) Förster resonance energy transfer (FRET) beacon for protein detection and distinct aptamer characterization. The beacon comprises a pair of aptamers, one intercalated with the cyanine dye (YOYO-3) and the other conjugated to a negatively charged, carboxyl-QD. When the target protein is present, structural folding and sandwich association of the two aptamers take place. As a consequence, YOYO-3 is displaced from the folded aptamer and transferred to the unblocked QD surface to yield a target concentration-dependent FRET signal. As a proof-of-principle, we demonstrate the detection of thrombin ranging from nanomolar to submicromolar concentrations and confirm the dye translocation using cylindrical illumination confocal spectroscopy (CICS). The proposed beacon provides a simple, rapid, signal-on FRET detection for protein as well as a potential platform for distinct aptamer screening.

  19. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  20. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

    PubMed Central

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  1. A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes

    PubMed Central

    Liu, TingYu; Yu, BuChin; Kakino, Mamoru; Fujimoto, Hitoshi; Ando, Yasutoshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity. PMID:27739494

  2. Adaptation of Clostridium difficile toxin A for use as a protein translocation system

    SciTech Connect

    Kern, Stephanie M.; Feig, Andrew L.

    2011-02-25

    Research highlights: {yields} Catalytic domain of TcdA was replaced by a luciferase reporter. {yields} Each functional domain retains activity in the context of the fusion protein. {yields} We provide evidence that reporter proteins are delivered into vero cells. {yields} System releases cargo into the cytosol, providing a powerful new biotechnology tool. -- Abstract: A cellular delivery system is a useful biotechnology tool, with many possible applications. Two derivatives of Clostridium difficile toxin A (TcdA) have been constructed (GFP-TcdA and Luc-TcdA), by fusing reporter genes to functional domains of TcdA, and evaluated for their ability to translocate their cargo into mammalian cells. The cysteine protease and receptor binding domains of TcdA have been examined and found to be functional when expressed in the chimeric construct. Whereas GFP failed to internalize in the context of the TcdA fusion, significant cellular luciferase activity was detected in vero cell lysates after treatment with Luc-TcdA. Treatment with bafilomycin A1, which inhibits endosomal acidification, traps the luciferase activity within endosomes. To further understand these results, clarified lysates were subjected to molecular weight sieving, demonstrating that active luciferase was released from Luc-TcdA after translocation and internal processing.

  3. Endocytosis Regulates Cell Soma Translocation and the Distribution of Adhesion Proteins in Migrating Neurons

    PubMed Central

    Shieh, Jennifer C.; Schaar, Bruce T.; Srinivasan, Karpagam; Brodsky, Frances M.; McConnell, Susan K.

    2011-01-01

    Newborn neurons migrate from their birthplace to their final location to form a properly functioning nervous system. During these movements, young neurons must attach and subsequently detach from their substrate to facilitate migration, but little is known about the mechanisms cells use to release their attachments. We show that the machinery for clathrin-mediated endocytosis is positioned to regulate the distribution of adhesion proteins in a subcellular region just proximal to the neuronal cell body. Inhibiting clathrin or dynamin function impedes the movement of migrating neurons both in vitro and in vivo. Inhibiting dynamin function in vitro shifts the distribution of adhesion proteins to the rear of the cell. These results suggest that endocytosis may play a critical role in regulating substrate detachment to enable cell body translocation in migrating neurons. PMID:21445347

  4. Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores

    NASA Astrophysics Data System (ADS)

    Wendell, David; Jing, Peng; Geng, Jia; Subramaniam, Varuni; Lee, Tae Jin; Montemagno, Carlo; Guo, Peixuan

    2009-11-01

    Biological pores have been used to study the transport of DNA and other molecules, but most pores have channels that allow only the movement of small molecules and single-stranded DNA and RNA. The bacteriophage phi29 DNA-packaging motor, which allows double-stranded DNA to enter the virus during maturation and exit during an infection, contains a connector protein with a channel that is between 3.6 and 6 nm wide. Here we show that a modified version of this connector protein, when reconstituted into liposomes and inserted into planar lipid bilayers, allows the translocation of double-stranded DNA. The measured conductance of a single connector channel was 4.8 nS in 1 M KCl. This engineered and membrane-adapted phage connector is expected to have applications in microelectromechanical sensing, microreactors, gene delivery, drug loading and DNA sequencing.

  5. Prediction of nuclear proteins using nuclear translocation signals proposed by probabilistic latent semantic indexing

    PubMed Central

    2012-01-01

    Background Identification of subcellular localization in proteins is crucial to elucidate cellular processes and molecular functions in a cell. However, given a tremendous amount of sequence data generated in the post-genomic era, determining protein localization based on biological experiments can be expensive and time-consuming. Therefore, developing prediction systems to analyze uncharacterised proteins efficiently has played an important role in high-throughput protein analyses. In a eukaryotic cell, many essential biological processes take place in the nucleus. Nuclear proteins shuttle between nucleus and cytoplasm based on recognition of nuclear translocation signals, including nuclear localization signals (NLSs) and nuclear export signals (NESs). Currently, only a few approaches have been developed specifically to predict nuclear localization using sequence features, such as putative NLSs. However, it has been shown that prediction coverage based on the NLSs is very low. In addition, most existing approaches only attained prediction accuracy and Matthew's correlation coefficient (MCC) around 54%~70% and 0.250~0.380 on independent test set, respectively. Moreover, no predictor can generate sequence motifs to characterize features of potential NESs, in which biological properties are not well understood from existing experimental studies. Results In this study, first we propose PSLNuc (Protein Subcellular Localization prediction for Nucleus) for predicting nuclear localization in proteins. First, for feature representation, a protein is represented by gapped-dipeptides and the feature values are weighted by homology information from a smoothed position-specific scoring matrix. After that, we incorporate probabilistic latent semantic indexing (PLSI) for feature reduction. Finally, the reduced features are used as input for a support vector machine (SVM) classifier. In addition to PSLNuc, we further identify gapped-dipeptide signatures for putative NLSs and NESs

  6. Tic21 Is an Essential Translocon Component for Protein Translocation across the Chloroplast Inner Envelope Membrane

    PubMed Central

    Teng, Yi-Shan; Su, Yi-shin; Chen, Lih-Jen; Lee, Yong Jik; Hwang, Inhwan; Li, Hsou-min

    2006-01-01

    An Arabidopsis thaliana mutant defective in chloroplast protein import was isolated and the mutant locus, cia5, identified by map-based cloning. CIA5 is a 21-kD integral membrane protein in the chloroplast inner envelope membrane with four predicted transmembrane domains, similar to another potential chloroplast inner membrane protein-conducting channel, At Tic20, and the mitochondrial inner membrane counterparts Tim17, Tim22, and Tim23. cia5 null mutants were albino and accumulated unprocessed precursor proteins. cia5 mutant chloroplasts were normal in targeting and binding of precursors to the chloroplast surface but were defective in protein translocation across the inner envelope membrane. Expression levels of CIA5 were comparable to those of major translocon components, such as At Tic110 and At Toc75, except during germination, at which stage At Tic20 was expressed at its highest level. A double mutant of cia5 At tic20-I had the same phenotype as the At tic20-I single mutant, suggesting that CIA5 and At Tic20 function similarly in chloroplast biogenesis, with At Tic20 functioning earlier in development. We renamed CIA5 as Arabidopsis Tic21 (At Tic21) and propose that it functions as part of the inner membrane protein-conducting channel and may be more important for later stages of leaf development. PMID:16891400

  7. Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3.

    PubMed

    Schrammeijer, Barbara; den Dulk-Ras, Amke; Vergunst, Annette C; Jurado Jácome, Esmeralda; Hooykaas, Paul J J

    2003-02-01

    Agrobacterium tumefaciens causes crown gall disease on a variety of plants. During the infection process Agrobacterium transfers a nucleoprotein complex, the VirD2 T-complex, and at least two Vir proteins, VirE2 and VirF, into the plant cell via the VirB/VirD4 type IV secretion system. Recently, we found that T-DNA could also be transferred from Agrobacterium to Saccharomyces cerevisiae. Here, we describe a novel method to also detect trans-kingdom Vir protein transfer from Agrobacterium to yeast, using the Cre/lox system. Protein fusions between Cre and VirE2 or VirF were expressed in AGROBACTERIUM: Transfer of the Cre-Vir fusion proteins from Agrobacterium to yeast was monitored by a selectable excision event resulting from site-specific recombination mediated by Cre on a lox-flanked transgene in yeast. The VirE2 and VirF proteins were transported to yeast via the virB-encoded transfer system in the presence of coupling factor VirD4, analogous to translocation into plant cells. The yeast system therefore provides a suitable and fast model system to study basic aspects of trans-kingdom protein transport from Agrobacterium into host cells. Using this method we showed that VirE2 and VirF protein transfer was inhibited by the presence of the Osa protein. Besides, we found evidence for a novel third effector protein, VirE3, which has a similar C-terminal signature to VirE2 and VirF. PMID:12560481

  8. Serotonin transporter (SERT) and translocator protein (TSPO) expression in the obese ob/ob mouse

    PubMed Central

    2011-01-01

    Background An ever growing body of evidences is emerging concerning metabolism hormones, neurotransmitters or stress-related biomarkers as effective modulators of eating behavior and body weight in mammals. The present study sought at examining the density and affinity of two proteins related to neurotransmission and cell metabolism, the serotonin transporter SERT and the cholesterol import-benzodiazepine site TSPO (translocator protein), in a rodent leptin-lacking mutant, the obese ob/ob mouse. Binding studies were thus carried out in brain or peripheral tissues, blood platelets (SERT) and kidneys (TSPO), of ob/ob and WT mice supplied with a standard diet, using the selective radiochemical ligands [3H]-paroxetine and [3H]-PK11195. Results We observed comparable SERT number or affinity in brain and platelets of ob/ob and WT mice, whilst a significantly higher [3H]-PK11195 density was reported in the brain of ob/ob animals. TSPO binding parameters were similar in the kidneys of all tested mice. By [3H]-PK11195 autoradiography of coronal hypothalamic-hippocampal sections, an increased TSPO signal was detected in the dentate gyrus (hippocampus) and choroids plexus of ob/ob mice, without appreciable changes in the cortex or hypothalamic-thalamic regions. Conclusions These findings show that TSPO expression is up-regulated in cerebral regions of ob/ob leptin-deficient mice, suggesting a role of the translocator protein in leptin-dependent CNS trophism and metabolism. Unchanged SERT in mutant mice is discussed herein in the context of previous literature as the forerunner to a deeper biochemical investigation. PMID:21299850

  9. Translocation of an 89-kDa periplasmic protein is associated with Holospora infection

    SciTech Connect

    Iwatani, Koichi; Dohra, Hideo; Lang, B. Franz; Burger, Gertraud; Hori, Manabu; Fujishima, Masahiro . E-mail: fujishim@yamaguchi-u.ac.jp

    2005-12-02

    The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside of the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus.

  10. Visualizing the Translocation and Localization of Bacterial Type III Effector Proteins by Using a Genetically Encoded Reporter System

    PubMed Central

    Gawthorne, Jayde A.; Audry, Laurent; McQuitty, Claire; Dean, Paul; Christie, John M.; Enninga, Jost

    2016-01-01

    Bacterial type III secretion system (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors. We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference. Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB. IpaB-LOV localized preferentially at bacterial poles before translocation. We observed the rapid translocation of IpaB-LOV in a T3SS-dependent manner into host cells, where it localized at the bacterial entry site within membrane ruffles. PMID:26921426

  11. Ethanol and diolein stimulate PKC (protein kinase C) translocation in astroglial cells

    SciTech Connect

    Skwish, S. ); Shain, W. New York State Department of Health, Albany )

    1990-01-01

    Ethanol exposure stimulates taurine release from astroglial cells. To determine if ethanol mediates this release using protein kinase C (PKC), PKC activity was measured using LRM55 astroglial cells. When ethanol or diolein was applied to cells for 30 seconds, PKC activity was observed to decrease in the cytosol and increase in the membrane fraction of the cell while the whole cell activity remained unchanged. The membrane-associated activity increased by almost 100%. When ethanol and diolein were applied simultaneously, membrane-associated activity increased to become 3-5 times greater than when either PKC activator was applied alone. These changes in PKC activity parallel changes in taurine release observed when cells are exposed to ethanol and the PKC activator diolein. Ethanol-stimulated release may be associated with the translocation of PKC activity from the cytosol to the membrane.

  12. Dynamin-Related Protein 1 Translocates from the Cytosol to Mitochondria during UV-Induced Apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Zhenzhen; Wu, Shengnan; Feng, Jie

    2011-01-01

    Mitochondria are dynamic structures that frequently divide and fuse with one another to form interconnecting network. This network disintegrates into punctiform organelles during apoptosis. However, the mechanisms involved in these processes are still not well characterized. In this study, we investigate the role of dynamin-related protein 1 (Drp1), a large GTPase that mediates outer mitochondrial membrane fission, in mitochondrial dynamics in response to UV irradiation in human lung adenocarcinoma cells (ASTC-α-1) and HeLa cells. Using time-lapse fluorescent imaging, we find that Drp1 primarily distributes in cytosol under physiological conditions. After UV treatment, Drp1 translocates from cytosol to mitochondria, indicating the enhancement of Drp1 mitochondrial accumulation. Our results suggest that Drp1 is involved in the regulation of transition from an interconnecting network to a punctiform mitochondrial phenotype during UV-induced apoptosis.

  13. Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation.

    PubMed

    Hartwig, J H; Davies, W A; Stossel, T P

    1977-12-01

    Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of beta-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle

  14. Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase.

    PubMed

    Kravats, Andrea; Jayasinghe, Manori; Stan, George

    2011-02-01

    Clp ATPases are ring-shaped AAA+ motors in the degradation pathway that perform critical actions of unfolding and translocating substrate proteins (SPs) through narrow pores to deliver them to peptidase components. These actions are effected by conserved diaphragm-forming loops found in the central channel of the Clp ATPase hexamer. Conformational changes, that take place in the course of repetitive ATP-driven cycles, result in mechanical forces applied by the central channel loops onto the SP. We use coarse-grained simulations to elucidate allostery-driven mechanisms of unfolding and translocation of a tagged four-helix bundle protein by the ClpY ATPase. Unfolding is initiated at the tagged C-terminal region via an obligatory intermediate. The resulting nonnative conformation is competent for translocation, which proceeds on a different time scale than unfolding and involves sharp stepped transitions. Completion of the translocation process requires assistance from the ClpQ peptidase. These mechanisms contrast nonallosteric mechanical unfolding of the SP. In atomic force microscopy experiments, multiple unfolding pathways are available and large mechanical forces are required to unravel the SP relative to those exerted by the central channel loops of ClpY. SP threading through a nonallosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.

  15. Development of a Split SNAP-CLIP Double Labeling System for Tracking Proteins Following Dissociation from Protein-Protein Complexes in Living Cells.

    PubMed

    Mie, Masayasu; Naoki, Tatsuhiko; Kobatake, Eiry

    2016-08-16

    The split SNAP-tag protein-fragment complementation assay (PCA) is a useful tool for imaging protein-protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein-protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein-protein complex. To track both proteins forming a protein-protein complex, each protein needs to be appropriately labeled. In this study, a split SNAP-CLIP double labeling system is developed and applied for tracking of each protein forming a protein-protein complex. As a proof-of concept, FM protein for PPIs and protein kinase C alpha (PKCα) for translocation are introduced to a split SNAP-CLIP double labeling system. The results show a split SNAP-CLIP double labeling system enables labeling of both proteins in a protein-protein complex and subsequent tracking of each of the proteins following dissociation from the protein-protein complexes in living cells.

  16. The Rnf Complex of Clostridium ljungdahlii Is a Proton-Translocating Ferredoxin:NAD+ Oxidoreductase Essential for Autotrophic Growth

    PubMed Central

    Tremblay, Pier-Luc; Zhang, Tian; Dar, Shabir A.; Leang, Ching; Lovley, Derek R.

    2012-01-01

    ABSTRACT It has been predicted that the Rnf complex of Clostridium ljungdahlii is a proton-translocating ferredoxin:NAD+ oxidoreductase which contributes to ATP synthesis by an H+-translocating ATPase under both autotrophic and heterotrophic growth conditions. The recent development of methods for genetic manipulation of C. ljungdahlii made it possible to evaluate the possible role of the Rnf complex in energy conservation. Disruption of the C. ljungdahlii rnf operon inhibited autotrophic growth. ATP synthesis, proton gradient, membrane potential, and proton motive force collapsed in the Rnf-deficient mutant with H2 as the electron source and CO2 as the electron acceptor. Heterotrophic growth was hindered in the absence of a functional Rnf complex, as ATP synthesis, proton gradient, and proton motive force were significantly reduced with fructose as the electron donor. Growth of the Rnf-deficient mutant was also inhibited when no source of fixed nitrogen was provided. These results demonstrate that the Rnf complex of C. ljungdahlii is responsible for translocation of protons across the membrane to elicit energy conservation during acetogenesis and is a multifunctional device also implicated in nitrogen fixation. PMID:23269825

  17. Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis.

    PubMed

    Li, Xinjian; Jiang, Yuhui; Meisenhelder, Jill; Yang, Weiwei; Hawke, David H; Zheng, Yanhua; Xia, Yan; Aldape, Kenneth; He, Jie; Hunter, Tony; Wang, Liwei; Lu, Zhimin

    2016-03-01

    It is unclear how the Warburg effect that exemplifies enhanced glycolysis in the cytosol is coordinated with suppressed mitochondrial pyruvate metabolism. We demonstrate here that hypoxia, EGFR activation, and expression of K-Ras G12V and B-Raf V600E induce mitochondrial translocation of phosphoglycerate kinase 1 (PGK1); this is mediated by ERK-dependent PGK1 S203 phosphorylation and subsequent PIN1-mediated cis-trans isomerization. Mitochondrial PGK1 acts as a protein kinase to phosphorylate pyruvate dehydrogenase kinase 1 (PDHK1) at T338, which activates PDHK1 to phosphorylate and inhibit the pyruvate dehydrogenase (PDH) complex. This reduces mitochondrial pyruvate utilization, suppresses reactive oxygen species production, increases lactate production, and promotes brain tumorigenesis. Furthermore, PGK1 S203 and PDHK1 T338 phosphorylation levels correlate with PDH S293 inactivating phosphorylation levels and poor prognosis in glioblastoma patients. This work highlights that PGK1 acts as a protein kinase in coordinating glycolysis and the tricarboxylic acid (TCA) cycle, which is instrumental in cancer metabolism and tumorigenesis.

  18. Mitochondria-translocated phosphoglycerate kinase 1 functions as a protein kinase to coordinate glycolysis and TCA cycle in tumorigenesis

    PubMed Central

    Li, Xinjian; Jiang, Yuhui; Meisenhelder, Jill; Yang, Weiwei; Hawke, David H.; Zheng, Yanhua; Xia, Yan; Aldape, Kenneth; He, Jie; Hunter, Tony; Wang, Liwei; Lu, Zhimin

    2016-01-01

    SUMMARY It is unclear how the Warburg effect that exemplifies enhanced glycolysis in the cytosol is coordinated with suppressed mitochondrial pyruvate metabolism. We demonstrate here that hypoxia, EGFR activation, and expression of K-Ras G12V and B-Raf V600E induce mitochondrial translocation of phosphoglycerate kinase 1 (PGK1); this is mediated by ERK-dependent PGK1 S203 phosphorylation and subsequent PIN1-mediated cis–trans isomerization. Mitochondrial PGK1 acts as a protein kinase to phosphorylate pyruvate dehydrogenase kinase 1 (PDHK1) at T338, which activates PDHK1 to phosphorylate and inhibit the pyruvate dehydrogenase (PDH) complex. This reduces mitochondrial pyruvate utilization, suppresses reactive oxygen species production, increases lactate production, and promotes brain tumorigenesis. Furthermore, PGK1 S203 and PDHK1 T338 phosphorylation levels correlate with PDH S293 inactivating phosphorylation levels and poor prognosis in glioblastoma patients. This work highlights that PGK1 act as a protein kinase in coordinating glycolysis and the TCA cycle, which is instrumental in cancer metabolism and tumorigenesis. PMID:26942675

  19. Signal-on Protein Detection via Dye Translocation between Aptamer and Quantum Dot.

    PubMed

    Lao, Yeh-Hsing; Chi, Chun-Wei; Friedrich, Sarah M; Peck, Konan; Wang, Tza-Huei; Leong, Kam W; Chen, Lin-Chi

    2016-05-18

    A unique interaction between the cyanine dye and negatively charged quantum dot is used to construct a signal-on biaptameric quantum dot (QD) Förster resonance energy transfer (FRET) beacon for protein detection and distinct aptamer characterization. The beacon comprises a pair of aptamers, one intercalated with the cyanine dye (YOYO-3) and the other conjugated to a negatively charged, carboxyl-QD. When the target protein is present, structural folding and sandwich association of the two aptamers take place. As a consequence, YOYO-3 is displaced from the folded aptamer and transferred to the unblocked QD surface to yield a target concentration-dependent FRET signal. As a proof-of-principle, we demonstrate the detection of thrombin ranging from nanomolar to submicromolar concentrations and confirm the dye translocation using cylindrical illumination confocal spectroscopy (CICS). The proposed beacon provides a simple, rapid, signal-on FRET detection for protein as well as a potential platform for distinct aptamer screening. PMID:27101438

  20. Structure-to-function relationships of bacterial translocator protein (TSPO): a focus on Pseudomonas.

    PubMed

    Leneveu-Jenvrin, Charlène; Connil, Nathalie; Bouffartigues, Emeline; Papadopoulos, Vassilios; Feuilloley, Marc G J; Chevalier, Sylvie

    2014-01-01

    The translocator protein (TSPO), which was previously designated as the peripheral-type benzodiazepine receptor, is a 3.5 billion year-old evolutionarily conserved protein expressed by most Eukarya, Archae and Bacteria, but its organization and functions differ remarkably. By taking advantage of the genomic data available on TSPO, we focused on bacterial TSPO and attempted to define functions of TSPO in Pseudomonas via in silico approaches. A tspo ortholog has been identified in several fluorescent Pseudomonas. This protein presents putative binding motifs for cholesterol and PK 11195, which is a specific drug ligand of mitochondrial TSPO. While it is a common surface distribution, the sense of insertion and membrane localization differ between α- and γ-proteobacteria. Experimental published data and STRING analysis of common TSPO partners in fluorescent Pseudomonas indicate a potential role of TSPO in the oxidative stress response, iron homeostasis and virulence expression. In these bacteria, TSPO could also take part in signal transduction and in the preservation of membrane integrity.

  1. Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation

    SciTech Connect

    Cambier, Linda; Pomies, Pascal

    2011-06-17

    Highlights: {yields} The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. {yields} smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. {yields} The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. {yields} The LIM domain of smALP is essential for the nuclear accumulation of the protein. {yields} smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.

  2. Nuclear translocation of IQGAP1 protein upon exposure to puromycin aminonucleoside in cultured human podocytes: ERK pathway involvement.

    PubMed

    Rigothier, Claire; Saleem, Moin Ahson; Bourget, Chantal; Mathieson, Peter William; Combe, Christian; Welsh, Gavin Iain

    2016-10-01

    IQGAP1, a protein that links the actin cytoskeleton to slit diaphragm proteins, is involved in podocyte motility and permeability. Its regulation in glomerular disease is not known. We have exposed human podocytes to puromycin aminonucleoside (PAN), an inducer of nephrotic syndrome in rats, and studied the effects on IQGAP1 biology and function. In human podocytes exposed to PAN, a nuclear translocation of IQGAP1 was observed by immunocytolocalization and confirmed by Western blot after selective nuclear/cytoplasmic extraction. In contrast to IQGAP1, IQGAP2 expression remained cytoplasmic. IQGAP1 nuclear translocation was associated with a significant decrease in its interaction with nephrin and podocalyxin. Activation of the ERK pathway was observed in PAN treated podocytes with a preponderant nuclear localization of the phosphorylated form of ERK (P-ERK). The interaction between IQGAP1 and P-ERK increased upon podocyte exposure to PAN. Inhibitors of ERK pathway activation blocked IQGAP1 nuclear translocation (p<0.02). Chromatin interaction protein assays demonstrated an interaction of IQGAP1 with chromatin and with Histone H3, which increased in response to PAN. In summary, PAN induces the ERK dependent translocation of IQGAP1 into the nuclei in human podocytes which leads to the interaction of IQGAP1 with chromatin and Histone H3, and decreased interactions between IQGAP1 and slit-diaphragm proteins. Therefore, IQGAP1 may have a role in podocyte gene regulation in glomerular disease.

  3. The Rnf Complex of Clostridium ljungdahlii Is a Proton-Translocating Ferredoxin:NAD(+) Oxidoreductase Essential for Autotrophic Growth

    SciTech Connect

    Tremblay, PL; Zhang, T; Dar, SA; Leang, C; Lovley, DR

    2012-12-26

    It has been predicted that the Rnf complex of Clostridium ljungdahlii is a proton-translocating ferredoxin: NAD(+) oxidoreductase which contributes to ATP synthesis by an H+-translocating ATPase under both autotrophic and heterotrophic growth conditions. The recent development of methods for genetic manipulation of C. ljungdahlii made it possible to evaluate the possible role of the Rnf complex in energy conservation. Disruption of the C. ljungdahlii rnf operon inhibited autotrophic growth. ATP synthesis, proton gradient, membrane potential, and proton motive force collapsed in the Rnf-deficient mutant with H-2 as the electron source and CO2 as the electron acceptor. Heterotrophic growth was hindered in the absence of a functional Rnf complex, as ATP synthesis, proton gradient, and proton motive force were significantly reduced with fructose as the electron donor. Growth of the Rnf-deficient mutant was also inhibited when no source of fixed nitrogen was provided. These results demonstrate that the Rnf complex of C. ljungdahlii is responsible for translocation of protons across the membrane to elicit energy conservation during acetogenesis and is a multifunctional device also implicated in nitrogen fixation. IMPORTANCE Mechanisms for energy conservation in the acetogen Clostridium ljungdahlii are of interest because of its potential value as a chassis for the production of biocommodities with novel electron donors such as carbon monoxide, syngas, and electrons derived from electrodes. Characterizing the components implicated in the chemiosmotic ATP synthesis during acetogenesis by C. ljungdahlii is a prerequisite for the development of highly productive strains. The Rnf complex has been considered the prime candidate to be the pump responsible for the formation of an ion gradient coupled with ATP synthesis in multiple acetogens. However, experimental evidence for a proton-pumping Rnf complex has been lacking. This study establishes the C. ljungdahlii Rnf complex as

  4. Protein-protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids.

    PubMed

    Lau, Julia B; Stork, Simone; Moog, Daniel; Schulz, Julian; Maier, Uwe G

    2016-04-01

    Most secondary plastids of red algal origin are surrounded by four membranes and nucleus-encoded plastid proteins have to traverse these barriers. Translocation across the second outermost plastid membrane, the periplastidal membrane (PPM), is facilitated by a ERAD-(ER-associated degradation) derived machinery termed SELMA (symbiont-specific ERAD-like machinery). In the last years, important subunits of this translocator have been identified, which clearly imply compositional similarities between SELMA and ERAD. Here we investigated, via protein-protein interaction studies, if the composition of SELMA is comparable to the known ERAD complex. As a result, our data suggest that the membrane proteins of SELMA, the derlin proteins, are linked to the soluble sCdc48 complex via the UBX protein sUBX. This is similar to the ERAD machinery whereas the additional SELMA components, sPUB und a second Cdc48 copy might indicate the influence of functional constraints in developing a translocation machinery from ERAD-related factors. In addition, we show for the first time that a rhomboid protease is a central interaction partner of the membrane proteins of the SELMA system in complex plastids.

  5. Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins across Membranes

    NASA Astrophysics Data System (ADS)

    Hoch, David H.; Romero-Mira, Miryam; Ehrlich, Barbara E.; Finkelstein, Alan; Dasgupta, Bibhuti R.; Simpson, Lance L.

    1985-03-01

    The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as ``tunnel proteins'' for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

  6. Molecular cloning of the breakpoints of a complex Philadelphia chromosome translocation: identification of a repeated region on chromosome 17.

    PubMed Central

    McKeithan, T W; Warshawsky, L; Espinosa, R; LeBeau, M M

    1992-01-01

    Complex translocations in chronic myelogenous leukemia involve various chromosomes, in addition to chromosomes 9 and 22, in a nonrandom fashion. We have analyzed the DNA from leukemia cells characterized by a complex translocation, t(9;22;10;17)(q34;q11;p13;q21), by using the techniques of Southern blot hybridization, in situ hybridization, and molecular cloning; one of the breakpoints is at 17q21, a band that is frequently involved in complex 9;22 translocations. All of the breakpoint junctions and the corresponding normal sequences from the four involved chromosomes have been molecularly cloned. Restriction mapping is consistent with a simple concerted exchange of chromosomal material among the four chromosomes, except that additional changes appeared to have occurred within the chromosome 17 sequences. The cloned sequences on chromosome 17 at band q21 were found to be repeated in normal cells. By fluorescence in situ hybridization, a strong signal is seen at 17q21, but a weaker signal is also present at 17q23. By comparison with other primate species, an inversion in chromosome 17 during evolution appears to be responsible for the splitting of the cluster of repeat units in normal human cells. Images PMID:1594595

  7. NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na+-Translocating NADH:Quinone Oxidoreductase

    PubMed Central

    Kostyrko, Vitaly A.; Bertsova, Yulia V.; Serebryakova, Marina V.; Baykov, Alexander A.

    2015-01-01

    ABSTRACT Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na+ translocation across the membrane. Na+-NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamed nqrM) that usually follows the apbE gene and is present only in Na+-NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli, which lacks its own Na+-NQR, resulted in an enzyme incapable of Na+-dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na+-NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na+-NQR, which could be recovered by an nqrM-containing plasmid. The Na+-NQR complex isolated from the nqrM-deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na+-NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane α-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na+-NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na+-NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys)4[Fe] center between subunits NqrD and NqrE. IMPORTANCE Na+-translocating NADH:quinone oxidoreductase complex (Na+-NQR) is a unique primary Na+ pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae, Vibrio

  8. PK11195 effect on steroidogenesis is not mediated through the translocator protein (TSPO).

    PubMed

    Tu, Lan N; Zhao, Amy H; Stocco, Douglas M; Selvaraj, Vimal

    2015-03-01

    Translocator protein (TSPO) is a mitochondrial outer membrane protein of unknown function with high physiological expression in steroidogenic cells. Using TSPO gene-deleted mice, we recently demonstrated that TSPO function is not essential for steroidogenesis. The first link between TSPO and steroidogenesis was established in studies showing modest increases in progesterone production by adrenocortical and Leydig tumor cell lines after treatment with PK11195. To reconcile discrepancies between physiological and pharmacological interpretations of TSPO function, we generated TSPO-knockout MA-10 mouse Leydig tumor cells (MA-10:TspoΔ/Δ) and examined their steroidogenic potential after exposure to either dibutyryl-cAMP or PK11195. Progesterone production in MA-10:TspoΔ/Δ after dibutyryl-cAMP was not different from control MA-10:Tspo+/+ cells, confirming that TSPO function is not essential for steroidogenesis. Interestingly, when treated with increasing concentrations of PK11195, both control MA-10:Tspo+/+ cells and MA-10:TspoΔ/Δ cells responded in a similar dose-dependent manner showing increases in progesterone production. These results show that the pharmacological effect of PK11195 on steroidogenesis is not mediated through TSPO.

  9. Translocation of capsular polysaccharides in pathogenic strains of Escherichia coli requires a 60-kilodalton periplasmic protein.

    PubMed Central

    Silver, R P; Aaronson, W; Vann, W F

    1987-01-01

    An 11.6-kilobase (kb) region of a 34-kb fragment of Escherichia coli DNA that encodes the K1 capsular polysaccharide genes is necessary for translocation of the K1 polysaccharide to the bacterial cell surface. This 11.6-kb region contains a gene, kpsD, encoding a 60-kilodalton protein. The kpsD gene was localized to a 2.4-kb PstI-BamHI fragment. Cells harboring a Tn1000 insertion in kpsD did not synthesize the 60-kilodalton protein and did not express polysaccharide on the cell surface. Immunodiffusion and rocket immunoelectrophoresis of cell extracts, however, demonstrated that K1 polysaccharide was synthesized by these cells. We present evidence that the kpsD gene product is synthesized as a precursor and that the processed form is located in the periplasmic space. Analysis of alkaline phosphatase activity of a kpsD-phoA fusion demonstrated that kpsD expression was under positive regulation. A 260-base-pair AluI fragment located within the kpsD coding sequence was used as a probe and was found to hybridize to chromosomal DNA from E. coli that synthesizes the K2, K5, K7, K12, and K13 capsular polysaccharides but not K3 and K100. These results suggest that the kpsD gene product may be required for export not only of K1 but for other K antigens as well. Images PMID:3119565

  10. Regulation of Translocator Protein 18 kDa (TSPO) Expression in Health and Disease States

    PubMed Central

    Batarseh, Amani; Papadopoulos, Vassilios

    2010-01-01

    Translocator protein (TSPO) is an 18-kDa high affinity cholesterol- and drug-binding protein found primarily in the outer mitochondrial membrane. Although TSPO is found in many tissue types, it is expressed at the highest levels under normal conditions in tissues that synthesize steroids. TSPO has been associated with cholesterol import into mitochondria, a key function in steroidogenesis, and directly or indirectly with multiple other cellular functions including apoptosis, cell proliferation, differentiation, anion transport, porphyrin transport, heme synthesis, and regulation of mitochondrial function. Aberrant expression of TSPO has been linked to multiple diseases, including cancer, brain injury, neurodegeneration, and ischemia reperfusion injury. There has been an effort during the last decade to understand the mechanisms regulating tissue- and disease-specific TSPO expression and to identify pharmacological means to control its expression. This review focuses on the current knowledge regarding the chemicals, hormones, and molecular mechanisms regulating Tspo gene expression under physiological conditions in a tissue- and disease-specific manner. The results described here provide evidence that the PKCε-ERK1/2-AP1/Stat3 signal transduction pathway is the primary regulator of Tspo gene expression in normal and pathological tissues expressing high levels of TSPO. PMID:20600583

  11. Protein-ligand and membrane-ligand interactions in pharmacology: the case of the translocator protein (TSPO).

    PubMed

    Hatty, Claire R; Banati, Richard B

    2015-10-01

    The targets of many small molecule drugs are membrane proteins, and traditionally the focus of pharmacology is on the interaction between such receptors and their small molecule drug ligands. However, the lipid membranes of cells and organelles are increasingly appreciated as diverse and dynamic structures that also specifically interact with small molecule drugs and peptides, causing profound changes in the properties of these membranes, and modulating the function of the membrane and the proteins within it. Drug-membrane interactions are likely to have a role in both the therapeutic and toxic activity of a variety of compounds, and their role in the overall pharmacological effect of a drug needs to be understood more clearly. This is the case for the 18 kDa translocator protein (TSPO) and its ligands, where functions that were established based on pharmacological studies are being called into question. Re-examining the putative functions of the TSPO and the effects of its ligands reveals a need to consider in more detail the interplay between protein-ligand and membrane-ligand interactions, and the modulatory relationship between TSPO and the lipid membrane.

  12. Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT).

    PubMed Central

    Reisz-Porszasz, S; Probst, M R; Fukunaga, B N; Hankinson, O

    1994-01-01

    The activated aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) bind DNA as a heterodimer. Both proteins represent a novel class of basic helix-loop-helix (bHLH)-containing transcription factors in that (i) activation of AHR requires the binding of ligand (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]), (ii) the xenobiotic responsive element (XRE) recognized by the AHR/ARNT heterodimer differs from the recognition sequence for nearly all other bHLH proteins, and (iii) both proteins contain a PAS homology region, which in the Drosophila PER and SIM proteins functions as a dimerization domain. A cDNA for mouse ARNT has been cloned, and potential functional domains of ARNT were investigated by deletion analysis. A mutant lacking all regions of ARNT other than the bHLH and PAS regions is unimpaired in TCDD-dependent dimerization and subsequent XRE binding and only modestly reduced in ability to complement an ARNT-deficient mutant cell line, c4, in vivo. Both the first and second alpha helices of the bHLH region are required for dimerization. The basic region is required for XRE binding but not for dimerization. Deletion of either the A or B segments of the PAS region slightly affects TCDD-induced heterodimerization, while deletion of the complete PAS region severely affects (but does not eliminate) dimerization. Thus, ARNT possesses multiple domains required for maximal heterodimerization. Mutants deleted for PAS A, PAS B, and the complete PAS region all retain some degree of XRE binding, yet none can rescue the c4 mutant. Therefore, both the PAS A and PAS B segments, besides contributing to dimerization, apparently fulfill additional, unknown functions required for biological activity of ARNT. Images PMID:8065341

  13. Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation*

    PubMed Central

    Zhu, Shaotong; Vik, Steven B.

    2015-01-01

    Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441–445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu2+ ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50–90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10–20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus. PMID:26134569

  14. The ciliary protein nephrocystin-4 translocates the canonical Wnt regulator Jade-1 to the nucleus to negatively regulate β-catenin signaling.

    PubMed

    Borgal, Lori; Habbig, Sandra; Hatzold, Julia; Liebau, Max C; Dafinger, Claudia; Sacarea, Ilinca; Hammerschmidt, Matthias; Benzing, Thomas; Schermer, Bernhard

    2012-07-20

    Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease and represents the most common genetic cause for end-stage renal disease in children and adolescents. It can be caused by the mutation of genes encoding for the nephrocystin proteins (NPHPs). All NPHPs localize to primary cilia, classifying this disease as a "ciliopathy." The primary cilium is a critical regulator of several cell signaling pathways. Cystogenesis in the kidney is thought to involve overactivation of canonical Wnt signaling, which is negatively regulated by the primary cilium and several NPH proteins, although the mechanism remains unclear. Jade-1 has recently been identified as a novel ubiquitin ligase targeting the canonical Wnt downstream effector β-catenin for proteasomal degradation. Here, we identify Jade-1 as a novel component of the NPHP protein complex. Jade-1 colocalizes with NPHP1 at the transition zone of primary cilia and interacts with NPHP4. Furthermore, NPHP4 stabilizes protein levels of Jade-1 and promotes the translocation of Jade-1 to the nucleus. Finally, NPHP4 and Jade-1 additively inhibit canonical Wnt signaling, and this genetic interaction is conserved in zebrafish. The stabilization and nuclear translocation of Jade-1 by NPHP4 enhances the ability of Jade-1 to negatively regulate canonical Wnt signaling. Loss of this repressor function in nephronophthisis might be an important factor promoting Wnt activation and contributing to cyst formation.

  15. Translocator Protein (TSPO) Affects Mitochondrial Fatty Acid Oxidation in Steroidogenic Cells.

    PubMed

    Tu, Lan N; Zhao, Amy H; Hussein, Mahmoud; Stocco, Douglas M; Selvaraj, Vimal

    2016-03-01

    Translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a highly conserved outer mitochondrial membrane protein present in specific subpopulations of cells within different tissues. In recent studies, the presumptive model depicting mammalian TSPO as a critical cholesterol transporter for steroidogenesis has been refuted by studies examining effects of Tspo gene deletion in vivo and in vitro, biochemical testing of TSPO cholesterol transport function, and specificity of TSPO-mediated pharmacological responses. Nevertheless, high TSPO expression in steroid-producing cells seemed to indicate an alternate function for this protein in steroidogenic mitochondria. To seek an explanation, we used CRISPR/Cas9-mediated TSPO knockout steroidogenic MA-10 Leydig cell (MA-10:TspoΔ/Δ) clones to examine changes to core mitochondrial functions resulting from TSPO deficiency. We observed that 1) MA-10:TspoΔ/Δ cells had a shift in substrate utilization for energy production from glucose to fatty acids with significantly higher mitochondrial fatty acid oxidation (FAO), and increased reactive oxygen species production; and 2) oxygen consumption rate, mitochondrial membrane potential, and proton leak were not different between MA-10:TspoΔ/Δ and MA-10:Tspo+/+ control cells. Consistent with this finding, TSPO-deficient adrenal glands from global TSPO knockout (Tspo(-/-)) mice also showed up-regulation of genes involved in FAO compared with the TSPO floxed (Tspo(fl/fl)) controls. These results demonstrate the first experimental evidence that TSPO can affect mitochondrial energy homeostasis through modulation of FAO, a function that appears to be consistent with high levels of TSPO expression observed in cell types active in lipid storage/metabolism.

  16. Targeting mitochondrial 18 kDa translocator protein (TSPO) regulates macrophage cholesterol efflux and lipid phenotype.

    PubMed

    Taylor, Janice M W; Allen, Anne-Marie; Graham, Annette

    2014-11-01

    The aim of the present study was to establish mitochondrial cholesterol trafficking 18 kDa translocator protein (TSPO) as a potential therapeutic target, capable of increasing macrophage cholesterol efflux to (apo)lipoprotein acceptors. Expression and activity of TSPO in human (THP-1) macrophages were manipulated genetically and by the use of selective TSPO ligands. Cellular responses were analysed by quantitative PCR (Q-PCR), immunoblotting and radiolabelling, including [3H]cholesterol efflux to (apo)lipoprotein A-I (apoA-I), high-density lipoprotein (HDL) and human serum. Induction of macrophage cholesterol deposition by acetylated low-density lipoprotein (AcLDL) increased expression of TSPO mRNA and protein, reflecting findings in human carotid atherosclerosis. Transient overexpression of TSPO enhanced efflux (E%) of [3H]cholesterol to apoA-I, HDL and human serum compared with empty vector (EV) controls, whereas gene knockdown of TSPO achieved the converse. Ligation of TSPO (using PK11195, FGIN-1-27 and flunitrazepam) triggered increases in [3H]cholesterol efflux, an effect that was amplified in TSPO-overexpressing macrophages. Overexpression of TSPO induced the expression of genes [PPARA (peroxisome-proliferator-activated receptor α), NR1H3 (nuclear receptor 1H3/liver X receptor α), ABCA1 (ATP-binding cassette A1), ABCG4 (ATP-binding cassette G4) and APOE (apolipoprotein E)] and proteins (ABCA1 and PPARα) involved in cholesterol efflux, reduced macrophage neutral lipid mass and lipogenesis and limited cholesterol esterification following exposure to AcLDL. Thus, targeting TSPO reduces macrophage lipid content and prevents macrophage foam cell formation, via enhanced cholesterol efflux to (apo)lipoprotein acceptors.

  17. Translocation of cellular prion protein to non-lipid rafts protects human prion-mediated neuronal damage.

    PubMed

    Jeong, Jae-Kyo; Moon, Myung-Hee; Lee, You-Jin; Seol, Jae-Won; Park, Sang-Youel

    2012-03-01

    Prions are the causative agents of transmissible spongiform encephalopathies, such as variant Creutzfeldt-Jakob disease in humans. Cellular prion proteins (PrPC) connect with cholesterol- and glycosphingolipid-rich lipid rafts through association of their glycosyl-phosphatidylinositol (GPI) anchor with saturated raft lipids and interaction of their N-terminal regions. Our previous study showed that cellular cholesterol enrichment prevented PrP(106-126)-induced neuronal death. We have now studied the influence of membrane cholesterol in PrP(106-126)-mediated neurotoxicity and identified membrane domains involved in this activity. We found that PrPC is normally distributed in lipid rafts, but high membrane cholesterol levels as a result of cholesterol treatment led to the translocation of PrPC from lipid rafts to non-lipid rafts. Moreover, cholesterol-mediated PrPC translocation protects PrP(106-126)-mediated apoptosis and p-38 activation and caspase-3 activation. In a mitochondrial functional assay including mitochondrial transmembrane potential, cholesterol treatment prevented the loss of mitochondrial potential, translocation of Bax and cytochrome c by prion protein fragment. Our results indicate that modulation of the PrPC location appears to protect against neuronal cell death caused by prion peptides. The results of this study suggest that regulation of membrane cholesterol affects the translocation of PrPC, which in turn regulates PrP(106-126)-induced mitochondrial dysfunction and neurotoxicity.

  18. SopB effector protein of Salmonella Typhimurium is translocated in mesenteric lymph nodes during murine salmonellosis.

    PubMed

    Giacomodonato, Mónica N; Sarnacki, Sebastián H; Llana, Mariángeles Noto; Cerquetti, María C

    2011-04-01

    Salmonella Typhimurium harbors two Salmonella pathogenicity islands (SPIs), each encoding a type three secretion system for virulence proteins. Although there is increasing evidence of postinvasion roles for SPI-1, it has been generally accepted that SPI-1 genes are downregulated following the invasion process. Here, we analyzed the expression and translocation of SopB in vitro, in cell culture and in vivo. To this end, a sopB-FLAG-tagged strain of Salmonella Typhimurium was obtained by epitope tagging. Tagged proteins were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. SopB expression was observed in vitro under cultured conditions that mimic the intestinal niche and different intracellular environments. In agreement, bacteria isolated from infected monolayers expressed and translocated SopB for at least 24 h postinoculation. For in vivo experiments, BALB/c mice were inoculated intraperitoneally with the tagged strain of Salmonella Typhimurium. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes. Our results showed that SopB continues to be synthesized in vivo during 5 days after inoculation. Interestingly, translocation of SopB was detected in the cytosol of cells isolated from lymph nodes 1 day after infection. Altogether, these findings indicate that the expression and translocation of SopB during Salmonella infection is not constrained to the initial host-bacteria encounter in the intestinal environment as defined previously.

  19. Lead Optimization of 2-Phenylindolylglyoxylyldipeptide Murine Double Minute (MDM)2/Translocator Protein (TSPO) Dual Inhibitors for the Treatment of Gliomas.

    PubMed

    Daniele, Simona; La Pietra, Valeria; Barresi, Elisabetta; Di Maro, Salvatore; Da Pozzo, Eleonora; Robello, Marco; La Motta, Concettina; Cosconati, Sandro; Taliani, Sabrina; Marinelli, Luciana; Novellino, Ettore; Martini, Claudia; Da Settimo, Federico

    2016-05-26

    In glioblastoma multiforme (GBM), translocator protein (TSPO) and murine double minute (MDM)2/p53 complex represent two druggable targets. We recently reported the first dual binder 3 possessing a higher anticancer effect in GBM cells than the standards PK11195 1 or Nutlin-3 2 singularly applied. Herein, through a structure-activity relationship study, we developed derivatives 4-10 with improved potencies toward both TSPO and MDM2. As a result, compound 9: (i) reactivated the p53 functionality; (ii) inhibited the viability of two human GBM cells; (iii) impaired the proliferation of glioma cancer stem cells (CSCs), more resistant to chemotherapeutics and responsible of GBM recurrence; (iv) sensitized GBM cells and CSCs to the activity of temozolomide; (v) directed its effects preferentially toward tumor cells with respect to healthy ones. Thus, 9 may represent a promising cytotoxic agent, which is worthy of being further developed for a therapeutic approach against GBM, where the downstream p53 signaling is intact and TSPO is overexpressed.

  20. Synchrotron Protein Footprinting Supports Substrate Translocation by ClpA via ATP-Induced Movements of the D2 Loop

    PubMed Central

    Bohon, Jen; Jennings, Laura D.; Phillips, Christine M.; Licht, Stuart; Chance, Mark R.

    2010-01-01

    SUMMARY Synchrotron x-ray protein footprinting is used to study structural changes upon formation of the ClpA hexamer. Comparative solvent accessibilities between ClpA monomer and ClpA hexamer samples are in agreement throughout most of the sequence with calculations based on two previously proposed hexameric models. The data differ substantially from the proposed models in two parts of the structure: the D1 sensor 1 domain and the D2 loop region. The results suggest that these two regions can access alternate conformations in which their solvent protection is greater than in the structural models based on crystallographic data. In combination with previously reported structural data, the footprinting data provide support for a revised model in which the D2 loop contacts the D1 sensor 1 domain in the ATP-bound form of the complex. These data provide the first direct experimental support for the nucleotide-dependent D2 loop conformational change previously proposed to mediate substrate translocation. PMID:18682217

  1. Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9.

    PubMed

    Macdonald, Colin J; Tozawa, Kaeko; Collins, Emily S; Penfold, Christopher N; James, Richard; Kleanthous, Colin; Clayden, Nigel J; Moore, Geoffrey R

    2004-09-01

    The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies (Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D (1)H-(13)C-(15)N NMR experiments, and the backbone dynamics of these regions investigated through measurement of (1)H-(15)N relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R(2) relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain. Abbreviations : Im9, the immunity protein for colicin E9; E9 DNase, the endonuclease domain of colicin E9; HSQC, heteronuclear single quantum coherence; ppm, parts per million; DSS, 2,2-(dimethylsilyl)propanesulfonic acid; TSP, sodium 3-trimethylsilypropionate; T(1 - 61)-DNase fusion protein, residues 1-61 of

  2. The complex translocation (9;14;14) involving IGH and CEBPE genes suggests a new subgroup in B-lineage acute lymphoblastic leukemia

    PubMed Central

    Zerrouki, Rachid; Benhassine, Traki; Bensaada, Mustapha; Lauzon, Patricia; Trabzi, Anissa

    2016-01-01

    Abstract Many subtypes of acute lymphoblastic leukemia (ALL) are associated with specific chromosomal rearrangements. The complex translocation t(9;14;14), a variant of the translocation (14;14)(q11;q32), is a rare but recurrent chromosomal abnormality involving the immunoglobulin heavy-chain (IGH) and CCAAT enhancer-binding protein (CEBPE) genes in B-lineage ALL (B-ALL) and may represent a new B-ALL subgroup. We report here the case of a 5-year-old girl with B-ALL, positive for CD19, CD38 and HLA-DR. A direct technique and G-banding were used for chromosomal analysis and fluorescentin situ hybridization (FISH) with BAC probes was used to investigate a possible rearrangement of the IGH andCEBPE genes. The karyotype exhibit the chromosomal aberration 46,XX,del(9)(p21),t(14;14)(q11;q32). FISH with dual-color break-apartIGH-specific and CEPBE-specific bacterial artificial chromosome (BAC) probes showed a complex t(9;14;14) associated with a deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A) and paired box gene 5 (PAX5) at 9p21-13 and duplication of the fusion gene IGH-CEBPE. PMID:27007892

  3. Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis.

    PubMed

    Zhao, Amy H; Tu, Lan N; Mukai, Chinatsu; Sirivelu, Madhu P; Pillai, Viju V; Morohaku, Kanako; Cohen, Roy; Selvaraj, Vimal

    2016-01-22

    Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo(-/-)) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

  4. Intracellular cholesterol changes induced by translocator protein (18 kDa) TSPO/PBR ligands.

    PubMed

    Falchi, Angela Maria; Battetta, Barbara; Sanna, Francesca; Piludu, Marco; Sogos, Valeria; Serra, Mariangela; Melis, Marta; Putzolu, Martina; Diaz, Giacomo

    2007-08-01

    One of the main functions of the translocator protein (18 kDa) or TSPO, previously known as peripheral-type benzodiazepine receptor, is the regulation of cholesterol import into mitochondria for steroid biosynthesis. In this paper we show that TSPO ligands induce changes in the distribution of intracellular cholesterol in astrocytes and fibroblasts. NBD-cholesterol, a fluorescent analog of cholesterol, was rapidly removed from membranes and accumulated into lipid droplets. This change was followed by a block of cholesterol esterification, but not by modification of intracellular cholesterol synthesis. NBD-cholesterol droplets were in part released in the medium, and increased cholesterol efflux was observed in [(3)H]cholesterol-prelabeled cells. TSPO ligands also induced a prominent shrinkage and depolarization of mitochondria and depletion of acidic vesicles with cytoplasmic acidification. Consistent with NBD-cholesterol changes, MTT assay showed enhanced accumulation of formazan into lipid droplets and inhibition of formazan exocytosis after treatment with TSPO ligands. The effects of specific TSPO ligands PK 11195 and Ro5-4864 were reproduced by diazepam, which binds with high affinity both TSPO and central benzodiazepine receptors, but not by clonazepam, which binds exclusively to GABA receptor, and other amphiphilic substances such as DIDS and propranolol. All these effects and the parallel immunocytochemical detection of TSPO in potentially steroidogenic cells (astrocytes) and non-steroidogenic cells (fibroblasts) suggest that TSPO is involved in the regulation and trafficking of intracellular cholesterol by means of mechanisms not necessarily related to steroid biosynthesis.

  5. TLR4 Signaling augments monocyte chemotaxis by regulating G protein-coupled receptor kinase 2 translocation.

    PubMed

    Liu, Zheng; Jiang, Yong; Li, Yuehua; Wang, Juan; Fan, Liyan; Scott, Melanie J; Xiao, Guozhi; Li, Song; Billiar, Timothy R; Wilson, Mark A; Fan, Jie

    2013-07-15

    Monocytes are critical effector cells of the innate immune system that protect the host by migrating to inflammatory sites, differentiating to macrophages and dendritic cells, eliciting immune responses, and killing pathogenic microbes. MCP-1, also known as CCL2, plays an important role in monocyte activation and migration. The chemotactic function of MCP-1 is mediated by binding to the CCR2 receptor, a member of the G protein-coupled receptor (GPCR) family. Desensitization of GPCR chemokine receptors is an important regulator of the intensity and duration of chemokine stimulation. GPCR kinases (GRKs) induce GPCR phosphorylation, and this leads to GPCR desensitization. Regulation of subcellular localization of GRKs is considered an important early regulatory mechanism of GRK function and subsequent GPCR desensitization. Chemokines and LPS are both present during Gram-negative bacterial infection, and LPS often synergistically exaggerates leukocyte migration in response to chemokines. In this study, we investigated the role and mechanism of LPS-TLR4 signaling on the regulation of monocyte chemotaxis. We demonstrate that LPS augments MCP-1-induced monocyte migration. We also show that LPS, through p38 MAPK signaling, induces phosphorylation of GRK2 at serine 670, which, in turn, suppresses GRK2 translocation to the membrane, thereby preventing GRK2-initiated internalization and desensitization of CCR2 in response to MCP-1. This results in enhanced monocyte migration. These findings reveal a novel function for TLR4 signaling in promoting innate immune cell migration.

  6. [Effects of sprinkler irrigation on the plant nitrogen accumulation and translocation and kernel protein content of winter wheat].

    PubMed

    Yao, Su-mei; Kang, Yue-hu; Ru, Zhen-gang; Liu, Ming-jiu; Yang, Wen-ping; Li, Gan

    2013-08-01

    Taking wheat cultivar Bainong AK58 as test material, a field experiment was conducted to study the plant nitrogen accumulation and translocation and kernel protein content of winter wheat under sprinkler irrigation and surface irrigation, aimed to understand the differences in the nitrogen metabolism characteristics of winter wheat under different irrigation regimes. At booting stage, no significant difference was observed in the total amount of plant nitrogen accumulation between sprinkler irrigation and surface irrigation; while from booting stage to maturing stage, the total amount of plant nitrogen accumulation under sprinkler irrigation was significantly higher. Under sprinkler irrigation, the translocation amount and contribution rate of the nitrogen stored in leaf, glume, stem and sheath at pre-anthesis to the kernel increased, while the contribution rate of the assimilated nitrogen after anthesis to the kernel nitrogen declined. Both the relative protein content and the total protein yield in the kernel increased significantly under sprinkler irrigation. In conclusion, sprinkler irrigation could significantly regulate the nitrogen translocation and kernel protein accumulation of winter wheat. PMID:24380339

  7. The Effect of Cigarette Smoke on the Translocator Protein (TSPO) in Cultured Lung Cancer Cells.

    PubMed

    Nagler, Rafael; Cohen, Shiri; Gavish, Moshe

    2015-12-01

    Lung cancer is prevalent in cigarette smokers. The mitochondrial membrane translocator protein (TSPO), is thought to protect cells from free radical damage. We examined the effect of cigarette smoke (CS) (containing free radicals) alone and in the presence of saliva (containing redox active free iron), on survival of H1299 lung cancer cells and on their mitochondrial characteristics, and whether TSPO binding was influenced by CS and by saliva. We exposed H1299 cells to CS in the presence/absence of saliva and also characterized TSPO binding in the cells using [3H]PK 11195 as a radioligand. CS induced a significant drop in mitochondrial potential (ΔΨm), while addition of saliva did not lead to further loss of ΔΨm (42.5% vs. 39.85%). Scatchard analysis of the saturation curve of [3H]PK 11195 binding (0.2-6 nM final concentration) yielded a straight-line plot (R =  0.9). Average Bmax value was 3274 ± 787 fmol/mg of protein, and average Kd value was 9.2 ± 1.3 nM. Benzodiazepine diazepam partially prevented decrease in cell survival following exposure to CS and redox active iron containing media (saliva) while benzodiazepine clonazepam did not, indicating that this effect is TSPO-specific. Exposure of cells to CS resulted in alternation of biomolecules expressed by CLs peroxidation, reduction of TSPO binding, and depletion of the mitochondrial potential. This irreversible damage was enhanced in the presence of saliva. All these modulations may result in cellular death increase following CS exposure, enhanced in the presence of saliva.

  8. Structural requirements to obtain highly potent and selective 18 kDa Translocator Protein (TSPO) Ligands.

    PubMed

    Taliani, Sabrina; Pugliesi, Isabella; Da Settimo, Federico

    2011-01-01

    The (18 kDa) Translocator Protein (TSPO), was initially identified in 1977 as peripheral binding site for the benzodiazepine diazepam and named "Peripheral-type benzodiazepine receptor (PBR)". It is an evolutionarily well-conserved protein particularly located at the outer/inner mitochondrial membrane contact sites, in closely association with the 32 kDa voltage-dependent anion channel (VDAC) and the 30 kDa adenine nucleotide translocase (ANT), thus forming the mitochondrial permeability transition pore (MPTP). TSPO is ubiquitary expressed in peripheral tissues (steroid producing tissues, liver, heart, kidney, lung, immune system) and in lower levels in the central nervous system, where it is mainly located in glial cells, and in neurons. TSPO is involved in a variety of biological processes such as cholesterol transport, steroidogenesis, calcium homeostasis, lipid metabolism, mitochondrial oxidation, cell growth and differentiation, apoptosis induction, and regulation of immune functions. In the last decade, many studies have reported that TSPO basal expression is altered in a number of human pathologies, such as cancer and neurodegenerative disorders (Huntington's and Alzheimer's diseases), as well as in various forms of brain injury and inflammation and anxiety. Consequently, TSPO has not only been suggested as a promising drug target for a number of therapeutic applications (anticonvulsant, anxiolytic, immunomodulating, etc.), but also as valid diagnostic marker for related-disease state and progression, prompting the development of specific labelled ligands as powerful tools for imaging techniques. A number of structurally different classes of ligands have been reported, showing high affinity and selectivity towards TSPO. Indeed, most of these ligands have been designed starting from selective CBR ligands which were structurally modified in order to shift their affinity towards TSPO. Extensive structure-activity relationship studies were performed allowing to

  9. Translocation of botulinum neurotoxin serotype a and associated proteins across the intestinal epithelia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins and considered to be a major venue of bioterrorist threat. BoNTs associate with neurotoxin associated proteins (NAPs), forming large complexes. NAPs have been shown to shield the BoNT holotoxin from the harsh environment of ...

  10. DNA translocation across protein channels: How does a polymer worm through a hole?

    NASA Astrophysics Data System (ADS)

    Muthukumar, M.

    2001-03-01

    Free energy barriers control the translocation of polymers through narrow channels. Based on an analogy with the classical nucleation and growth process, we have calculated the translocation time and its dependencies on the length, stiffness, and sequence of the polymer, solution conditions, and the strength of the driving electrochemical potential gradient. Our predictions will be compared with experimental results and prospects of reading polymer sequences.

  11. Response to Comment on "Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism".

    PubMed

    Li, Fei; Liu, Jian; Zheng, Yi; Garavito, R Michael; Ferguson-Miller, Shelagh

    2015-10-30

    Wang comments that the diffraction data for the structure of the A139T mutant of translocator protein TSPO from Rhodobacter sphaeroides should be used to 1.65 instead of 1.8 angstroms and that the density interpreted as porphyrin and monoolein is better fitted as polyethylene glycol. Although different practices of data processing exist, in this case they do not substantially influence the final map. Additional data are presented supporting the fit of a porphyrin and monooleins. PMID:26516277

  12. A single Sec61-complex functions as a protein-conducting channel.

    PubMed

    Kalies, Kai-Uwe; Stokes, Vivica; Hartmann, Enno

    2008-12-01

    During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61alpha-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.

  13. Protein import and the origin of red complex plastids.

    PubMed

    Gould, Sven B; Maier, Uwe-G; Martin, William F

    2015-06-15

    The number and nature of endosymbioses involving red algal endosymbionts are debated. Gene phylogenies have become the most popular tool to untangle this issue, but they deliver conflicting results. As gene and lineage sampling has increased, so have both the number of conflicting trees and the number of suggestions in the literature for multiple tertiary, and even quaternary, symbioses that might reconcile the tree conflicts. Independent lines of evidence that can address the issue are needed. Here we summarize the mechanism and machinery of protein import into complex red plastids. The process involves protein translocation machinery, known as SELMA, that arose once in evolution, that facilitates protein import across the second outermost of the four plastid membranes, and that is always targeted specifically to that membrane, regardless of where it is encoded today. It is widely accepted that the unity of protein import across the two membranes of primary plastids is strong evidence for their single cyanobacterial origin. Similarly, the unity of SELMA-dependent protein import across the second outermost plastid membrane constitutes strong evidence for the existence of a single red secondary endosymbiotic event at the common origin of all red complex plastids. We furthermore propose that the two outer membranes of red complex plastids are derived from host endoplasmic reticulum in the initial red secondary endosymbiotic event. PMID:26079086

  14. Protein import and the origin of red complex plastids.

    PubMed

    Gould, Sven B; Maier, Uwe-G; Martin, William F

    2015-06-15

    The number and nature of endosymbioses involving red algal endosymbionts are debated. Gene phylogenies have become the most popular tool to untangle this issue, but they deliver conflicting results. As gene and lineage sampling has increased, so have both the number of conflicting trees and the number of suggestions in the literature for multiple tertiary, and even quaternary, symbioses that might reconcile the tree conflicts. Independent lines of evidence that can address the issue are needed. Here we summarize the mechanism and machinery of protein import into complex red plastids. The process involves protein translocation machinery, known as SELMA, that arose once in evolution, that facilitates protein import across the second outermost of the four plastid membranes, and that is always targeted specifically to that membrane, regardless of where it is encoded today. It is widely accepted that the unity of protein import across the two membranes of primary plastids is strong evidence for their single cyanobacterial origin. Similarly, the unity of SELMA-dependent protein import across the second outermost plastid membrane constitutes strong evidence for the existence of a single red secondary endosymbiotic event at the common origin of all red complex plastids. We furthermore propose that the two outer membranes of red complex plastids are derived from host endoplasmic reticulum in the initial red secondary endosymbiotic event.

  15. Oridonin induces NPM mutant protein translocation and apoptosis in NPM1c+ acute myeloid leukemia cells in vitro

    PubMed Central

    Li, Fei-fei; Yi, Sha; Wen, Lu; He, Jing; Yang, Li-jing; Zhao, Jie; Zhang, Ben-ping; Cui, Guo-hui; Chen, Yan

    2014-01-01

    Aim: Skewed cytoplasmic accumulation of NPM mutant protein (NPM1c+) is close related to leukemia pathogenesis. The aim of this study was to investigate whether oridonin, a diterpenoid isolated from the Chinese traditional medicine Rabdosia rubescens, was able to interfere with NPM1c+ protein trafficking and induce apoptosis in NPM1c+ acute myeloid leukemia cells in vitro. Methods: OCI-AML3 cell line harboring a NPM1 gene mutation was examined. Cell growth was detected by MTT assay. Cell apoptosis was evaluated using flow cytometry and Hoechst 33258 staining. The expression and subcellular localization of relevant proteins were detected by Western blot and immunofluorescent staining. The mRNA expression was detected by RT-PCR. Results: Oridonin (2–12 μmol/L) dose-dependently inhibited the viability of OCI-AML3 cells (the IC50 value was 3.27±0.23 μmol/L at 24 h). Moreover, oridonin induced OCI-AML3 cell apoptosis accompanied by activation of caspase-3 and nuclear translocation of NPM1c+ protein. Oridonin did not change the expression of Crm1 (the export receptor for nuclear export signal-containing proteins), but induced nuclear translocation of Crm1. Oridonin markedly increased the expression of nucleoporin98 (Nup98), which had an important role in Crm1-mediated nuclear protein export, and induced nuclear accumulation of Nup98. Furthermore, oridonin markedly increased the expression of p14arf and p53. Conclusion: In NPM1c+ leukemia cells, oridonin induces NPM1c+ protein translocation into the nucleus possibly via nuclear accumulation of Crm1; the compound markedly increases p53 and p14arf expression, which may contribute to cell apoptosis. PMID:24902788

  16. Proteins Connecting the Nuclear Pore Complex with the Nuclear Interior

    PubMed Central

    Strambio-de-Castillia, Caterina; Blobel, Günter; Rout, Michael P.

    1999-01-01

    While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC. PMID:10085285

  17. Structure Prediction of Protein Complexes

    NASA Astrophysics Data System (ADS)

    Pierce, Brian; Weng, Zhiping

    Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.

  18. Translocator protein (Tspo) gene promoter-driven green fluorescent protein synthesis in transgenic mice: an in vivo model to study Tspo transcription

    PubMed Central

    Wang, Hui-Jie; Fan, Jinjiang; Papadopoulos, Vassilios

    2013-01-01

    Translocator protein (TSPO), previously known as the peripheral-type benzodiazepine receptor, is a ubiquitous drug- and cholesterol-binding protein primarily found in the outer mitochondrial membrane as part of a mitochondrial cholesterol transport complex. TSPO is present at higher levels in steroid-synthesizing and rapidly proliferating tissues, and its biological role has been mainly linked to mitochondrial function, steroidogenesis, and cell proliferation/apoptosis. Aberrant TSPO levels have been linked to multiple diseases, including cancer, endocrine disorders, brain injury, neurodegeneration, ischemia-reperfusion injury, and inflammatory diseases. Investigation of the functions of this protein in vitro and in vivo have been mainly carried out using high-affinity drug ligands, such as isoquinoline carboxamides and benzodiazepines, and more recently, gene silencing methods. To establish a model to study the regulation of Tspo transcription in vivo, we generated a transgenic mouse model expressing green fluorescent protein (GFP) from Aequorea coerulescens under control of the Tspo promoter region (Tspo-AcGFP). The expression profiles of Tspo-AcGFP, endogenous TSPO, and Tspo mRNA were found to be well correlated. Tspo-AcGFP synthesis in the transgenic mice was seen in almost every tissue examined, and as with TSPO in wild-type mice, Tspo-AcGFP was highly expressed in steroidogenic cells of the endocrine and reproductive systems, epithelial cells of the digestive system, skeletal muscle, and other organs. In summary, this transgenic Tspo-AcGFP mouse model recapitulates endogenous Tspo expression patterns and could be a useful, tractable tool for monitoring the transcriptional regulation and function of Tspo in live animal experiments. PMID:22868914

  19. Solid-state nanopore detection of protein complexes: applications in healthcare and protein kinetics.

    PubMed

    Freedman, Kevin J; Bastian, Arangassery R; Chaiken, Irwin; Kim, Min Jun

    2013-03-11

    Protein conjugation provides a unique look into many biological phenomena and has been used for decades for molecular recognition purposes. In this study, the use of solid-state nanopores for the detection of gp120-associated complexes are investigated. They exhibit monovalent and multivalent binding to anti-gp120 antibody monomer and dimers. In order to investigate the feasibility of many practical applications related to nanopores, detection of specific protein complexes is attempted within a heterogeneous protein sample, and the role of voltage on complexed proteins is researched. It is found that the electric field within the pore can result in unbinding of a freely translocating protein complex within the transient event durations measured experimentally. The strong dependence of the unbinding time with voltage can be used to improve the detection capability of the nanopore system by adding an additional level of specificity that can be probed. These data provide a strong framework for future protein-specific detection schemes, which are shown to be feasible in the realm of a 'real-world' sample and an automated multidimensional method of detecting events.

  20. The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum.

    PubMed

    Lipschutz, Joshua H; Lingappa, Vishwanath R; Mostov, Keith E

    2003-06-01

    We previously showed that the exocyst complex specifically affected the synthesis and delivery of secretory and basolateral plasma membrane proteins. Significantly, the entire spectrum of secreted proteins was increased when the hSec10 (human Sec10) component of the exocyst complex was overexpressed, suggestive of post-transcriptional regulation (Lipschutz, J. H., Guo, W., O'Brien, L. E., Nguyen, Y. H., Novick, P., and Mostov, K. E. (2000) Mol. Biol. Cell 11, 4259-4275). Here, using an exogenously transfected basolateral protein, the polymeric immunoglobulin receptor (pIgR), and a secretory protein, gp80, we show that pIgR and gp80 protein synthesis and delivery are increased in cells overexpressing Sec10 despite the fact that mRNA levels are unchanged, which is highly indicative of post-transcriptional regulation. To test specificity, we also examined the synthesis and delivery of an exogenous apical protein, CNT1 (concentrative nucleoside transporter 1), and found no increase in CNT1 protein synthesis, delivery, or mRNA levels in cells overexpressing Sec10. Sec10-GFP-overexpressing cell lines were created, and staining was seen in the endoplasmic reticulum. It was demonstrated previously in yeast that high levels of expression of SEB1, the Sec61beta homologue, suppressed sec15-1, an exocyst mutant (Toikkanen, J., Gatti, E., Takei, K., Saloheimo, M., Olkkonen, V. M., Soderlund, H., De Camilli, P., and Keranen, S. (1996) Yeast 12, 425-438). Sec61beta is a member of the Sec61 heterotrimer, which is the main component of the endoplasmic reticulum translocon. By co-immunoprecipitation we show that Sec10, which forms an exocyst subcomplex with Sec15, specifically associates with the Sec61beta component of the translocon and that Sec10 overexpression increases the association of other exocyst complex members with Sec61beta. Proteosome inhibition does not appear to be the mechanism by which increased protein synthesis occurs in the face of equivalent amounts of m

  1. Reciprocal translocations

    SciTech Connect

    1993-12-31

    Chapter 26, describes reciprocal translocations of chromosomes: their occurrence, breakpoints, and multiple rearrangements. In addition, phenotypes of balanced and unbalanced translocation carriers and fetal death are discussed. Examples of translocation families are given. Meiosis and genetic risk in translocation carriers is presented. Finally, sperm chromosomes in meiotic segregation analysis is mentioned. 39 refs., 3 figs., 1 tab.

  2. Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.

    PubMed

    Bumba, Ladislav; Masin, Jiri; Macek, Pavel; Wald, Tomas; Motlova, Lucia; Bibova, Ilona; Klimova, Nela; Bednarova, Lucie; Veverka, Vaclav; Kachala, Michael; Svergun, Dmitri I; Barinka, Cyril; Sebo, Peter

    2016-04-01

    Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.

  3. Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.

    PubMed

    Bumba, Ladislav; Masin, Jiri; Macek, Pavel; Wald, Tomas; Motlova, Lucia; Bibova, Ilona; Klimova, Nela; Bednarova, Lucie; Veverka, Vaclav; Kachala, Michael; Svergun, Dmitri I; Barinka, Cyril; Sebo, Peter

    2016-04-01

    Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens. PMID:27058787

  4. Complexation of Arsenite with Phytochelatins Reduces Arsenite Efflux and Translocation from Roots to Shoots in Arabidopsis1[W

    PubMed Central

    Liu, Wen-Ju; Wood, B. Alan; Raab, Andrea; McGrath, Steve P.; Zhao, Fang-Jie; Feldmann, Jörg

    2010-01-01

    Complexation of arsenite [As(III)] with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray ionization-mass spectrometry coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis (Arabidopsis thaliana) exposed to arsenate [As(V)]. As(V) was efficiently reduced to As(III) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC4, As(III)-PC3, and As(III)-(PC2)2. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to As(V) than the wild type. In cad1-3 roots, only 8% of As was complexed with GSH as As(III)-(GS)3 and no As(III)-PCs were detected, while in cad2-1 roots, As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 to 12 times higher shoot-to-root As concentration ratio than the wild type. Roots also effluxed a substantial proportion of the As(V) taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to l-buthionine sulfoximine or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops. PMID:20130102

  5. The planar cell polarity (PCP) protein Diversin translocates to the nucleus to interact with the transcription factor AF9

    SciTech Connect

    Haribaskar, Ramachandran; Puetz, Michael; Schupp, Birte; Skouloudaki, Kassiani; Bietenbeck, Andreas; Walz, Gerd; Schaefer, Tobias

    2009-09-11

    The planar cell polarity (PCP) pathway, a {beta}-catenin-independent branch of the Wnt signaling pathway, orients cells and their appendages with respect to the body axes. Diversin, the mammalian homolog of the Drosophila PCP protein Diego, acts as a molecular switch that blocks {beta}-catenin-dependent and promotes {beta}-catenin-independent Wnt signaling. We report now that Diversin, containing several nuclear localization signals, translocates to the nucleus, where it interacts with the transcription factor AF9. Both Diversin and AF9 block canonical Wnt signaling; however, this occurs independently of each other, and does not require nuclear Diversin. In contrast, AF9 strongly augments the Diversin-driven activation of c-Jun N-terminal kinase (JNK)-dependent gene expression in the nucleus, and this augmentation largely depends on the presence of nuclear Diversin. Thus, our findings reveal that components of the PCP cascade translocate to the nucleus to participate in transcriptional regulation and PCP signaling.

  6. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  7. Investigation of a protein complex network

    NASA Astrophysics Data System (ADS)

    Mashaghi, A. R.; Ramezanpour, A.; Karimipour, V.

    2004-09-01

    The budding yeast Saccharomyces cerevisiae is the first eukaryote whose genome has been completely sequenced. It is also the first eukaryotic cell whose proteome (the set of all proteins) and interactome (the network of all mutual interactions between proteins) has been analyzed. In this paper we study the structure of the yeast protein complex network in which weighted edges between complexes represent the number of shared proteins. It is found that the network of protein complexes is a small world network with scale free behavior for many of its distributions. However we find that there are no strong correlations between the weights and degrees of neighboring complexes. To reveal non-random features of the network we also compare it with a null model in which the complexes randomly select their proteins. Finally we propose a simple evolutionary model based on duplication and divergence of proteins.

  8. Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus

    PubMed Central

    Woolston, Benjamin M.; Schlagnhaufer, Carl; Wilkinson, Jack; Larsen, Jeffrey; Shi, Zhixin; Mayer, Kimberly M.; Walters, Donald S.; Curtis, Wayne R.; Romaine, C. Peter

    2011-01-01

    Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long

  9. Syndromal frontonasal dysostosis in a child with a complex translocation involving chromosomes 3, 7, and 11

    SciTech Connect

    Stevens, C.A.; Qumsiyeh, M.B. |

    1995-02-13

    We report on a 4-year-old boy with typical frontonasal dysostosis and an apparently balanced de novo translocation involving chromosomes 3, 7, and 11, and four breakpoints. The karyotype was 46,XY,t(7;3)(3;11) (7pter{r_arrow}7q21.3::3q27{r_arrow}3qter;3pter{r_arrow}3q23::11q21{r_arrow}11qter;11pter{r_arrow}11q21::3q23{r_arrow}3q27::7q21.3{r_arrow}7qter). In situ hybridization with a chromosome 3 painting probe confirmed the interpretation from GTG banding. The child had a widow`s peak, marked hypertelorism, absence of the nasal tip, and widely separated nares. He also had an atrial septal defect, micropenis, small testes, clubfeet, scoliosis, block C2-4, and structural brain abnormalities on MRI. In review we found two other cases of frontonasal dysostosis with chromosome abnormalities, neither of which was similar to our case. The presence of a de novo (apparently) balanced translocation in our patient may help to locate the gene(s) for frontonasal dysplasia and perhaps other midline craniofacial malformations. 16 refs., 4 figs.

  10. Structural Studies of Protein-Surfactant Complexes

    SciTech Connect

    Chodankar, S. N.; Aswal, V. K.; Wagh, A. G.

    2008-03-17

    The structure of protein-surfactant complexes of two proteins bovine serum albumin (BSA) and lysozyme in presence of anionic surfactant sodium dodecyl sulfate (SDS) has been studied using small-angle neutron scattering (SANS). It is observed that these two proteins form different complex structures with the surfactant. While BSA protein undergoes unfolding on addition of surfactant, lysozyme does not show any unfolding even up to very high surfactant concentrations. The unfolding of BSA protein is caused by micelle-like aggregation of surfactant molecules in the complex. On the other hand, for lysozyme protein there is only binding of individual surfactant molecules to protein. Lysozyme in presence of higher surfactant concentrations has protein-surfactant complex structure coexisting with pure surfactant micelles.

  11. Crystal structure of the effector AvrLm4-7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins.

    PubMed

    Blondeau, Karine; Blaise, Françoise; Graille, Marc; Kale, Shiv D; Linglin, Juliette; Ollivier, Bénédicte; Labarde, Audrey; Lazar, Noureddine; Daverdin, Guillaume; Balesdent, Marie-Hélène; Choi, Danielle H Y; Tyler, Brett M; Rouxel, Thierry; van Tilbeurgh, Herman; Fudal, Isabelle

    2015-08-01

    The avirulence gene AvrLm4-7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4-7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4-7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well-conserved among AvrLm4-7 homologs. Loss of recognition of AvrLm4-7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well-conserved C-terminal motif or close to the glycine involved in Rlm4-mediated recognition, resulting in the loss of Rlm7-mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4-7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4-7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region. PMID:26082394

  12. Nuclear translocation of p21(WAF1/CIP1) protein prior to its cytosolic degradation by UV enhances DNA repair and survival.

    PubMed

    Lee, Ji Young; Kim, Hee Suk; Kim, Joo Young; Sohn, Jeongwon

    2009-12-25

    We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis. These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.

  13. Nuclear translocation of p21{sup WAF1/CIP1} protein prior to its cytosolic degradation by UV enhances DNA repair and survival

    SciTech Connect

    Lee, Ji Young; Kim, Hee Suk; Kim, Joo Young; Sohn, Jeongwon

    2009-12-25

    We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis. These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.

  14. FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex

    PubMed Central

    Fournes, Florian; Crozat, Estelles; Pages, Carine; Tardin, Catherine; Salomé, Laurence; Cornet, François

    2016-01-01

    In bacteria, the FtsK/Xer/dif (chromosome dimer resolution site) system is essential for faithful vertical genetic transmission, ensuring the resolution of chromosome dimers during their segregation to daughter cells. This system is also targeted by mobile genetic elements that integrate into chromosomal dif sites. A central question is thus how Xer/dif recombination is tuned to both act in chromosome segregation and stably maintain mobile elements. To explore this question, we focused on pathogenic Neisseria species harboring a genomic island in their dif sites. We show that the FtsK DNA translocase acts differentially at the recombination sites flanking the genomic island. It stops at one Xer/dif complex, activating recombination, but it does not stop on the other site, thus dismantling it. FtsK translocation thus permits cis discrimination between an endogenous and an imported Xer/dif recombination complex. PMID:27317749

  15. Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

    PubMed Central

    Sommer, Maik S.; Schleiff, Enrico

    2014-01-01

    With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity. PMID:25085907

  16. A small molecule inhibitor of redox-regulated protein translocation into mitochondria.

    PubMed

    Dabir, Deepa V; Hasson, Samuel A; Setoguchi, Kiyoko; Johnson, Meghan E; Wongkongkathep, Piriya; Douglas, Colin J; Zimmerman, Johannes; Damoiseaux, Robert; Teitell, Michael A; Koehler, Carla M

    2013-04-15

    The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small translocase of the inner membrane (Tim) proteins and cysteine-rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, mitochondrial protein import blockers from the Carla Koehler laboratory (MitoBloCK-6), attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the translocase of the outer membrane complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6-exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis.

  17. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  18. Co-translational assembly of protein complexes.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2015-12-01

    The interaction of biological macromolecules is a fundamental attribute of cellular life. Proteins, in particular, often form stable complexes with one another. Although the importance of protein complexes is widely recognized, we still have only a very limited understanding of the mechanisms underlying their assembly within cells. In this article, we review the available evidence for one such mechanism, namely the coupling of protein complex assembly to translation at the polysome. We discuss research showing that co-translational assembly can occur in both prokaryotic and eukaryotic organisms and can have important implications for the correct functioning of the complexes that result. Co-translational assembly can occur for both homomeric and heteromeric protein complexes and for both proteins that are translated directly into the cytoplasm and those that are translated into or across membranes. Finally, we discuss the properties of proteins that are most likely to be associated with co-translational assembly.

  19. Carbenoxolone induces permeability transition pore opening in rat mitochondria via the translocator protein TSPO and connexin43.

    PubMed

    Azarashvili, Tamara; Baburina, Yulia; Grachev, Dmitry; Krestinina, Olga; Papadopoulos, Vassilios; Lemasters, John J; Odinokova, Irina; Reiser, Georg

    2014-09-15

    Ca(2+)-induced permeability transition pore (mPTP) opening in isolated rat brain mitochondria is promoted through targeting of connexin43. After a threshold Ca(2+) load, mitochondrial membrane potential drops and efflux of accumulated Ca(2+) from the mitochondrial matrix occurs, indicating the mPTP opening. Specific antibodies were used to assess the role of the translocator protein (18kDa; TSPO) and connexin43 in swelling of isolated rat liver and brain mitochondria induced by carbenoxolone and the endogenous TSPO ligand protoporphyrin IX. Mitochondrial membrane potential, Ca(2+) transport and oxygen consumption were determined using selective electrodes. All the parameters were detected simultaneously in a chamber with the selective electrodes. The phosphorylation state of mitochondrial protein targets was assessed. We report that Ca(2+)-induced mitochondrial swelling was strengthened in the presence of both carbenoxolone and protoporphyrin IX. The carbenoxolone- and protoporphyrin IX-accelerated mPTP induction in brain mitochondria was completely prevented by antibodies specific for the mitochondrial translocator protein (TSPO). The anti-TSPO antibodies were more effective than anti-сonnexin43 antibodies. Moreover, carbenoxolone-stimulated phosphorylation of mitochondrial proteins was inhibited by anti-TSPO antibodies. Taken together, the data suggests that, in addition to acting via connexion43, carbenoxolone may exert its effect on mPTP via mitochondrial outer membrane TSPO.

  20. Complex, compound inversion/translocation polymorphism in an ape: presumptive intermediate stage in the karyotypic evolution of the agile gibbon Hylobates agilis.

    PubMed

    Van Tuinen, P; Mootnick, A R; Kingswood, S C; Hale, D W; Kumamoto, A T

    1999-10-01

    Karyotypic variation in five gibbon species of the subgenus Hylobates (2n = 44) was assessed in 63 animals, 23 of them wild born. Acquisition of key specimens of Hylobates agilis (agile gibbon), whose karyotype had been problematic due to unresolved structural polymorphisms, led to disclosure of a compound inversion/translocation polymorphism. A polymorphic region of chromosome 8 harboring two pericentric inversions, one nested within the other, was in turn bissected by one breakpoint of a reciprocal translocation. In double-inversion + translocation heterozygotes, the theoretical meiotic pairing configuration is a double inversion loop, with four arms of a translocation quadrivalent radiating from the loop. Electron-microscopic analysis of synaptonemal complex configurations consistently revealed translocation quadrivalents but no inversion loops. Rather, nonhomologous pairing was evident in the inverted region, a condition that should preclude crossing over and the subsequent production of duplication-deficiency gametes. This is corroborated by the existence of normal offspring of compound heterozygotes, indicating that fertility may not be reduced despite the topological complexity of this polymorphic system. The distribution of inversion and translocation morphs in these taxa suggests application of cytogenetics in identifying gibbon specimens and avoiding undesirable hybridization in captive breeding efforts.

  1. The kinetics of translocation and cellular quantity of protein kinase C in human leukocytes are modified during spaceflight

    NASA Technical Reports Server (NTRS)

    Hatton, J. P.; Gaubert, F.; Lewis, M. L.; Darsel, Y.; Ohlmann, P.; Cazenave, J. P.; Schmitt, D.

    1999-01-01

    Protein kinase C (PKC) is a family of serine/threonine kinases that play an important role in mediating intracellular signal transduction in eukaryotes. U937 cells were exposed to microgravity during a space shuttle flight and stimulated with a radiolabeled phorbol ester ([3H]PDBu) to both specifically label and activate translocation of PKC from the cytosol to the particulate fraction of the cell. Although significant translocation of PKC occurred at all g levels, the kinetics of translocation in flight were significantly different from those on the ground. In addition, the total quantity of [3H]PDBu binding PKC was increased in flight compared to cells at 1 g on the ground, whereas the quantity in hypergravity (1.4 g) was decreased with respect to 1 g. Similarly, in purified human peripheral blood T cells the quantity of PKCdelta varied in inverse proportion to the g level for some experimental treatments. In addition to these novel findings, the results confirm earlier studies which showed that PKC is sensitive to changes in gravitational acceleration. The mechanisms of cellular gravisensitivity are poorly understood but the demonstrated sensitivity of PKC to this stimulus provides us with a useful means of measuring the effect of altered gravity levels on early cell activation events.

  2. The Bacterial Twin-Arginine Translocation Pathway

    PubMed Central

    Lee, Philip A.; Tullman-Ercek, Danielle; Georgiou, George

    2009-01-01

    The twin-arginine translocation (Tat) pathway is responsible for the export of folded proteins across the cytoplasmic membrane of bacteria. Substrates for the Tat pathway include redox enzymes requiring cofactor insertion in the cytoplasm, multimeric proteins that have to assemble into a complex prior to export, certain membrane proteins, and proteins whose folding is incompatible with Sec export. These proteins are involved in a diverse range of cellular activities including anaerobic metabolism, cell envelope biogenesis, metal acquisition and detoxification, and virulence. The Escherichia coli translocase consists of the TatA, TatB, and TatC proteins, but little is known about the precise sequence of events that leads to protein translocation, the energetic requirements, or the mechanism that prevents the export of misfolded proteins. Owing to the unique characteristics of the pathway, it holds promise for biotechnological applications. PMID:16756481

  3. Identification of Streptomyces lividans proteins secreted by the twin-arginine translocation pathway following growth with different carbon sources.

    PubMed

    Guimond, Julien; Morosoli, Rolf

    2008-07-01

    Genome-based signal peptide predictions classified Streptomyces coelicolor as the microorganism that secretes the most proteins through the twin-arginine translocation (Tat)-dependent secretion pathway. Availability of a DeltatatC mutant of the closely related strain Streptomyces lividans impaired Tat-dependent protein secretion and enabled identification of many extracellular proteins that are secreted via the Tat pathway. Proteomic techniques were applied to analyze proteins from the supernatants of log-phase cultures. Since the bacterial secretome depends mainly on the carbon sources available during growth, xylose, glucose, chitin, and soil extracts were used. A total of 63 proteins were identified, among which 7 were predicted by the TATscan program, and 20 were not predicted but contained a potential Tat signal motif. Thirteen proteins having no signal sequence could be co-transported by Tat-dependent proteins because the genes that encode these proteins are in close proximity in the genome. Finally, the presence of 23 proteins lacking signal peptides was difficult to explain. More secreted proteins could be identified as Tat substrates in varying carbon sources.

  4. The Salmonella enterica serovar typhimurium-encoded type III secretion systems can translocate Chlamydia trachomatis proteins into the cytosol of host cells.

    PubMed

    Ho, Theresa D; Starnbach, Michael N

    2005-02-01

    Chlamydia trachomatis is an obligate, intracellular pathogen that is a major cause of preventable blindness and infertility worldwide. Although the published genome sequence suggests that C. trachomatis encodes a type III secretion system, the lack of genetic tools for studying Chlamydia has hindered the examination of this potentially important class of virulence genes. We have developed a technique to identify Chlamydia proteins that can be translocated into the host cell cytoplasm by a type III secretion system. We have selected several Chlamydia proteins and tagged them with a multiple peptide motif element called F8M4. Epitopes contained in the F8M4 tag allow us to use tools corresponding to different arms of the adaptive immune system to detect the expression and translocation of these proteins by Salmonella enterica serovar Typhimurium. In particular, CD8(+)-T-cell reactivity can be used to detect the translocation of F8M4-tagged proteins into the cytoplasm of host cells. We have found that CD8(+)-T-cell activity assays are sensitive enough to detect translocation of even a small amount of F8M4-tagged protein. We have used CD8(+)-T-cell activity to show that CopN, a Chlamydia protein previously shown to be translocated by Yersinia type III secretion, can be translocated by the Salmonella pathogenicity island 1 (SPI-1) type III secretion system. Additionally, we demonstrate that CopD and Pkn5, two Chlamydia proteins hypothesized to be substrates of a type III secretion system, are translocated via the SPI-2 type III secretion system of serovar Typhimurium. The epitope tag system described here can be used more generally to examine the expression and subcellular compartmentalization of bacterial proteins deployed during the interaction of pathogens with mammalian cells.

  5. Complex Reconstitution from Individual Protein Modules.

    PubMed

    Basquin, Jérôme; Taschner, Michael; Lorentzen, Esben

    2016-01-01

    Cellular function relies on protein complexes that work as nano-machines. The structure and function of protein complexes is an outcome of the specific combination of protein subunits, or modules, within the complex. A major focus of molecular biology is thus to understand how protein subunits assemble to form complexes with distinct biological function. To this end, in vitro reconstitution of complexes from individual subunits to study their assembly, structure and activity is of central importance. With purified individual subunits and sub-modules at hand one can systematically dissect the hierarchical assembly of larger complexes using direct protein-protein interaction assays. Furthermore, activity assays can be carried out with individual subunits or smaller sub-complexes and compared to those of the fully assembled complex to precisely map functional sites and provide a molecular basis for in vivo observations. In this chapter we review methods for protein complex assembly from individual subunits and provide examples of advantages and potential pitfalls to this approach. PMID:27165333

  6. Phylogenomic Analysis and Predicted Physiological Role of the Proton-Translocating NADH:Quinone Oxidoreductase (Complex I) Across Bacteria

    PubMed Central

    Spero, Melanie A.; Aylward, Frank O.; Currie, Cameron R.

    2015-01-01

    ABSTRACT The proton-translocating NADH:quinone oxidoreductase (complex I) is a multisubunit integral membrane enzyme found in the respiratory chains of both bacteria and eukaryotic organelles. Although much research has focused on the enzyme’s central role in the mitochondrial respiratory chain, comparatively little is known about its role in the diverse energetic lifestyles of different bacteria. Here, we used a phylogenomic approach to better understand the distribution of complex I across bacteria, the evolution of this enzyme, and its potential roles in shaping the physiology of different bacterial groups. By surveying 970 representative bacterial genomes, we predict complex I to be present in ~50% of bacteria. While this includes bacteria with a wide range of energetic schemes, the presence of complex I is associated with specific lifestyles, including aerobic respiration and specific types of phototrophy (bacteria with only a type II reaction center). A phylogeny of bacterial complex I revealed five main clades of enzymes whose evolution is largely congruent with the evolution of the bacterial groups that encode complex I. A notable exception includes the gammaproteobacteria, whose members encode one of two distantly related complex I enzymes predicted to participate in different types of respiratory chains (aerobic versus anaerobic). Comparative genomic analyses suggest a broad role for complex I in reoxidizing NADH produced from various catabolic reactions, including the tricarboxylic acid (TCA) cycle and fatty acid beta-oxidation. Together, these findings suggest diverse roles for complex I across bacteria and highlight the importance of this enzyme in shaping diverse physiologies across the bacterial domain. PMID:25873378

  7. Ontology integration to identify protein complex in protein interaction networks

    PubMed Central

    2011-01-01

    Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO) annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches. PMID:22165991

  8. Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. Studies with chemical modifiers of amino acid residues.

    PubMed

    Lorusso, M; Gatti, D; Boffoli, D; Bellomo, E; Papa, S

    1983-12-15

    Possible involvement of polypeptides of b-c1 complex of beef-heart mitochondria in its redox and protonmotive activity has been investigated, by means of chemical modification of amino acid residues in the soluble as well as in the phospholipid-reconstituted b-c1 complex. Treatment of the enzyme with tetranitromethane (C(NO2)4) or with ethoxyformic anhydride (EFA), that modify reversibly tyrosyl and hystidyl residues respectively, resulted in a marked inhibition of electron transport from reduced quinols to cytochrome c. This was accompanied, in b-c1 reconstituted into phospholipid vesicles, by a parallel inhibition of respiratory-linked proton translocation; the H+/e- stoichiometry remained unchanged. Treatment of b-c1 complex with DCCD, that specifically modifies carboxylic groups of glutammic or aspartic residues caused a marked depression of proton translocation in b-c1 vesicles, under conditions where the rate of electron flow in the coupled state, was enhanced. As a consequence the H+/e- stoichiometry was lowered. SDS gel electrophoresis and [14C]DCCD-labelling of the polypeptides of the b-c1 complex showed a major binding of 14C-DCCD to the 8-kDa subunit of the complex and possible cross-linking, induced by DCCD treatment, of polypeptide(s) in the 8-kDa band and the 12-kDa band, with the Fe-s protein of the complex, with the appearance of a new polypeptide band with an apparent molecular mass of about 40 kDa. Involvement of polypeptides of low molecular mass, for which no functional role was so far described, and possibly of the Fe-S protein in the redox-linked proton translocation in b-c1 complex is suggested.

  9. 3D complex: a structural classification of protein complexes.

    PubMed

    Levy, Emmanuel D; Pereira-Leal, Jose B; Chothia, Cyrus; Teichmann, Sarah A

    2006-11-17

    Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.

  10. Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

    PubMed Central

    Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Radford, Sheena E; Tuma, Roman; Collinson, Ian

    2016-01-01

    The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids. DOI: http://dx.doi.org/10.7554/eLife.15598.001 PMID:27183269

  11. Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.

    PubMed

    Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Baldwin, Steve A; Radford, Sheena E; Tuma, Roman; Collinson, Ian

    2016-05-16

    The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.

  12. Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein.

    PubMed

    Stocki, Pawel; Sawicki, Maxime; Mays, Charles E; Hong, Seo Jung; Chapman, Daniel C; Westaway, David; Williams, David B

    2016-03-01

    Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrP(C)) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrP(C) expression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrP(C) expression. We show that FK506 treatment results in a profound reduction in PrP(C) expression due to a defect in the translocation of PrP(C) into the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrP(C) signal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrP(C). However, this occurred at a later stage, after translocation of PrP(C) into the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrP(Sc) propagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrP(C) biogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases. PMID:26764098

  13. Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein

    PubMed Central

    Stocki, Pawel; Sawicki, Maxime; Mays, Charles E.; Hong, Seo Jung; Chapman, Daniel C.; Westaway, David; Williams, David B.

    2016-01-01

    Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrPC) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrPC expression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrPC expression. We show that FK506 treatment results in a profound reduction in PrPC expression due to a defect in the translocation of PrPC into the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrPC signal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrPC. However, this occurred at a later stage, after translocation of PrPC into the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrPSc propagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrPC biogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases. PMID:26764098

  14. Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport.

    PubMed

    Cleland, P J; Appleby, G J; Rattigan, S; Clark, M G

    1989-10-25

    Contraction-induced translocation of protein kinase C (Richter E.A., Cleland, P.J.F., Rattigan, S., and Clark, M.G. (1987) FEBS Lett. 217, 232-236) implies a role for this enzyme in muscle contraction or the associated metabolic adjustments. In the present study, this role is further examined particularly in relation to changes in glucose transport. Electrical stimulation of the sciatic nerve of the anesthetized rat in vivo led to a time-dependent translocation of protein kinase C and a 2-fold increase in the concentrations of both diacylglycerol and phosphatidic acid. Maximum values for the latter were reached at 2 min and preceded the maximum translocation of protein kinase C (10 min). Stimulation of muscles in vitro increased the rate of glucose transport, but this required 20 min to reach maximum. There was no reversal of translocation or decrease in the concentrations of diacylglycerol and phosphatidic acid even after 30 min of rest following a 5-min period of stimulation in vivo. Translocation was not influenced by variations in applied load at maximal fiber recruitment but was dependent on the frequency of nontetanic stimuli, reaching a maximum at 4 Hz. The relationship between protein kinase C and glucose transport was also explored by varying the number of tetanic stimuli. Whereas only one train of stimuli (200 ms, 100 Hz) was required for maximal effects on protein kinase C, diacylglycerol, and phosphatidic acid, more than 35 trains of stimuli were required to activate glucose transport. It is concluded that the production of diacylglycerol and the translocation of protein kinase C may be causally related. However, if the translocated protein kinase C is involved in the activation of glucose transport during muscle contractions, an accumulated exposure to Ca2+, resulting from multiple contractions, would appear to be necessary.

  15. A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells.

    PubMed

    Nagai, Hiroki; Cambronne, Eric D; Kagan, Jonathan C; Amor, Juan Carlos; Kahn, Richard A; Roy, Craig R

    2005-01-18

    The Legionella pneumophila Dot/Icm system is a type IV secretion apparatus that transfers bacterial proteins into eukaryotic host cells. The RalF protein is a substrate engaged and translocated into host cells by the Dot/Icm system. In this study, the mechanism of Dot/Icm-mediated translocation of RalF has been investigated. It was determined that RalF translocation into host cells occurs before bacterial internalization. Sequences essential for RalF translocation were located at the C terminus of the RalF protein. A fusion protein consisting of a 20-aa C-terminal RalF peptide appended to the calmodulin-dependent adenylate cyclase domain of the Bordetella pertussis adenylate cyclase protein was translocated into host cells by the Dot/Icm system. A leucine (L372) residue at the -3 position in relation to the RalF C terminus was critical for translocation. Consistent with RalF L372 playing an important role in substrate recognition by the Dot/Icm system, most other Dot/Icm substrates were found to have amino acid residues with similar physical properties at their -3 or -4 C-terminal positions. These data demonstrate that the Dot/Icm system can transfer bacterial proteins that modulate host cellular functions before uptake and indicate that substrate recognition involves a C-terminal translocation signal. Thus, Legionella has the ability to engage synthesized substrate proteins and transfer them into host cells on contact, enabling Legionella to rapidly alter transport of the vacuole in which it resides. PMID:15613486

  16. Endoproteolytic Cleavage of TUG Protein Regulates GLUT4 Glucose Transporter Translocation*

    PubMed Central

    Bogan, Jonathan S.; Rubin, Bradley R.; Yu, Chenfei; Löffler, Michael G.; Orme, Charisse M.; Belman, Jonathan P.; McNally, Leah J.; Hao, Mingming; Cresswell, James A.

    2012-01-01

    To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake. PMID:22610098

  17. Electrochemical impedance spectroscopy for graphene surface modification and protein translocation through the chemically modified graphene nanopore

    NASA Astrophysics Data System (ADS)

    Tiwari, Purushottam; Shan, Yuping; Wang, Xuewen; Darici, Yesim; He, Jin

    2014-03-01

    The multilayer graphene surface has been modified using mercaptohexadecanoic acid (MHA) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (DPPE-PEG750). The surface modifications are evaluated using electrochemical impedance spectroscopy (EIS). EIS measurements show the better graphene surface passivation with DPPE-PEG750 than with MHA. After modification with ferritin, the MHA modified surface shows greater charge transfer resistance (Rct) change than DPPE-PEG750 modified surface. Based on these results the translocations of ferritin through modified graphene nanopore with diameter 5-20 nm are studied. The translocation is more successful through DPPE-PEG750 modified graphene nanopore. This concludes that that the attachment of ferritin to DPPE-PEG750 modified graphene nanopore is not significant compared to MHA modified pore for the ferritin translocation hindrance. These results nicely correlate with the EIS data for respective Rct change of ferritin modified surfaces. P. Tiwari would like to thank FIU School of Integrated Science & Humanity, College Arts & Sciences for the research assistantship.

  18. AIRE-induced apoptosis is associated with nuclear translocation of stress sensor protein GAPDH

    SciTech Connect

    Liiv, Ingrid; Haljasorg, Uku; Kisand, Kai; Maslovskaja, Julia; Laan, Martti; Peterson, Paert

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer AIRE induces apoptosis in epithelial cells. Black-Right-Pointing-Pointer CARD domain of AIRE is sufficient for apoptosis induction. Black-Right-Pointing-Pointer AIRE induced apoptosis involves GAPDH translocation to the nuclei. Black-Right-Pointing-Pointer Deprenyl inhibits AIRE induced apoptosis. -- Abstract: AIRE (Autoimmune Regulator) has a central role in the transcriptional regulation of self-antigens in medullary thymic epithelial cells, which is necessary for negative selection of autoreactive T cells. Recent data have shown that AIRE can also induce apoptosis, which may be linked to cross-presentation of these self-antigens. Here we studied AIRE-induced apoptosis using AIRE over-expression in a thymic epithelial cell line as well as doxycycline-inducible HEK293 cells. We show that the HSR/CARD domain in AIRE together with a nuclear localization signal is sufficient to induce apoptosis. In the nuclei of AIRE-positive cells, we also found an increased accumulation of a glycolytic enzyme, glyceraldehyde-3-phosphate (GAPDH) reflecting cellular stress and apoptosis. Additionally, AIRE-induced apoptosis was inhibited with an anti-apoptotic agent deprenyl that blocks GAPDH nitrosylation and nuclear translocation. We propose that the AIRE-induced apoptosis pathway is associated with GAPDH nuclear translocation and induction of NO-induced cellular stress in AIRE-expressing cells.

  19. Arabidopsis ABCG14 protein controls the acropetal translocation of root-synthesized cytokinins

    NASA Astrophysics Data System (ADS)

    Zhang, Kewei; Novak, Ondrej; Wei, Zhaoyang; Gou, Mingyue; Zhang, Xuebin; Yu, Yong; Yang, Huijun; Cai, Yuanheng; Strnad, Miroslav; Liu, Chang-Jun

    2014-02-01

    Cytokinins are a major group of phytohormones regulating plant growth, development and stress responses. However, in contrast to the well-defined polar transport of auxins, the molecular basis of cytokinin transport is poorly understood. Here we show that an ATP-binding cassette transporter in Arabidopsis, AtABCG14, is essential for the acropetal (root to shoot) translocation of the root-synthesized cytokinins. AtABCG14 is expressed primarily in the pericycle and stelar cells of roots. Knocking out AtABCG14 strongly impairs the translocation of trans-zeatin (tZ)-type cytokinins from roots to shoots, thereby affecting the plant’s growth and development. AtABCG14 localizes to the plasma membrane of transformed cells. In planta feeding of C14 or C13-labelled tZ suggests that it acts as an efflux pump and its presence in the cells directly correlates with the transport of the fed cytokinin. Therefore, AtABCG14 is a transporter likely involved in the long-distance translocation of cytokinins in planta.

  20. Protein complexes that control renal epithelial polarity

    PubMed Central

    Pieczynski, Jay

    2011-01-01

    Establishment of epithelial apicobasal polarity is crucial for proper kidney development and function. In recent years, there have been important advances in our understanding of the factors that mediate the initiation of apicobasal polarization. Key among these are the polarity complexes that are evolutionarily conserved from simple organisms to humans. Three of these complexes are discussed in this review: the Crumbs complex, the Par complex, and the Scribble complex. The apical Crumbs complex consists of three proteins, Crumbs, PALS1, and PATJ, whereas the apical Par complex consists of Par-3, Par-6, and atypical protein kinase C. The lateral Scribble complex consists of Scribble, discs large, and lethal giant larvae. These complexes modulate kinase and small G protein activity such that the apical and basolateral complexes signal antagonistically, leading to the segregation of the apical and basolateral membranes. The polarity complexes also serve as scaffolds to direct and retain proteins at the apical membrane, the basolateral membrane, or the intervening tight junction. There is plasticity in apicobasal polarity, and this is best seen in the processes of epithelial-to-mesenchymal transition and the converse mesenchymal-to-epithelial transition. These transitions are important in kidney disease as well as kidney development, and modulation of the polarity complexes are critical for these transitions. PMID:21228104

  1. Three-way complex variant translocation involving short arm chromosome (1;9;22)(p36;q34;q11) in a chronic myeloid leukemia patient

    PubMed Central

    ASIF, MUHAMMAD; HUSSAIN, ABRAR; MALIK, ARIF; RASOOL, MAHMOOD

    2015-01-01

    Chronic myeloid leukemia (CML) is a disease of the clonal hematopoietic stem cells caused by a balanced translocation between the long arms of chromosomes 9 and 22. Overall, 90–95% of CML patients present with a Philadelphia (Ph) chromosome t(9;22)(q34;q11) translocation and in addition, variant complex translocations, involving a third chromosome, are observed in 5–8% of CML patients. Cytogenic testing using bone marrow sample was performed and the FISH test was used for the detection of BCR-ABL fusion gene and complete blood analysis of CML patient was also performed. Results of hematological analysis showed the induced values of white blood cells (168,5000/mm3) and platelets (300,000/mm3) and FISH analysis test showed that 98% cells were positive for BCR/ABL gene translocation. The present study describes a three-way (1;9;22)(p36;q34;q11) Ph chromosome translocation in a 24-year-old female with CML. The patient, who was in the chronic phase of the disease, was treated with daily dose of 400 mg/dl with imatinib mesylateand was monitored constantly at various intervals over a 6-month period. Many studies reported that certain CML patents with variant translocation responded poorly to imatinib. In the current case report, the CML patient exhibited a suboptimal response to imatinib, denoting a poor prognosis. PMID:26622740

  2. Strandwise translocation of a DNA glycosylase on undamaged DNA

    SciTech Connect

    Qi, Yan; Nam, Kwangho; Spong, Marie C.; Banerjee, Anirban; Sung, Rou-Jia; Zhang, Michael; Karplus, Martin; Verdine, Gregory L.

    2012-05-14

    Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA.

  3. Translocation of the ABC transporter ABCD4 from the endoplasmic reticulum to lysosomes requires the escort protein LMBD1

    PubMed Central

    Kawaguchi, Kosuke; Okamoto, Takumi; Morita, Masashi; Imanaka, Tsuneo

    2016-01-01

    We previously demonstrated that ABCD4 does not localize to peroxisomes but rather, the endoplasmic reticulum (ER), because it lacks the NH2-terminal hydrophilic region required for peroxisomal targeting. It was recently reported that mutations in ABCD4 result in a failure to release vitamin B12 from lysosomes. A similar phenotype is caused by mutations in LMBRD1, which encodes the lysosomal membrane protein LMBD1. These findings suggested to us that ABCD4 translocated from the ER to lysosomes in association with LMBD1. In this report, it is demonstrated that ABCD4 interacts with LMBD1 and then localizes to lysosomes, and this translocation depends on the lysosomal targeting ability of LMBD1. Furthermore, endogenous ABCD4 was localized to both lysosomes and the ER, and its lysosomal localization was disturbed by knockout of LMBRD1. To the best of our knowledge, this is the first report demonstrating that the subcellular localization of the ABC transporter is determined by its association with an adaptor protein. PMID:27456980

  4. Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus.

    PubMed

    Nayar, Saraswati; Kapoor, Meenu; Kapoor, Sanjay

    2014-10-01

    OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine(23) and arginine(24)) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins.

  5. Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus

    PubMed Central

    Nayar, Saraswati; Kapoor, Meenu; Kapoor, Sanjay

    2014-01-01

    OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine23 and arginine24) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins. PMID:25096923

  6. Disruption of the three cytoskeletal networks in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock.

    PubMed Central

    Welch, W J; Feramisco, J R

    1985-01-01

    Mammalian cells show a complex series of transcriptional and translational switching events in response to heat shock treatment which ultimately lead to the production and accumulation of a small number of proteins, the so-called heat shock (or stress) proteins. We investigated the heat shock response in both qualitative and quantitative ways in cells that were pretreated with drugs that specifically disrupt one or more of the three major cytoskeletal networks. (These drugs alone, cytochalasin E and colcemid, do not result in induction of the heat shock response.) Our results indicated that disruption of the actin microfilaments, the vimentin-containing intermediate filaments, or the microtubules in living cells does not hinder the ability of the cell to undergo an apparently normal heat shock response. Even when all three networks were simultaneously disrupted (resulting in a loose, baglike appearance of the cells), the cells still underwent a complete heat shock response as assayed by the appearance of the heat shock proteins. In addition, the major induced 72-kilodalton heat shock protein was efficiently translocated from the cytoplasm into its proper location in the nucleus and nucleolus irrespective of the condition of the three cytoskeletal elements. Images PMID:4040602

  7. How can proteins enter the interior of a MOF? Investigation of cytochrome c translocation into a MOF consisting of mesoporous cages with microporous windows.

    PubMed

    Chen, Yao; Lykourinou, Vasiliki; Vetromile, Carissa; Hoang, Tran; Ming, Li-June; Larsen, Randy W; Ma, Shengqian

    2012-08-15

    It has been demonstrated for the first time that the heme protein cytochrome c (Cyt c) can enter the interior of a MOF despite the larger molecular dimension of the protein relative to the access pore sizes. Mechanistic studies suggest that the Cyt c molecules must undergo a significant conformational change during translocation into the MOF interior through the relatively small nanopores.

  8. Complementary Proteomic Analysis of Protein Complexes

    PubMed Central

    Greco, Todd M.; Miteva, Yana; Conlon, Frank L.; Cristea, Ileana M.

    2013-01-01

    Proteomic characterization of protein complexes leverages the versatile platform of liquid chromatography-tandem mass spectrometry to elucidate molecular and cellular signaling processes underlying the dynamic regulation of macromolecular assemblies. Here, we describe a complementary proteomic approach optimized for immunoisolated protein complexes. As the relative complexity, abundance, and physiochemical properties of proteins can vary significantly between samples, we have provided (1) complementary sample preparation workflows, (2) detailed steps for HPLC and mass spectrometric method development, and (3) a bioinformatic workflow that provides confident peptide/protein identification paired with unbiased functional gene ontology analysis. This protocol can also be extended for characterization of larger complexity samples from whole cell or tissue Xenopus proteomes. PMID:22956100

  9. The Claudin Megatrachea Protein Complex*

    PubMed Central

    Jaspers, Martin H. J.; Nolde, Kai; Behr, Matthias; Joo, Seol-hee; Plessmann, Uwe; Nikolov, Miroslav; Urlaub, Henning; Schuh, Reinhard

    2012-01-01

    Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates. PMID:22930751

  10. Interaction proteomics of synapse protein complexes

    PubMed Central

    Klemmer, Patricia; Smit, August B.

    2010-01-01

    The brain integrates complex types of information, and executes a wide range of physiological and behavioral processes. Trillions of tiny organelles, the synapses, are central to neuronal communication and information processing in the brain. Synaptic transmission involves an intricate network of synaptic proteins that forms the molecular machinery underlying transmitter release, activation, and modulation of transmitter receptors and signal transduction cascades. These processes are dynamically regulated and underlie neuroplasticity, crucial to learning and memory formation. In recent years, interaction proteomics has increasingly been used to elucidate the constituents of synaptic protein complexes. Unlike classic hypothesis-based assays, interaction proteomics detects both known and novel interactors without bias. In this trend article, we focus on the technical aspects of recent proteomics to identify synapse protein complexes, and the complementary methods used to verify the protein–protein interaction. Moreover, we discuss the experimental feasibility of performing global analysis of the synapse protein interactome. PMID:20361179

  11. AIRE-induced apoptosis is associated with nuclear translocation of stress sensor protein GAPDH.

    PubMed

    Liiv, Ingrid; Haljasorg, Uku; Kisand, Kai; Maslovskaja, Julia; Laan, Martti; Peterson, Pärt

    2012-06-22

    AIRE (Autoimmune Regulator) has a central role in the transcriptional regulation of self-antigens in medullary thymic epithelial cells, which is necessary for negative selection of autoreactive T cells. Recent data have shown that AIRE can also induce apoptosis, which may be linked to cross-presentation of these self-antigens. Here we studied AIRE-induced apoptosis using AIRE over-expression in a thymic epithelial cell line as well as doxycycline-inducible HEK293 cells. We show that the HSR/CARD domain in AIRE together with a nuclear localization signal is sufficient to induce apoptosis. In the nuclei of AIRE-positive cells, we also found an increased accumulation of a glycolytic enzyme, glyceraldehyde-3-phosphate (GAPDH) reflecting cellular stress and apoptosis. Additionally, AIRE-induced apoptosis was inhibited with an anti-apoptotic agent deprenyl that blocks GAPDH nitrosylation and nuclear translocation. We propose that the AIRE-induced apoptosis pathway is associated with GAPDH nuclear translocation and induction of NO-induced cellular stress in AIRE-expressing cells.

  12. Complex Oncogenic Translocations with Gene Amplification are Initiated by Specific DNA Breaks in Lymphocytes

    PubMed Central

    Wright, Sarah M.; Woo, Yong H.; Alley, Travis L.; Shirley, Bobbi-Jo; Akeson, Ellen C.; Snow, Kathy J.; Maas, Sarah A.; Elwell, Rachel L.; Foreman, Oded; Mills, Kevin D.

    2009-01-01

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. While chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now demonstrate that specific DNA double strand breaks, occurring within a narrow segment of Igh are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Eμ are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability, and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors. PMID:19435904

  13. Complex oncogenic translocations with gene amplification are initiated by specific DNA breaks in lymphocytes.

    PubMed

    Wright, Sarah M; Woo, Yong H; Alley, Travis L; Shirley, Bobbi-Jo; Akeson, Ellen C; Snow, Kathy J; Maas, Sarah A; Elwell, Rachel L; Foreman, Oded; Mills, Kevin D

    2009-05-15

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. Whereas chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double-strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now show that specific DNA double-strand breaks, occurring within a narrow segment of Igh, are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Emu are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors.

  14. Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe.

    PubMed

    Eliahoo, Elad; Litovco, Phyana; Ben Yosef, Ron; Bendalak, Keren; Ziv, Tamar; Manor, Haim

    2014-04-01

    Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.

  15. Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

    SciTech Connect

    Kim, Hye-Mi; Kang, Dong-Ku; Kim, Hak Yong; Kang, Sang Sun; Chang, Soo-Ik . E-mail: sichang@cbnu.ac.kr

    2007-01-12

    Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.

  16. Cytoplasmic translocation of high-mobility group box-1 protein is induced by diabetes and high glucose in retinal pericytes

    PubMed Central

    Kim, Junghyun; Kim, Chan-Sik; Sohn, Eunjin; Kim, Jin Sook

    2016-01-01

    The aim of the present study was to assess the involvement of the high-mobility group box-1 (HMGB1) protein, receptor for advanced glycation end products (RAGE) and nuclear factor (NF)-κB signaling pathway in the development of diabetic retinopathy. Rat primary retinal pericytes were exposed to 25 mmol/l D-glucose for 48 h. Diabetic retinal vessels were prepared from streptozotocin-induced diabetic rats 12 weeks following the induction of diabetes. The expression of HMGB1 was detected using immunofluorescence staining. The expression of RAGE and the activity of NF-κB were analyzed using western blot and electrophoretic mobility shift assays, respectively. The results showed that HMGB1 was translocated to the cytoplasm of the high glucose-treated pericytes and diabetic retinal pericytes, whereas, in the control cells and the normal retinas, HMGB1 was expressed in the cell nuclei only. The expression of RAGE, a potential receptor for HMGB1, and the activity of NF-κB were also increased in the high glucose-treated pericytes, compared with the normal control cells. In addition, high glucose increased the binding of NF-κB to the RAGE promoter. These findings suggested that the cytoplasmic translocation of HMGB1 may be caused by diabetes and high glucose in retinal pericytes, and that the pathogenic role of HMGB1 may be dependent on the expression of RAGE and activation of NF-κB. PMID:27599553

  17. Cytoplasmic translocation of high-mobility group box-1 protein is induced by diabetes and high glucose in retinal pericytes.

    PubMed

    Kim, Junghyun; Kim, Chan-Sik; Sohn, Eunjin; Kim, Jin Sook

    2016-10-01

    The aim of the present study was to assess the involvement of the high-mobility group box-1 (HMGB1) protein, receptor for advanced glycation end products (RAGE) and nuclear factor (NF)-κB signaling pathway in the development of diabetic retinopathy. Rat primary retinal pericytes were exposed to 25 mmol/l D‑glucose for 48 h. Diabetic retinal vessels were prepared from streptozotocin-induced diabetic rats 12 weeks following the induction of diabetes. The expression of HMGB1 was detected using immunofluorescence staining. The expression of RAGE and the activity of NF‑κB were analyzed using western blot and electrophoretic mobility shift assays, respectively. The results showed that HMGB1 was translocated to the cytoplasm of the high glucose‑treated pericytes and diabetic retinal pericytes, whereas, in the control cells and the normal retinas, HMGB1 was expressed in the cell nuclei only. The expression of RAGE, a potential receptor for HMGB1, and the activity of NF‑κB were also increased in the high glucose‑treated pericytes, compared with the normal control cells. In addition, high glucose increased the binding of NF‑κB to the RAGE promoter. These findings suggested that the cytoplasmic translocation of HMGB1 may be caused by diabetes and high glucose in retinal pericytes, and that the pathogenic role of HMGB1 may be dependent on the expression of RAGE and activation of NF‑κB. PMID:27599553

  18. Complex coacervation of supercharged proteins with polyelectrolytes.

    PubMed

    Obermeyer, Allie C; Mills, Carolyn E; Dong, Xue-Hui; Flores, Romeo J; Olsen, Bradley D

    2016-04-21

    Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase separated with strong polycations when the ratio of negatively charged residues to positively charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of positive charge in the coacervate phase. The composition of protein and polymer in the coacervate phase was determined using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromolecular charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temperatures.

  19. Clustering of T cell ligands on artificial APC membranes influences T cell activation and protein kinase C theta translocation to the T cell plasma membrane.

    PubMed

    Giannoni, Francesca; Barnett, Joellen; Bi, Kun; Samodal, Rodrigo; Lanza, Paola; Marchese, Patrizia; Billetta, Rosario; Vita, Randi; Klein, Mark R; Prakken, Berent; Kwok, William W; Sercarz, Eli; Altman, Amnon; Albani, Salvatore

    2005-03-15

    T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase theta, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.

  20. Progesterone receptor-NFκB complex formation is required for progesterone-induced NFκB nuclear translocation and binding onto the p53 promoter.

    PubMed

    Hsu, Sung-Po; Yang, Ho-Ching; Kuo, Chun-Ting; Wen, Heng-Ching; Chen, Li-Ching; Huo, Yen-Nien; Lee, Wen-Sen

    2015-01-01

    We previously demonstrated that progesterone (P4) up-regulates p53 expression in human umbilical venous endothelial cells (HUVECs) through P4 receptor (PR) activation of extranuclear signaling pathways. However, the involvement of nuclear PR in P4-increased p53 expression is still unclear. Here, the molecular mechanism underlying PR-regulated p53 expression in HUVECs was investigated. Treatment with P4 increased nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α phosphorylation (IκBα and nuclear factor-κB (NFκB) nuclear translocation. Interestingly, P4 also increased PR-A, but not PR-B, nuclear translocation in HUVECs. Immunoprecipitation assay illustrated that P4 increased the formation of PR-A-NFκB complex in both the cytosol and the nucleus of HUVEC. Chromatin immunoprecipitation assay showed an interaction between PR and the NFκB binding motif on the p53 promoter. Ablation of the NFκB binding motif in the p53 promoter completely abolished P4-increased p53 promoter activity. In the absence of P4, overexpression of NFκB did not increase NFκB nuclear translocation. In contrast, treatment of NFκB-overexpressing HUVECs with P4 for only 4 hours, which is much shorter than the time (21.5 h) required for P4-induced IκBα phosphorylation, increased NFκB nuclear translocation. Blockade of PR activity abolished this effect. Taken together, these results uncover a novel role of PR for P4-induced NFκB nuclear translocation and suggest that PR-A-NFκB complex formation is required for NFκB nuclear translocation and binding onto the p53 promoter in HUVECs. Our data indicate that both nuclear and extranuclear signaling pathways of PR are involved in P4-regulated p53 expression in HUVECs. PMID:25353185

  1. Progesterone receptor-NFκB complex formation is required for progesterone-induced NFκB nuclear translocation and binding onto the p53 promoter.

    PubMed

    Hsu, Sung-Po; Yang, Ho-Ching; Kuo, Chun-Ting; Wen, Heng-Ching; Chen, Li-Ching; Huo, Yen-Nien; Lee, Wen-Sen

    2015-01-01

    We previously demonstrated that progesterone (P4) up-regulates p53 expression in human umbilical venous endothelial cells (HUVECs) through P4 receptor (PR) activation of extranuclear signaling pathways. However, the involvement of nuclear PR in P4-increased p53 expression is still unclear. Here, the molecular mechanism underlying PR-regulated p53 expression in HUVECs was investigated. Treatment with P4 increased nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α phosphorylation (IκBα and nuclear factor-κB (NFκB) nuclear translocation. Interestingly, P4 also increased PR-A, but not PR-B, nuclear translocation in HUVECs. Immunoprecipitation assay illustrated that P4 increased the formation of PR-A-NFκB complex in both the cytosol and the nucleus of HUVEC. Chromatin immunoprecipitation assay showed an interaction between PR and the NFκB binding motif on the p53 promoter. Ablation of the NFκB binding motif in the p53 promoter completely abolished P4-increased p53 promoter activity. In the absence of P4, overexpression of NFκB did not increase NFκB nuclear translocation. In contrast, treatment of NFκB-overexpressing HUVECs with P4 for only 4 hours, which is much shorter than the time (21.5 h) required for P4-induced IκBα phosphorylation, increased NFκB nuclear translocation. Blockade of PR activity abolished this effect. Taken together, these results uncover a novel role of PR for P4-induced NFκB nuclear translocation and suggest that PR-A-NFκB complex formation is required for NFκB nuclear translocation and binding onto the p53 promoter in HUVECs. Our data indicate that both nuclear and extranuclear signaling pathways of PR are involved in P4-regulated p53 expression in HUVECs.

  2. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    PubMed

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  3. Cdc42-Interacting Protein 4 Represses E-Cadherin Expression by Promoting β-Catenin Translocation to the Nucleus in Murine Renal Tubular Epithelial Cells.

    PubMed

    Xu, Chuou; Zhou, Qiaodan; Liu, Lili; Liu, Ping; Pei, Guangchang; Zeng, Rui; Han, Min; Xu, Gang

    2015-08-14

    Renal fibrosis is an inevitable outcome of end-stage chronic kidney disease. During this process, epithelial cells lose E-cadherin expression. β-Catenin may act as a mediator by accumulation and translocation to the nucleus. Studies have suggested that CIP4, a Cdc42 effector protein, is associated with β-catenin. However, whether CIP4 contributes to E-cadherin loss in epithelial cells by regulating β-catenin translocation is unclear. In this study, we investigated the involvement of CIP4 in β-catenin translocation. Expression of CIP4 was upregulated in renal tissues of 5/6 nephrectomized rats and mainly distributed in renal tubular epithelia. In TGF-β1-treated NRK-52E cells, upregulation of CIP4 expression was accompanied by reduced expression of E-cadherin. CIP4 overexpression promoted the translocation of β-catenin to the nucleus, which was accompanied by reduced expression of E-cadherin even without TGF-β1 stimulation. In contrast, CIP4 depletion by using siRNA inhibited the translocation of β-catenin to the nucleus and reversed the decrease in expression of E-cadherin. The interaction between CIP4 and β-catenin was detected. We also show that β-catenin depletion could restore the expression of E-cadherin that was suppressed by CIP4 overexpression. In conclusion, these results suggest that CIP4 overexpression represses E-cadherin expression by promoting β-catenin translocation to the nucleus.

  4. Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

    NASA Technical Reports Server (NTRS)

    Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

    2002-01-01

    Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

  5. Tumour necrosis factor-alpha induces translocation of protein kinase C in tumour necrosis factor-sensitive cell lines.

    PubMed Central

    Matsubara, N; Fuchimoto, S; Orita, K

    1991-01-01

    In this study we investigated whether the anti-proliferative effect of tumour necrosis factor-alpha (TNF-alpha) was associated with the activation of protein kinase C (PKC), using PANC-1 cells (TNF-alpha sensitive) and LoVo cells (TNF-alpha resistant). In combination with 12-0-tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, TNF-alpha caused marked inhibition of the growth of LoVo cells. Inhibition of PANC-1 cell growth by TNF-alpha was blocked by pretreatment with TPA for 24 hr, along with down-regulation of PKC activity. Intracellular translocation of PKC from cytosol to membrane was induced by TNF-alpha treatment in PANC-1 cells but not in LoVo cells. PMID:1916896

  6. Protein domain of unknown function 3233 is a translocation domain of autotransporter secretory mechanism in gamma proteobacteria.

    PubMed

    Prakash, Ananth; Yogeeshwari, S; Sircar, Sanchari; Agrawal, Shipra

    2011-01-01

    Vibrio cholerae, the enteropathogenic gram negative bacteria is one of the main causative agents of waterborne diseases like cholera. About 1/3(rd) of the organism's genome is uncharacterised with many protein coding genes lacking structure and functional information. These proteins form significant fraction of the genome and are crucial in understanding the organism's complete functional makeup. In this study we report the general structure and function of a family of hypothetical proteins, Domain of Unknown Function 3233 (DUF3233), which are conserved across gram negative gammaproteobacteria (especially in Vibrio sp. and similar bacteria). Profile and HMM based sequence search methods were used to screen homologues of DUF3233. The I-TASSER fold recognition method was used to build a three dimensional structural model of the domain. The structure resembles the transmembrane beta-barrel with an axial N-terminal helix and twelve antiparallel beta-strands. Using a combination of amphipathy and discrimination analysis we analysed the potential transmembrane beta-barrel forming properties of DUF3233. Sequence, structure and phylogenetic analysis of DUF3233 indicates that this gram negative bacterial hypothetical protein resembles the beta-barrel translocation unit of autotransporter Va secretory mechanism with a gene organisation that differs from the conventional Va system. PMID:22073138

  7. Optimization of the electrostatic interactions in protein-protein complexes

    NASA Astrophysics Data System (ADS)

    Alexov, Emil; Brock, Kelly; Kundrotas, Petras

    2007-03-01

    Electrostatic energy is one of the driving forces of protein-protein association. Understanding the role of the energy components on the energetics of protein-protein association will help us in engineering protein-protein interactions and could lead to development of scoring functions that can rank alternative models and decoys. Here we investigate whether the components of the electrostatic energy of protein-protein complexes is optimized in respect to random distribution of the charged residues. We report a clear tendency that coulombic electrostatic interactions are optimized, while the reaction field energy is inversely optimized. It was found that the maximum of the coulombic energy Z-score is shifted 3 units away from the origin and the maximum of the reaction field energy by 2 units. Such a large shift of the Z-score of both coulombic and reaction field energies indicates that wild-type protein-protein interactions are in most cases optimized in terms of coulombic interactions while compromising reaction field energy. Based on these finding a scoring function was developed as a linear combination of the Z-score of the coulombic interactions minus Z-score of the reaction field energy. The scoring function was tested against the decoy sets and it was shown that in majority of the cases we can identify the wild-type complex among hundreds of decoys.

  8. Ca2+-dependent translocation of the calcyclin-binding protein in neurons and neuroblastoma NB-2a cells.

    PubMed

    Filipek, Anna; Jastrzebska, Beata; Nowotny, Marcin; Kwiatkowska, Katarzyna; Hetman, Michal; Surmacz, Liliana; Wyroba, Elzbieta; Kuznicki, Jacek

    2002-06-01

    The calcyclin-binding protein (CacyBP) binds calcyclin (S100A6) at physiological levels of [Ca(2+)] and is highly expressed in brain neurons. Subcellular localization of CacyBP was examined in neurons and neuroblastoma NB-2a cells at different [Ca(2+)](i). Immunostaining indicates that CacyBP is present in the cytoplasm of unstimulated cultured neurons in which resting [Ca(2+)](i) is known to be approximately 50 nm. When [Ca(2+)](i) was increased to above 300 nm by KCl treatment, the immunostaining was mainly apparent as a ring around the nucleus. Such perinuclear localization of CacyBP was observed in untreated neuroblastoma NB-2a cells in which [Ca(2+)](i) is approximately 120 nm. An additional increase in [Ca(2+)](i) to above 300 nm by thapsigargin treatment did not change CacyBP localization. However, when [Ca(2+)](i) in NB-2a cells dropped to 70 nm, because of BAPTA/AM treatment, perinuclear localization was diminished. Ca(2+)-induced translocation of CacyBP was confirmed by immunogold electron microscopy and by fluorescence of NB-2a cells transfected with an EGFP-CacyBP vector. Recombinant CacyBP can be phosphorylated by protein kinase C in vitro. In untreated neuroblastoma NB-2a cells, CacyBP is phosphorylated on a serine residue(s), but exists in the dephosphorylated form in BAPTA/AM-treated cells. Thus, phosphorylation of CacyBP occurs in the same [Ca(2+)](i) range that leads to its perinuclear translocation.

  9. Protein-protein interaction networks (PPI) and complex diseases

    PubMed Central

    Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

    2014-01-01

    The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

  10. Measuring the functional sequence complexity of proteins

    PubMed Central

    Durston, Kirk K; Chiu, David KY; Abel, David L; Trevors, Jack T

    2007-01-01

    Background Abel and Trevors have delineated three aspects of sequence complexity, Random Sequence Complexity (RSC), Ordered Sequence Complexity (OSC) and Functional Sequence Complexity (FSC) observed in biosequences such as proteins. In this paper, we provide a method to measure functional sequence complexity. Methods and Results We have extended Shannon uncertainty by incorporating the data variable with a functionality variable. The resulting measured unit, which we call Functional bit (Fit), is calculated from the sequence data jointly with the defined functionality variable. To demonstrate the relevance to functional bioinformatics, a method to measure functional sequence complexity was developed and applied to 35 protein families. Considerations were made in determining how the measure can be used to correlate functionality when relating to the whole molecule and sub-molecule. In the experiment, we show that when the proposed measure is applied to the aligned protein sequences of ubiquitin, 6 of the 7 highest value sites correlate with the binding domain. Conclusion For future extensions, measures of functional bioinformatics may provide a means to evaluate potential evolving pathways from effects such as mutations, as well as analyzing the internal structural and functional relationships within the 3-D structure of proteins. PMID:18062814

  11. Protein-protein interactions in the synaptonemal complex.

    PubMed Central

    Tarsounas, M; Pearlman, R E; Gasser, P J; Park, M S; Moens, P B

    1997-01-01

    In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II. Images PMID:9285814

  12. Impact of the Diffusion of Microtubule-Associated Protein EB1 on Kinesin Translocation in Vitro

    NASA Astrophysics Data System (ADS)

    Lopez, Benjamin; Valentine, Megan

    2014-03-01

    Using the slowly hydrolyzable GTP analog GMPCPP, we polymerize microtubules that recapitulate the end binding behavior of EB1 along their entire length, and investigate the impact of EB1 on kinesin translocation. Through direct observation of single molecules of EB1 fused to GFP, we find that EB1 diffuses along the microtubule lattice, and that the presence of taxol affects the rate of diffusion. To test whether EB1 presence and diffusion has an effect on kinesin-driven cargo transport, we observe quantum dot labeled kinesins walking on microtubules assembled with GMPCPP and taxol and coated with EB1. We find that the addition of EB1 significantly reduces kinesin speed compared to the no EB1 condition, but when microtubules stabilized by both taxol and GMPCPP are used, the speed reduction is nearly abolished. Our data suggest a new possible mechanism for the regulation of kinesin function by EB1 in which kinesin speed is directly modulated through the interference of EB1 diffusion. Our results also raise important questions about the effects of taxol on microtubule-MAP interactions.

  13. Viral and cellular SOS-regulated motor proteins: dsDNA translocation mechanisms with divergent functions.

    PubMed

    Wolfe, Annie; Phipps, Kara; Weitao, Tao

    2014-01-01

    DNA damage attacks on bacterial cells have been known to activate the SOS response, a transcriptional response affecting chromosome replication, DNA recombination and repair, cell division and prophage induction. All these functions require double-stranded (ds) DNA translocation by ASCE hexameric motors. This review seeks to delineate the structural and functional characteristics of the SOS response and the SOS-regulated DNA translocases FtsK and RuvB with the phi29 bacteriophage packaging motor gp16 ATPase as a prototype to study bacterial motors. While gp16 ATPase, cellular FtsK and RuvB are similarly comprised of hexameric rings encircling dsDNA and functioning as ATP-driven DNA translocases, they utilize different mechanisms to accomplish separate functions, suggesting a convergent evolution of these motors. The gp16 ATPase and FtsK use a novel revolution mechanism, generating a power stroke between subunits through an entropy-DNA affinity switch and pushing dsDNA inward without rotation of DNA and the motor, whereas RuvB seems to employ a rotation mechanism that remains to be further characterized. While FtsK and RuvB perform essential tasks during the SOS response, their roles may be far more significant as SOS response is involved in antibiotic-inducible bacterial vesiculation and biofilm formation as well as the perspective of the bacteria-cancer evolutionary interaction.

  14. Long lasting MDM2/Translocator protein modulator: a new strategy for irreversible apoptosis of human glioblastoma cells

    PubMed Central

    Zappelli, Elisa; Marinelli, Luciana; Novellino, Ettore; Da Settimo, Federico; Taliani, Sabrina; Trincavelli, Maria L.; Martini, Claudia

    2016-01-01

    The development of multi-target drugs and irreversible modulators of deregulated signalling proteins is the major challenge for improving glioblastoma multiforme (GBM) treatment. Reversible single-target drugs are not sufficient to sustain a therapeutic effect over time and may favour the activation of alternative signalling pathways and the onset of resistance phenomena. Thus, a multi-target compound that has a long-lasting mechanism of action might have a greater and longer life span of anti-proliferative activity. Recently, a dual-target indol-3ylglyoxyldipeptide derivative, designed to bind to the Translocator Protein (TSPO) and reactivate p53 function via dissociation from its physiological inhibitor, murine double minute 2 (MDM2), has been developed as a potent GBM pro-apoptotic agent. In this study, this derivative was chemically modified to irreversibly bind MDM2 and TSPO. The new compound elicited a TSPO-mediated mitochondrial membrane dissipation and restored p53 activity, triggering a long-lasting apoptosis of GBM cells. These effects were sustained over time, involved a stable activation of extracellular signal regulated kinases and were specifically observed in cancer cells, in which these protein kinases are deregulated. Dual-targeting and irreversible binding properties combined in the same molecule may represent a useful strategy to overcome the time-limited effects elicited by classical chemotherapies. PMID:26761214

  15. REST/NRSF-Interacting LIM Domain Protein, a Putative Nuclear Translocation Receptor

    PubMed Central

    Shimojo, Masahito; Hersh, Louis B.

    2003-01-01

    The transcriptional repressor REST/NRSF (RE-1 silencing transcription factor/neuron-restrictive silencer factor) and the transcriptional regulator REST4 share an N-terminal zinc finger domain structure involved in nuclear targeting. Using this domain as bait in a yeast two-hybrid screen, a novel protein that contains three LIM domains, putative nuclear localization sequences, protein kinase A phosphorylation sites, and a CAAX prenylation motif was isolated. This protein, which is localized around the nucleus, is involved in determining the nuclear localization of REST4 and REST/NRSF. We propose the name RILP, for REST/NRSF-interacting LIM domain protein, to label this novel protein. RILP appears to serve as a nuclear receptor for REST/NRSF, REST4, and possibly other transcription factors. PMID:14645515

  16. Coarse-Grained Simulations of Topology-Dependent Mechanisms of Protein Unfolding and Translocation Mediated by ClpY ATPase Nanomachines.

    PubMed

    Kravats, Andrea N; Tonddast-Navaei, Sam; Stan, George

    2016-01-01

    Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an α/β model protein. We contrast this mechanism with our previous studies which used an all-α SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation. PMID:26734937

  17. Coarse-Grained Simulations of Topology-Dependent Mechanisms of Protein Unfolding and Translocation Mediated by ClpY ATPase Nanomachines.

    PubMed

    Kravats, Andrea N; Tonddast-Navaei, Sam; Stan, George

    2016-01-01

    Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an α/β model protein. We contrast this mechanism with our previous studies which used an all-α SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation.

  18. Coarse-Grained Simulations of Topology-Dependent Mechanisms of Protein Unfolding and Translocation Mediated by ClpY ATPase Nanomachines

    PubMed Central

    Kravats, Andrea N.; Tonddast-Navaei, Sam; Stan, George

    2016-01-01

    Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an α/β model protein. We contrast this mechanism with our previous studies which used an all-α SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation. PMID:26734937

  19. Complex micropatterning of proteins within microfluidic channels.

    PubMed

    Kim, Miju; Doh, Junsang

    2014-01-01

    Microfluidic channels containing protein micropatterned surfaces are useful in many bioanalytical and biological applications. In this study, we developed a new method to integrate microfluidics and protein micropatterning by attaching poly(dimethylsiloxane) (PDMS) microfluidic channels to bio-friendly photoresist films via poly(dopamine) (PDA) adhesive. A bio-friendly photoresist poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP) was synthesized and used. By performing microscope projection photolithography (MPP) to the PDMP thin films within PDMS microchannels, complex micropatterns of proteins were successfully generated within microfluidic channels. PMID:25570075

  20. Activated Rac1 regulates the degradation of IκBα and the nuclear translocation of STAT3–NFκB complexes in starved cancer cells

    PubMed Central

    Kim, Sung Joo; Yoon, Sarah

    2016-01-01

    In several human tumors, signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB (NFκB) are activated and interact; how these STAT3–NFκB complexes are transported to the nucleus is not fully understood. In this study, we found that Rac1 was activated in starved cancer cells and that activated Rac1 coexisted with STAT3 and NFκB. Rac1 knockdown and overexpression of the dominant-negative mutant Rac1N19 inhibited the degradation of IκBα, an inhibitor of NFκB. MG132, an inhibitor of the ubiquitin proteasome pathway, increased the amount of non-phosphorylated IκBα, but not serine-phosphorylated IκBα, indicating that IκBα degradation by Rac1 in starved cancer cells is independent of IκBα serine phosphorylation by IKK. Rac1 knockdown also inhibited the nuclear translocation of STAT3–NFκB complexes, indicating that this translocation requires activated Rac1. We also demonstrated that the mutant STAT3 Y705F could form complexes with NFκB, and these unphosphorylated STAT3–NFκB complexes translocated into the nucleus and upregulated the activity of NFκB in starved cancer cells, suggesting that phosphorylation of STAT3 is not essential for its translocation. To our knowledge, this is the first study demonstrating the crucial role of Rac1 in the function of STAT3–NFκB complexes in starved cancer cells and implies that targeting Rac1 may have future therapeutic significance in cancer therapy. PMID:27151455

  1. Translocation of a store of maternal cytoplasmic c-myc protein into nuclei during early development.

    PubMed Central

    Gusse, M; Ghysdael, J; Evan, G; Soussi, T; Méchali, M

    1989-01-01

    The c-myc proto-oncogene is expressed as a maternal protein during oogenesis in Xenopus laevis, namely, in nondividing cells. A delayed translation of c-myc mRNA accumulated in early oocytes results in the accumulation of the protein during late oogenesis. The oocyte c-myc protein is unusually stable and is located in the cytoplasm, contrasting with its features in somatic cells. A mature oocyte contains a maternal c-myc protein stockpile of 4 x 10(5) to 6 x 10(5) times the level in a somatic growing cell. This level of c-myc protein is preserved only during the cleavage stage of the embryo. Fertilization triggers its rapid migration into the nuclei of the cleaving embryo and a change in the phosphorylation state of the protein. The c-myc protein content per nucleus decreases exponentially during the cleavage stage until a stoichiometric titration by the embryonic nuclei is reached during a 0.5-h period at the midblastula stage. Most of the maternal c-myc store is degraded by the gastrula stage. These observations implicate the participation of c-myc in the events linked to early embryonic development and the midblastula transition. Images PMID:2685563

  2. New Anthocyanin-Human Salivary Protein Complexes.

    PubMed

    Ferrer-Gallego, Raúl; Soares, Susana; Mateus, Nuno; Rivas-Gonzalo, Julián; Escribano-Bailón, M Teresa; de Freitas, Victor

    2015-08-01

    The interaction between phenolic compounds and salivary proteins is considered the basis of the poorly understood phenomenon of astringency. Furthermore, this interaction is an important factor in relation to their bioavailability. In this work, interactions between anthocyanin and human salivary protein fraction were studied by mass spectrometry (MALDI-TOF-MS and FIA-ESI-MS) and saturation-transfer difference (STD) NMR spectroscopy. Anthocyanins were able to interact with saliva proteins. The dissociation constant (KD) between malvidin 3-glucoside and salivary proline-rich proteins was 1.92 mM for the hemiketal form (pH 3.4) and 1.83 mM for the flavylium cation (pH 1.0). New soluble complexes between these salivary proteins and malvidin 3-glucoside were identified for the first time.

  3. Peroxisome protein import: a complex journey

    PubMed Central

    Baker, Alison; Hogg, Thomas Lanyon; Warriner, Stuart L.

    2016-01-01

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor–cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. PMID:27284042

  4. Rapid regulation of nuclear proteins by rapamycin-induced translocation in fission yeast.

    PubMed

    Ding, Lin; Laor, Dana; Weisman, Ronit; Forsburg, Susan L

    2014-07-01

    Genetic analysis of protein function requires a rapid means of inactivating the gene under study. Typically, this exploits temperature-sensitive mutations or promoter shut-off techniques. We report the adaptation to Schizosaccharomyces pombe of the anchor-away technique, originally designed in budding yeast by Laemmli lab. This method relies on a rapamycin-mediated interaction between the FRB- and FKBP12-binding domains to relocalize nuclear proteins of interest to the cytoplasm. We demonstrate a rapid nuclear depletion of abundant proteins as proof of principle.

  5. On protein abundance distributions in complex mixtures

    PubMed Central

    2013-01-01

    Mass spectrometry, an analytical technique that measures the mass-to-charge ratio of ionized atoms or molecules, dates back more than 100 years, and has both qualitative and quantitative uses for determining chemical and structural information. Quantitative proteomic mass spectrometry on biological samples focuses on identifying the proteins present in the samples, and establishing the relative abundances of those proteins. Such protein inventories create the opportunity to discover novel biomarkers and disease targets. We have previously introduced a normalized, label-free method for quantification of protein abundances under a shotgun proteomics platform (Griffin et al., 2010). The introduction of this method for quantifying and comparing protein levels leads naturally to the issue of modeling protein abundances in individual samples. We here report that protein abundance levels from two recent proteomics experiments conducted by the authors can be adequately represented by Sichel distributions. Mathematically, Sichel distributions are mixtures of Poisson distributions with a rather complex mixing distribution, and have been previously and successfully applied to linguistics and species abundance data. The Sichel model can provide a direct measure of the heterogeneity of protein abundances, and can reveal protein abundance differences that simpler models fail to show. PMID:23360617

  6. First Demonstration of Positive Allosteric-like Modulation at the Human Wild Type Translocator Protein (TSPO).

    PubMed

    Narlawar, Rajeshwar; Werry, Eryn L; Scarf, Alana M; Hanani, Raphy; Chua, Sook Wern; King, Victoria A; Barron, Melissa L; Martins, Ralph N; Ittner, Lars M; Rendina, Louis M; Kassiou, Michael

    2015-11-12

    We show that changing the number and position of nitrogen atoms in the heteroatomic core of a pyrazolopyrimidine acetamide is sufficient to induce complex binding to wild type human TSPO. Only compounds with this complex binding profile lacked intrinsic effect on glioblastoma proliferation but positively modulated the antiproliferative effects of a synthetic TSPO ligand. To the best of our knowledge this is the first demonstration of allosteric-like interaction at the wild type human TSPO.

  7. Mutations in the Yersinia pseudotuberculosis Type III Secretion System Needle Protein, YscF, That Specifically Abrogate Effector Translocation into Host Cells▿ †

    PubMed Central

    Davis, Alison J.; Mecsas, Joan

    2007-01-01

    The trafficking of effectors, termed Yops, from Yersinia spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD) yscF mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two yscF TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I yscF TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II yscF TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface. PMID:17071752

  8. Dietary long-chain n-3 fatty acids modify blood and cardiac phospholipids and reduce protein kinase-C-delta and protein kinase-C-epsilon translocation.

    PubMed

    Judé, Sébastien; Martel, Eric; Vincent, Fanny; Besson, Pierre; Couet, Charles; Ogilvie, Gregory K; Pinault, Michelle; De Chalendar, Catherine; Bougnoux, Philippe; Richard, Serge; Champeroux, Pascal; Crozatier, Bertrand; Le Guennec, Jean-Yves

    2007-12-01

    The effects of an n-3 PUFA-enriched diet on cardiac cell membrane phospholipid fraction compositions and associated protein kinase-C (PKC) translocation modification have never been studied in higher mammals. This is of importance since membrane fatty acid composition has been shown to influence PKC signalling pathways. In the present study, we have tested whether the incorporation of n-3 PUFA in cardiac membrane phospholipids correlated with changes in the fatty acid composition of diacylglycerols (DAG) and led to a differential translocation of PKC isoforms. Two groups of five dogs were fed the standard diet supplemented with palm oil or fish oil for 8 weeks. Dogs fed a fish oil-enriched diet showed a preferential incorporation of EPA and, to a lesser extent, of DHA, at the expense of arachidonic acid, in the circulating TAG, plasma phospholipids, erythrocyte phospholipids and cardiomyocyte phospholipid fractions. Analysis of 1,2-DAG fatty acid composition also indicated a preferential enrichment of EPA compared with DHA. Associated with these results, a reduction in the expression of PKC-delta and PKC-epsilon isoforms in the particulate fractions was observed whereas no effect was seen for PKC-alpha and PKC-zeta. We conclude that a fish oil-enriched diet induces a modification in fatty acid composition of cardiac membrane phospholipids, associated with a differential translocation of PKC isoforms. These results can be explained by the production of structurally different DAG that may participate in some of the protective effects of n-3 PUFA against various chronic diseases.

  9. Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

    PubMed Central

    O'Driscoll, K R; Teng, K K; Fabbro, D; Greene, L A; Weinstein, I B

    1995-01-01

    The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells. Images PMID:7626808

  10. Coexistence of translocated cytochrome c and nitrated protein in neurons of the rat cerebral cortex after oxygen and glucose deprivation.

    PubMed

    Alonso, D; Encinas, J M; Uttenthal, L O; Boscá, L; Serrano, J; Fernández, A P; Castro-Blanco, S; Santacana, M; Bentura, M L; Richart, A; Fernández-Vizarra, P; Rodrigo, J

    2002-01-01

    Changes in the distribution of immunoreactive cytochrome c and protein nitration were studied in the rat cerebral cortex after oxygen and glucose deprivation by bright field, confocal and electron microscopy. In control cerebral cortex, nitrotyrosine immunoreactivity indicating protein nitration was found mostly in the neuronal nuclear region, with only a small amount distributed in the cytosol, whereas cytochrome c immunoreactivity was found at the inner membrane and in the intermembrane space of the mitochondria. During the recovery phase after oxygen and glucose deprivation, cytochrome c immunoreactivity was released from the intermembrane space of swollen mitochondria into the surrounding cytosol. The cytosol now also displayed nitrotyrosine immunoreactivity, which had diminished in the nuclear region. Both immunoreactivities were dispersed throughout the soma and processes of the cortical neurons. These changes were largely prevented by the administration of cyclosporin A, which inhibits both the mitochondrial permeability transition and the neuronal isoform of nitric oxide synthase while blocking the induction of the inducible isoform. Ischemia/reperfusion injury increases the production of nitric oxide, reactive oxygen species and intracellular factors that damage the mitochondria and liberate apoptotic factors. We suggest that translocation of cytochrome c from the mitochondria to the cytosol, which has been shown to precede the mitochondrial permeability transition, could result from peroxynitrite-mediated nitration. This phenomenon is attenuated by cyclosporin A administration, suggesting a neuroprotective role for this agent.

  11. The 18-kDa Translocator Protein (TSPO) Disrupts Mammary Epithelial Morphogenesis and Promotes Breast Cancer Cell Migration

    PubMed Central

    Wu, Xiaoting; Gallo, Kathleen A.

    2013-01-01

    Mitochondria play important roles in cancer progression and have emerged as viable targets for cancer therapy. Increasing levels of the outer mitochondrial membrane protein, 18-kDa translocator protein (TSPO), are associated with advancing breast cancer stage. In particular, higher TSPO levels are found in estrogen receptor (ER)-negative breast tumors, compared with ER-positive tumors. In this study, we sought to define the roles of TSPO in the acquisition of breast cancer malignancy. Using a three-dimensional Matrigel culture system, we determined the impact of elevated TSPO levels on mammary epithelial morphogenesis. Our studies demonstrate that stable overexpression of TSPO in mammary epithelial MCF10A acini drives proliferation and provides partial resistance to luminal apoptosis, resulting in enlarged acinar structures with partially filled lumen that resemble early stage breast lesions leading to breast cancer. In breast cancer cell lines, TSPO silencing or TSPO overexpression significantly altered the migratory activity. In addition, we found that combination treatment with the TSPO ligands (PK 11195 or Ro5-4864) and lonidamine, a clinical phase II drug targeting mitochondria, decreased viability of ER-negative breast cancer cell lines. Taken together, these data demonstrate that increases in TSPO levels at different stages of breast cancer progression results in the acquisition of distinct properties associated with malignancy. Furthermore, targeting TSPO, particularly in combination with other mitochondria-targeting agents, may prove useful for the treatment of ER-negative breast cancer. PMID:23967175

  12. Investigating the interactions of the 18kDa translocator protein and its ligand PK11195 in planar lipid bilayers.

    PubMed

    Hatty, Claire R; Le Brun, Anton P; Lake, Vanessa; Clifton, Luke A; Liu, Guo Jun; James, Michael; Banati, Richard B

    2014-03-01

    The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.

  13. Black pepper and piperine reduce cholesterol uptake and enhance translocation of cholesterol transporter proteins.

    PubMed

    Duangjai, Acharaporn; Ingkaninan, Kornkanok; Praputbut, Sakonwun; Limpeanchob, Nanteetip

    2013-04-01

    Black pepper (Piper nigrum L.) lowers blood lipids in vivo and inhibits cholesterol uptake in vitro, and piperine may mediate these effects. To test this, the present study aimed to compare actions of black pepper extract and piperine on (1) cholesterol uptake and efflux in Caco-2 cells, (2) the membrane/cytosol distribution of cholesterol transport proteins in these cells, and (3) the physicochemical properties of cholesterol micelles. Piperine or black pepper extract (containing the same amount of piperine) dose-dependently reduced cholesterol uptake into Caco-2 cells in a similar manner. Both preparations reduced the membrane levels of NPC1L1 and SR-BI proteins but not their overall cellular expression. Micellar cholesterol solubility of lipid micelles was unaffected except by 1 mg/mL concentration of black pepper extract. These data suggest that piperine is the active compound in black pepper and reduces cholesterol uptake by internalizing the cholesterol transporter proteins.

  14. Loss of retinoschisin (RS1) cell surface protein in maturing mouse rod photoreceptors elevates the luminance threshold for light-driven translocation of transducin but not arrestin.

    PubMed

    Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bush, Ronald A; Sieving, Paul A

    2012-09-19

    Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1-KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1-KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1-KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1-KO retinas was 10-fold higher than WT, but it decreased to <2.5-fold higher by P60. Light-activated arrestin translocation and re-translocation of transducin in the dark were not affected. Rs1-KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1-KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1-KO mice at P21 but not at P60. Expression of transducin was 15-30% lower in P21 Rs1-KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1-KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1-KO photoreceptors.

  15. Phosphatidylserine translocation at the yeast trans-Golgi network regulates protein sorting into exocytic vesicles

    PubMed Central

    Hankins, Hannah M.; Sere, Yves Y.; Diab, Nicholas S.; Menon, Anant K.; Graham, Todd R.

    2015-01-01

    Sorting of plasma membrane proteins into exocytic vesicles at the yeast trans-Golgi network (TGN) is believed to be mediated by their coalescence with specific lipids, but how these membrane-remodeling events are regulated is poorly understood. Here we show that the ATP-dependent phospholipid flippase Drs2 is required for efficient segregation of cargo into exocytic vesicles. The plasma membrane proteins Pma1 and Can1 are missorted from the TGN to the vacuole in drs2∆ cells. We also used a combination of flippase mutants that either gain or lose the ability to flip phosphatidylserine (PS) to determine that PS flip by Drs2 is its critical function in this sorting event. The primary role of PS flip at the TGN appears to be to control the oxysterol-binding protein homologue Kes1/Osh4 and regulate ergosterol subcellular distribution. Deletion of KES1 suppresses plasma membrane–missorting defects and the accumulation of intracellular ergosterol in drs2 mutants. We propose that PS flip is part of a homeostatic mechanism that controls sterol loading and lateral segregation of protein and lipid domains at the TGN. PMID:26466678

  16. Protein and Quality Characterization of Triticale Translocation Lines in Bread Making

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction of high molecular weight glutenin subunits (HMW-GS) from the Glu-Did locus of wheat into triticale restores the genetic constitution of storage protein loci to that of wheat and subsequently improves the bread making quality of triticale. One means to achieve such restoration of the gen...

  17. Protein Translocation: SecA-SecY Conformational Crosstalk Opens Channel.

    PubMed

    Kuhn, Andreas; Dalbey, Ross E

    2016-09-12

    A new study of the bacterial Sec translocase complex reports that ADP/ATP binding to SecA triggers multiple conformational changes in the SecYEG channel that may allow the passive directional movement of the polypeptide chain. PMID:27623265

  18. Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells

    PubMed Central

    Manku, Gurpreet; Culty, Martine

    2016-01-01

    Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology. PMID:27608010

  19. Anxiolytic-like effects of translocator protein (TSPO) ligand ZBD-2 in an animal model of chronic pain.

    PubMed

    Wang, Dong-Sheng; Tian, Zhen; Guo, Yan-Yan; Guo, Hong-Liang; Kang, Wen-Bo; Li, Shuo; Den, Ya-Ting; Li, Xu-Bo; Feng, Bing; Feng, Dan; Zhao, Jian-Ning; Liu, Gang; Zhao, Ming-Gao

    2015-03-26

    The activation of Translocator protein (18 kDa) (TSPO) has been demonstrated to mediate rapid anxiolytic efficacy in stress response and stress-related disorders. This protein is involved in the synthesis of endogenous neurosteroids that promote γ-aminobutyric acid (GABA)-mediated neurotransmission in the central neural system. However, little is known about the functions and the underlying mechanisms of TSPO in chronic pain-induced anxiety-like behaviors. The novel TSPO ligand N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2) was used in the present study. We found that ZBD-2 (0.15 or 1.5 mg/kg) significantly attenuated anxiety-like behaviors in mice with chronic inflammatory pain induced by hindpaw injection of complete Freund's adjuvant (CFA). However, the treatment did not alter the nociceptive threshold or inflammation in the hindpaw. Hindpaw injection of CFA induced the upregulation of TSPO, GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and NR2B-containing N-methyl-D-aspartate (NMDA) receptors in the basolateral amygdala (BLA). ZBD-2 administration reversed the alterations of the abovementioned proteins in the BLA of the CFA-injected mice. Electrophysiological recording revealed that ZBD-2 could prevent an imbalance between excitatory and inhibitory transmissions in the BLA synapses of CFA-injected mice. Therefore, as the novel ligand of TSPO, ZBD-2 induced anxiolytic effects, but did not affect the nociceptive threshold of mice under chronic pain. The anxiolytic effects of ZBD-2 were related to the regulation of the balance between excitatory and inhibitory transmissions in the BLA.

  20. Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells.

    PubMed

    Manku, Gurpreet; Culty, Martine

    2016-09-06

    Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology.

  1. Adenosine monophosphate-activated protein kinase activation, substrate transporter translocation, and metabolism in the contracting hyperthyroid rat heart.

    PubMed

    Heather, Lisa C; Cole, Mark A; Atherton, Helen J; Coumans, Will A; Evans, Rhys D; Tyler, Damian J; Glatz, Jan F C; Luiken, Joost J F P; Clarke, Kieran

    2010-01-01

    Thyroid hormones can modify cardiac metabolism via multiple molecular mechanisms, yet their integrated effect on overall substrate metabolism is poorly understood. Here we determined the effect of hyperthyroidism on substrate metabolism in the isolated, perfused, contracting rat heart. Male Wistar rats were injected for 7 d with T(3) (0.2 mg/kg x d ip). Plasma free fatty acids increased by 97%, heart weights increased by 33%, and cardiac rate pressure product, an indicator of contractile function, increased by 33% in hyperthyroid rats. Insulin-stimulated glycolytic rates and lactate efflux rates were increased by 33% in hyperthyroid rat hearts, mediated by an increased insulin-stimulated translocation of the glucose transporter GLUT4 to the sarcolemma. This was accompanied by a 70% increase in phosphorylated AMP-activated protein kinase (AMPK) and a 100% increase in phosphorylated acetyl CoA carboxylase, confirming downstream signaling from AMPK. Fatty acid oxidation rates increased in direct proportion to the increased heart weight and rate pressure product in the hyperthyroid heart, mediated by synchronized changes in mitochondrial enzymes and respiration. Protein levels of the fatty acid transporter, fatty acid translocase (FAT/CD36), were reduced by 24% but were accompanied by a 19% increase in the sarcolemmal content of fatty acid transport protein 1 (FATP1). Thus, the relationship between fatty acid metabolism, cardiac mass, and contractile function was maintained in the hyperthyroid heart, associated with a sarcolemmal reorganization of fatty acid transporters. The combined effects of T(3)-induced AMPK activation and insulin stimulation were associated with increased sarcolemmal GLUT4 localization and glycolytic flux in the hyperthyroid heart. PMID:19940039

  2. Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells.

    PubMed

    Manku, Gurpreet; Culty, Martine

    2016-01-01

    Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology. PMID:27608010

  3. Phospholipid translocation and miltefosine potency require both L. donovani miltefosine transporter and the new protein LdRos3 in Leishmania parasites.

    PubMed

    Pérez-Victoria, F Javier; Sánchez-Cañete, María P; Castanys, Santiago; Gamarro, Francisco

    2006-08-18

    The antitumor drug miltefosine has been recently approved as the first oral drug active against visceral leishmaniasis. We have previously identified the L. donovani miltefosine transporter (LdMT) as a P-type ATPase involved in phospholipid translocation at the plasma membrane of Leishmania parasites. Here we show that this protein is essential but not sufficient for the phospholipid translocation activity and, thus, for the potency of the drug. Based on recent findings in yeast, we have identified the putative beta subunit of LdMT, named LdRos3, as another protein factor required for the translocation activity. LdRos3 belongs to the CDC50/Lem3 family, proposed as likely beta subunits for P4-ATPases. The phenotype of LdRos3-defective parasites was identical to that of the LdMT-/-, including a defect in the uptake of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-phosphatidylserine, generally considered as not affected in Lem3p-deficient yeast. Both LdMT and LdRos3 normally localized to the plasma membrane but were retained inside the endoplasmic reticulum in the absence of the other protein or when inactivating point mutations were introduced in LdMT. Modulating the expression levels of either protein independently, we show that any one of them could behave as the protein limiting the level of flippase activity. Thus, LdMT and LdRos3 seem to form part of the same translocation machinery that determines flippase activity and miltefosine sensitivity in Leishmania, further supporting the consideration of CDC50/Lem3 proteins as beta subunits required for the normal functioning of P4-ATPases. PMID:16785229

  4. Dynamic interactions of proteins in complex networks

    SciTech Connect

    Appella, E.; Anderson, C.

    2009-10-01

    Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact with the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing

  5. Isolation of proteins and protein complexes by immunoprecipitation.

    PubMed

    Kaboord, Barbara; Perr, Maria

    2008-01-01

    Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures. Three different approaches for performing IP will be discussed; traditional (classical) method, oriented affinity method and direct affinity method. The traditional method of incubating the IP antibody with the sample and sequentially binding to Protein A or G agarose beads (resin) facilitates the most efficient target antigen recovery. However, this approach results in the target protein becoming contaminated with the IP antibody that can interfere with downstream analyses. The orientated affinity method uses Protein A or G beads to serve as an anchor to which the IP antibody is crosslinked thereby preventing the antibody from co-eluting with the target protein. Similarly, the direct affinity method also immobilizes the IP antibody except in this case it is directly attached to a chemically activated support. Both methods prevent co-elution of the IP antibody enabling reuse of the immunomatrix. All three approaches have unique advantages and can also be used for co-immunoprecipitation to study protein:protein interactions and investigate the functional proteome.

  6. Calcium translocation mechanism in sarcoplasmic reticulum vesicles, deduced from location studies of protein-bound spin labels.

    PubMed Central

    Champeil, P; Rigaud, J L; Gary-Bobo, C M

    1980-01-01

    Sarcoplasmic reticulum vesicles were exposed to various thiol-directed spin labels, and the position of the label on the inner or outer vesicle surface was investigated as a function of the ATPase (adenosinetriphosphatase; ATP phosphohydrolase, EC 3.6.1.3) chemical state. Previous measurements of label accessibility to externally added ascorbate had been considered to suggest an external-internal transition of protein-bound labels, coupled with ion translocation [Tonomura, Y. & Morales, M.F. (1974) Proc. Natl. Acad. Sci. USA 71, 3687-3691]. We show that these ascorbate studies do not lead to convincing conclusions. We demonstrate, on the contrary, that transition ions (nickel and ferricyanide) can be used as selective line-broadening agents for the signals arising from external labels. No significant difference in nickel- or ferricyanide-label interaction can be attributed to a different orientation of the label in any of the enzyme chemical states tested. Our results therefore contradict the current interpretation of ascorbate quenching experiments in terms of calcium ATPase rotatory motion; rather they are consistent with ion transport models involving only limited conformational rearrangements of the pump. PMID:6446710

  7. Overexpression of Vesicle-associated Membrane Protein (VAMP) 3, but Not VAMP2, Protects Glucose Transporter (GLUT) 4 Protein Translocation in an in Vitro Model of Cardiac Insulin Resistance*

    PubMed Central

    Schwenk, Robert W.; Angin, Yeliz; Steinbusch, Laura K. M.; Dirkx, Ellen; Hoebers, Nicole; Coumans, Will A.; Bonen, Arend; Broers, Jos L. V.; van Eys, Guillaume J. J. M.; Glatz, Jan F. C.; Luiken, Joost J. F. P.

    2012-01-01

    Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart. PMID:22936810

  8. Overexpression of vesicle-associated membrane protein (VAMP) 3, but not VAMP2, protects glucose transporter (GLUT) 4 protein translocation in an in vitro model of cardiac insulin resistance.

    PubMed

    Schwenk, Robert W; Angin, Yeliz; Steinbusch, Laura K M; Dirkx, Ellen; Hoebers, Nicole; Coumans, Will A; Bonen, Arend; Broers, Jos L V; van Eys, Guillaume J J M; Glatz, Jan F C; Luiken, Joost J F P

    2012-10-26

    Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.

  9. Expanding the Limits of Posterior Aortic Translocation: Biventricular Correction of Complex Transposition With Complete Atrioventricular Septal Defect and Heterotaxy.

    PubMed

    Sugiura, Junya; Bierbach, Benjamin; Hraska, Viktor

    2016-02-01

    This case report describes successful repair of d-transposition of the great arteries with severe left ventricular outflow tract obstruction and complete atrioventricular septal defect associated with heterotaxy by the use of posterior aortic translocation combined with repair of the atrioventricular septal defect and systemic venous anomalies. PMID:26777936

  10. Drug Ligand-Induced Activation of Translocator Protein (TSPO) Stimulates Steroid Production by Aged Brown Norway Rat Leydig Cells

    PubMed Central

    Chung, J.-Y.; Chen, H.; Midzak, A.; Burnett, A. L.; Papadopoulos, V.

    2013-01-01

    Translocator protein (TSPO; 18 kDA) is a high-affinity cholesterol-binding protein that is integrally involved in cholesterol transfer from intracellular stores into mitochondria, the rate-determining step in steroid formation. Previous studies have shown that TSPO drug ligands are able to activate steroid production by MA-10 mouse Leydig tumor cells and by mitochondria isolated from steroidogenic cells. We hypothesized herein that the direct, pharmacological activation of TSPO might induce aged Leydig cells, which are characterized by reduced T production, to produce significantly higher levels of T both in vitro and in vivo. To test this, we first examined the in vitro effects of the TSPO selective and structurally distinct drug ligands N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27) and benzodiazepine 4′-chlorodiazepam (Ro5-4864) on steroidogenesis by Leydig cells isolated from aged (21-24 months old) and young adult (3-6 months old) Brown Norway rats. The ligands stimulated Leydig cell T production significantly, and equivalently, in cells of both ages, an effect that was significantly inhibited by the specific TSPO inhibitor 5-androsten-3,17,19-triol (19-Atriol). Additionally, we examined the in vivo effects of administering FGIN-1-27 to young and aged rats. In both cases, serum T levels increased significantly, consistent with the in vitro results. Indeed, serum T levels in aged rats administered FGIN-1-27 were equivalent to T levels in the serum of control young rats. Taken together, these results indicate that although there are reduced amounts of TSPO in aged Leydig cells, its direct activation is able to increase T production. We suggest that this approach might serve as a therapeutic means to increase steroid levels in vivo in cases of primary hypogonadism. PMID:23525219

  11. Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis.

    PubMed

    Tu, Lan N; Morohaku, Kanako; Manna, Pulak R; Pelton, Susanne H; Butler, W Ronald; Stocco, Douglas M; Selvaraj, Vimal

    2014-10-01

    Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knock-out mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo(-/-)) mice. Contrary to the early report, Tspo(-/-) mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo(-/-) mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo(-/-) mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo(-/-) mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine.

  12. A voltage-gated pore for translocation of tRNA

    SciTech Connect

    Koley, Sandip; Adhya, Samit

    2013-09-13

    Highlights: •A tRNA translocating complex was assembled from purified proteins. •The complex translocates tRNA at a membrane potential of ∼60 mV. •Translocation requires Cys and His residues in the Fe–S center of RIC6 subunit. -- Abstract: Very little is known about how nucleic acids are translocated across membranes. The multi-subunit RNA Import Complex (RIC) from mitochondria of the kinetoplastid protozoon Leishmania tropica induces translocation of tRNAs across artificial or natural membranes, but the nature of the translocation pore remains unknown. We show that subunits RIC6 and RIC9 assemble on the membrane in presence of subunit RIC4A to form complex R3. Atomic Force Microscopy of R3 revealed particles with an asymmetric surface groove of ∼20 nm rim diameter and ∼1 nm depth. R3 induced translocation of tRNA into liposomes when the pH of the medium was lowered to ∼6 in the absence of ATP. R3-mediated tRNA translocation could also be induced at neutral pH by a K{sup +} diffusion potential with an optimum of 60–70 mV. Point mutations in the Cys{sub 2}–His{sub 2} Fe-binding motif of RIC6, which is homologous to the respiratory Complex III Fe–S protein, abrogated import induced by low pH but not by K{sup +} diffusion potential. These results indicate that the R3 complex forms a pore that is gated by a proton-generated membrane potential and that the Fe–S binding region of RIC6 has a role in proton translocation. The tRNA import complex of L. tropica thus contains a novel macromolecular channel distinct from the mitochondrial protein import pore that is apparently involved in tRNA import in some species.

  13. Border control: selectivity of chloroplast protein import and regulation at the TOC-complex.

    PubMed

    Demarsy, Emilie; Lakshmanan, Ashok M; Kessler, Felix

    2014-01-01

    Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC-TIC (translocon of outer membrane complex-translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms.

  14. Protein Kinase D and Gβγ Subunits Mediate Agonist-evoked Translocation of Protease-activated Receptor-2 from the Golgi Apparatus to the Plasma Membrane.

    PubMed

    Jensen, Dane D; Zhao, Peishen; Jimenez-Vargas, Nestor N; Lieu, TinaMarie; Gerges, Marina; Yeatman, Holly R; Canals, Meritxell; Vanner, Stephen J; Poole, Daniel P; Bunnett, Nigel W

    2016-05-20

    Agonist-evoked endocytosis of G protein-coupled receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled receptors from intracellular stores are unexplored. Protease-activated receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca(2+) signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases. PMID:27030010

  15. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    SciTech Connect

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  16. Radiation damage to DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Spotheim-Maurizot, M.; Davídková, M.

    2011-01-01

    We review here the advances in understanding the effects of ionizing radiations on DNA, proteins and their complexes, resulting from the collaboration of the authors' teams. It concerns the preponderant indirect effect of low LET ionizing radiations, thus the attack of the macromolecules in aqueous solution by the most aggressive product of water radiolysis, the hydroxyl radical. A model of simulation of the reaction of these radicals with the macromolecules (called RADACK) was developed and was used for calculating the probabilities of damage of each constituent of DNA or proteins (nucleotide or amino-acid). The calculations allowed to draw conclusions from electrophoresis, mutagenesis, spectroscopic (fluorescence, circular dichroïsm) and mass spectrometry experiments. Thus we have shown that the extent and location of the lesions are strongly dependent on the 3D structure of the macromolecules, which in turns is modulated by their sequence and by the binding of some ligands. Molecular dynamics simulation completed our studies in showing the consequences of each lesion on the stability and structure of the proteins and their complexes with DNA.

  17. Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY.

    PubMed

    Cannon, Kurt S; Or, Eran; Clemons, William M; Shibata, Yoko; Rapoport, Tom A

    2005-04-25

    During their biosynthesis, many proteins pass through the membrane via a hydrophilic channel formed by the heterotrimeric Sec61/SecY complex. Whether this channel forms at the interface of multiple copies of Sec61/SecY or is intrinsic to a monomeric complex, as suggested by the recently solved X-ray structure of the Methanococcus jannaschii SecY complex, is a matter of contention. By introducing a single cysteine at various positions in Escherichia coli SecY and testing its ability to form a disulfide bond with a single cysteine in a translocating chain, we provide evidence that translocating polypeptides pass through the center of the SecY complex. The strongest cross-links were observed with residues that would form a constriction in an hourglass-shaped pore. This suggests that the channel makes only limited contact with a translocating polypeptide, thus minimizing the energy required for translocation. PMID:15851514

  18. Water-Soluble Coenzyme Q10 Inhibits Nuclear Translocation of Apoptosis Inducing Factor and Cell Death Caused by Mitochondrial Complex I Inhibition

    PubMed Central

    Li, Haining; Chen, Guisheng; Ma, Wanrui; Li, Ping-An Andy

    2014-01-01

    The objectives of the study were to explore the mechanism of rotenone-induced cell damage and to examine the protective effects of water-soluble Coenzyme Q10 (CoQ10) on the toxic effects of rotenone. Murine hippocampal HT22 cells were cultured with mitochondrial complex I inhibitor rotenone. Water-soluble CoQ10 was added to the culture media 3 h prior to the rotenone incubation. Cell viability was determined by alamar blue, reactive oxygen species (ROS) production by dihydroethidine (DHE) and mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM). Cytochrome c, caspase-9 and apoptosis-inducing factor (AIF) were measured using Western blotting after 24 h rotenone incubation. Rotenone caused more than 50% of cell death, increased ROS production, AIF nuclear translocation and reduction in mitochondrial membrane potential, but failed to cause mitochondrial cytochrome c release and caspase-9 activation. Pretreatment with water-soluble CoQ10 enhanced cell viability, decreased ROS production, maintained mitochondrial membrane potential and prevented AIF nuclear translocation. The results suggest that rotenone activates a mitochondria-initiated, caspase-independent cell death pathway. Water-soluble CoQ10 reduces ROS accumulation, prevents the fall of mitochondrial membrane potential, and inhibits AIF translocation and subsequent cell death. PMID:25089873

  19. Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system.

    PubMed

    Schröder, H C; Rottmann, M; Bachmann, M; Müller, W E; McDonald, A R; Agutter, P S

    1986-08-15

    Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.

  20. Quality Control of a Cytoplasmic Protein Complex

    PubMed Central

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H.

    2015-01-01

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2. PMID:25564609

  1. Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish.

    PubMed

    Lu, Jin Fang; Wang, Wei Na; Wang, Gai Ling; Zhang, He; Zhou, Ying; Gao, Zhi Peng; Nie, Pin; Xie, Hai Xia

    2016-01-01

    The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens. PMID:26459509

  2. Cigarette smoke-induced reduction in binding of the salivary translocator protein is not mediated by free radicals.

    PubMed

    Nagler, R; Savulescu, D; Gavish, M

    2016-02-01

    Oral cancer is the most common malignancy of the head and neck and its main inducer is exposure to cigarette smoke (CS) in the presence of saliva. It is commonly accepted that CS contributes to the pathogenesis of oral cancer via reactive free radicals and volatile aldehydes. The 18 kDa translocator protein (TSPO) is an intracellular receptor involved in proliferation and apoptosis, and has been linked to various types of cancer. The presence of TSPO in human saliva has been linked to oral cancer, and its binding affinity to its ligand is reduced following exposure to CS. In the present study we wished to further investigate the mechanism behind the CS-induced reduction of TSPO binding by exploring the possible mediatory role of reactive oxygen species (ROS) and volatile aldehydes in this process. We first analyzed TSPO binding in control saliva and in saliva exposed to CS in the presence and absence of various antioxidants. These experiments found that TSPO binding ability was not reversed by any of the antioxidants added, suggesting that CS exerts its effect on TSPO via mechanisms that do not involve volatile aldehydes and free radicals tested. Next, we analyzed TSPO binding in saliva following addition of exogenous ROS in the form of H2O2. These experiments found that TSPO binding was enhanced due to the treatment, once again showing that the CS-induced TSPO binding reduction is not mediated by this common form of ROS. However, the previously reported CS-induced reduction in salivary TSPO binding together with the role of TSPO in cells and its link to cancer strongly suggest that TSPO has a critical role in the pathogenesis of CS-induced oral cancer. The importance of further elucidating the mechanisms behind it should be emphasized.

  3. Prioritizing protein complexes implicated in human diseases by network optimization

    PubMed Central

    2014-01-01

    Background The detection of associations between protein complexes and human inherited diseases is of great importance in understanding mechanisms of diseases. Dysfunctions of a protein complex are usually defined by its member disturbance and consequently result in certain diseases. Although individual disease proteins have been widely predicted, computational methods are still absent for systematically investigating disease-related protein complexes. Results We propose a method, MAXCOM, for the prioritization of candidate protein complexes. MAXCOM performs a maximum information flow algorithm to optimize relationships between a query disease and candidate protein complexes through a heterogeneous network that is constructed by combining protein-protein interactions and disease phenotypic similarities. Cross-validation experiments on 539 protein complexes show that MAXCOM can rank 382 (70.87%) protein complexes at the top against protein complexes constructed at random. Permutation experiments further confirm that MAXCOM is robust to the network structure and parameters involved. We further analyze protein complexes ranked among top ten for breast cancer and demonstrate that the SWI/SNF complex is potentially associated with breast cancer. Conclusions MAXCOM is an effective method for the discovery of disease-related protein complexes based on network optimization. The high performance and robustness of this approach can facilitate not only pathologic studies of diseases, but also the design of drugs targeting on multiple proteins. PMID:24565064

  4. Acute leukemias of different lineages have similar MLL gene fusions encoding related chimeric proteins resulting from chromosomal translocation

    SciTech Connect

    Corral, J.; Forster, A.; Thompson, S.; Rabbitts, T.H. ); Lampert, F. ); Kaneko, Y. ); Slater, R.; Kroes, W.G. ); Van Der Schoot, C.E. ); Ludwig, W.D. ); Karpas, A. ); Pocock, C.; Cotter, F. )

    1993-09-15

    The MLL gene, on human chromosome 11q23, undergoes chromosomal translocation in acute leukemias, resulting in gene fusion with AF4 (chromosome 4) and ENL (chromosome 19). The authors report here translocation of MLL with nine different chromosomes and two paracentric chromosome 11 deletions in early B cell, B- or T-cell lineage, or nonlymphocytic acute leukemias. The mRNA translocation junction from 22t(4;11) patients, including six adult leukemias, and nine t(11;19) tumors reveals a remarkable conservation of breakpoints within MLL, AF4, or ENL genes, irrespective of tumor phenotype. Typically, the breakpoints are upstream of the zinc-finger region of MLL, and deletion of this region can accompany translocation, supporting the der(11) chromosome as the important component in leukemogenesis. Partial sequence of a fusion between MLL and the AFX1 gene from chromosome X shows the latter to be rich in Ser/Pro codons, like the ENL mRNA. These data suggest that the heterogeneous 11q23 abnormalities might cause attachment of Ser/Pro-rich segments to the NH[sub 2] terminus of MLL, lacking the zinc-finger region, and that translocation occurs in early hematopoietic cells, before commitment to distinct lineages. 36 refs., 2 figs.

  5. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking.

    PubMed

    Heo, Lim; Lee, Hasup; Seok, Chaok

    2016-01-01

    Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex. PMID:27535582

  6. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking

    PubMed Central

    Heo, Lim; Lee, Hasup; Seok, Chaok

    2016-01-01

    Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex. PMID:27535582

  7. Probing nanoparticle effect in protein-surfactant complexes

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Aswal, V. K.; Kohlbrecher, J.

    2015-06-01

    SANS experiments have been carried to probe the role of anionic silica nanoparticles in the anionic BSA protein-cationic DTAB surfactant complexes. In protein-surfactant complex, surfactant molecules aggregate to form micelle-like clusters along the unfolded polypeptide chains of the protein. The nanoparticle aggregation mediated by oppositely charged protein-surfactant complex coexists with the free protein-surfactant complexes in the nanoparticle-protein-surfactant system. There is rearrangement of micelles in adsorbed protein-surfactant complex on nanoparticles in leading to their (nanoparticle) aggregation. On the other hand, the unfolding of protein in free protein-surfactant complex is found to be significantly enhanced in presence of nanoparticles.

  8. Loss of Retinoschisin (RS1) Cell Surface Protein in Maturing Mouse Rod Photoreceptors Elevates the Luminance Threshold for Light-Driven Translocation of Transducin But Not Arrestin

    PubMed Central

    Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bush, Ronald A.

    2012-01-01

    Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1–KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1–KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1–KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1–KO retinas was 10-fold higher than WT, but it decreased to <2.5-fold higher by P60. Light-activated arrestin translocation and re-translocation of transducin in the dark were not affected. Rs1–KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1–KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1–KO mice at P21 but not at P60. Expression of transducin was 15–30% lower in P21 Rs1–KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1–KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1–KO photoreceptors. PMID:22993419

  9. Translocation of Incoming Pseudorabies Virus Capsids to the Cell Nucleus Is Delayed in the Absence of Tegument Protein pUL37▿

    PubMed Central

    Krautwald, Mirjam; Fuchs, Walter; Klupp, Barbara G.; Mettenleiter, Thomas C.

    2009-01-01

    After fusion of the envelope of herpesvirus particles with the host cell plasma membrane, incoming nucleocapsids are transported to nuclear pores. Inner tegument proteins pUL36, pUL37, and pUS3 remain attached to the nucleocapsid after entry and therefore might mediate interactions between the nucleocapsid and cellular microtubule-associated motor proteins during transport. To assay for the role of pUL37 in this process, we constructed a pUL37-deleted pseudorabies virus mutant, PrV-ΔUL37/UL35GFP, which expresses a fusion protein of green fluorescent protein (GFP) and the nonessential small capsid protein pUL35, resulting in the formation of fluorescently labeled capsids. Confocal laser-scanning microscopy of rabbit kidney cells infected with PrV-ΔUL37/UL35GFP revealed that, whereas penetration was not affected in the absence of pUL37, nuclear translocation of incoming particles was delayed by approximately 1 h compared to PrV-UL35GFP, but not abolished. In contrast, phenotypically complemented pUL37-containing virions of PrV-ΔUL37/UL35GFP exhibited wild type-like entry kinetics. Thus, the presence of pUL37 is required for rapid nuclear translocation of incoming nucleocapsids. PMID:19144717

  10. Translocation of incoming pseudorabies virus capsids to the cell nucleus is delayed in the absence of tegument protein pUL37.

    PubMed

    Krautwald, Mirjam; Fuchs, Walter; Klupp, Barbara G; Mettenleiter, Thomas C

    2009-04-01

    After fusion of the envelope of herpesvirus particles with the host cell plasma membrane, incoming nucleocapsids are transported to nuclear pores. Inner tegument proteins pUL36, pUL37, and pUS3 remain attached to the nucleocapsid after entry and therefore might mediate interactions between the nucleocapsid and cellular microtubule-associated motor proteins during transport. To assay for the role of pUL37 in this process, we constructed a pUL37-deleted pseudorabies virus mutant, PrV-DeltaUL37/UL35GFP, which expresses a fusion protein of green fluorescent protein (GFP) and the nonessential small capsid protein pUL35, resulting in the formation of fluorescently labeled capsids. Confocal laser-scanning microscopy of rabbit kidney cells infected with PrV-DeltaUL37/UL35GFP revealed that, whereas penetration was not affected in the absence of pUL37, nuclear translocation of incoming particles was delayed by approximately 1 h compared to PrV-UL35GFP, but not abolished. In contrast, phenotypically complemented pUL37-containing virions of PrV-DeltaUL37/UL35GFP exhibited wild type-like entry kinetics. Thus, the presence of pUL37 is required for rapid nuclear translocation of incoming nucleocapsids. PMID:19144717

  11. RNA interference targeting rye secalins alters flour protein composition in a wheat variety carrying a 1Bl.1RS translocation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat varieties carrying chromosome translocations from rye are part of the international wheat breeding pool, despite being associated with defects in dough processing quality. Among the proposed causes for the quality defects of flours from such wheats is the presence of the secalins, encoded by ...

  12. Engineering of complex protein sialylation in plants.

    PubMed

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-08-23

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  13. Engineering of complex protein sialylation in plants

    PubMed Central

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-01-01

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  14. An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

    PubMed

    Lee, Hyeon-Cheol; Portnoff, Alyse D; Rocco, Mark A; DeLisa, Matthew P

    2014-12-22

    The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

  15. Robertsonian translocations

    SciTech Connect

    1993-12-31

    Chapter 27, describes the occurrence of Robertsonian translocations (RTs), which refer to the recombination of whole chromosome arms, in both monocentric and dicentric chromosomes. The nonrandom participation of acrocentric chromosomes in RTs is documented by various methods, including unbiased ascertainment and ascertainment through trisomy, infertility, unspecified mental retardation, and Prader-Willi syndrome. Causes of nonrandom participation of chromosomes in RTs is presented, as are the following topics: segregation in carriers of RTs and segregation in sperm cells of RT carriers, interchromosomal effects and conclusions. 48 refs., 3 figs., 2 tabs.

  16. Crystallographic snapshot of cellulose synthesis and membrane translocation

    PubMed Central

    Morgan, Jacob L.W.; Strumillo, Joanna; Zimmer, Jochen

    2012-01-01

    Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the multi-spanning catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-B translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time. PMID:23222542

  17. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    PubMed

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks.

  18. Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

    PubMed Central

    Fiester, Steven E.; Nwugo, Chika C.; Penwell, William F.; Neary, John M.; Beckett, Amber C.; Arivett, Brock A.; Schmidt, Robert E.; Geiger, Sarah C.; Connerly, Pamela L.; Menke, Sharon M.; Tomaras, Andrew P.

    2015-01-01

    Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen that causes pneumonia and soft tissue and systemic infections. Screening of a transposon insertion library of A. baumannii ATCC 19606T resulted in the identification of the 2010 derivative, which, although capable of growing well in iron-rich media, failed to prosper under iron chelation. Genetic, molecular, and functional assays showed that 2010's iron utilization-deficient phenotype is due to an insertion within the 3′ end of secA, which results in the production of a C-terminally truncated derivative of SecA. SecA plays a critical role in protein translocation through the SecYEG membrane channel. Accordingly, the secA mutation resulted in undetectable amounts of the ferric acinetobactin outer membrane receptor protein BauA while not affecting the production of other acinetobactin membrane protein transport components, such as BauB and BauE, or the secretion of acinetobactin by 2010 cells cultured in the presence of subinhibitory concentrations of the synthetic iron chelator 2,2′-dipyridyl. Outer membrane proteins involved in nutrient transport, adherence, and biofilm formation were also reduced in 2010. The SecA truncation also increased production of 30 different proteins, including proteins involved in adaptation/tolerance responses. Although some of these protein changes could negatively affect the pathobiology of the 2010 derivative, its virulence defect is mainly due to its inability to acquire iron via the acinetobactin-mediated system. These results together indicate that although the C terminus of the A. baumannii ATCC 19606T SecA is not essential for viability, it plays a critical role in the production and translocation of different proteins and virulence. PMID:25605767

  19. Heteroplasmy and Ancient Translocation of Mitochondrial DNA to the Nucleus in the Chinese Horseshoe Bat (Rhinolophus sinicus) Complex

    PubMed Central

    Hua, Panyu; He, Guimei; Zhang, Shuyi; Rossiter, Stephen J.

    2014-01-01

    The utility and reliability of mitochondrial DNA sequences in phylogenetic and phylogeographic studies may be compromised by widespread and undetected nuclear mitochondrial copies (numts) as well as heteroplasmy within individuals. Both numts and heteroplasmy are likely to be common across diverse taxa yet few studies have characterised their frequencies and variation at the intra-specific level. Here we report the presence of both numts and heteroplasmy in the mitochondrial control region of the Chinese horseshoe bat Rhinolophus sinicus. In total we generated 123 sequences from 18 bats, which contained two different numt clades (i.e. Numt-1 and Numt-2) and one mtDNA clade. The sequence divergence between Numt-1 and Numt-2 was 16.8% and each numt type was found in all four R. sinicus taxa, suggesting either two ancient translocations of mitochondrial DNA into the nucleus from the same source taxon, or a single translocation from different source taxa that occurred before the split of R. sinicus into different lineages. Within the mtDNA clade, phylogenetic relationships among the four taxa of R. sinicus were similar to those seen in previous results. Based on PCR comparisons, heteroplasmy was inferred between almost all individuals of R. sinicus with respect to sequence variation. Consistent with introgression of mtDNA between Central sinicus and septentrionalis, individuals from these two taxa exhibited similar signatures of repeated sequences in the control region. Our study highlights the importance of testing for the presence of numts and heteroplasmy when applying mtDNA markers to phylogenetic studies. PMID:24842827

  20. Simulations of Polymer Translocation

    NASA Astrophysics Data System (ADS)

    Vocks, H.

    2008-07-01

    Transport of molecules across membranes is an essential mechanism for life processes. These molecules are often long, and the pores in the membranes are too narrow for the molecules to pass through as a single unit. In such circumstances, the molecules have to squeeze -- i.e., translocate -- themselves through the pores. DNA, RNA and proteins are such naturally occuring long molecules in a variety of biological processes. Understandably, the process of translocation has been an active topic of current research: not only because it is a cornerstone of many biological processes, but also due to its relevance for practical applications. Translocation is a complicated process in living organisms -- the presence of chaperone molecules, pH, chemical potential gradients, and assisting molecular motors strongly influence its dynamics. Consequently, the translocation process has been empirically studied in great variety in biological literature. Study of translocation as a biophysical process is more recent. Herein, the polymer is simplified to a sequentially connected string of N monomers as it passes through a narrow pore on a membrane. The quantities of interest are the typical time scale for the polymer to leave a confining cell (the ``escape of a polymer from a vesicle'' time scale), and the typical time scale the polymer spends in the pore (the ``dwell'' time scale) as a function of N and other parameters like membrane thickness, membrane adsorption, electrochemical potential gradient, etc. Our research is focused on computer simulations of translocation. Since our main interest is in the scaling properties, we use a highly simplified description of the translocation process. The polymer is described as a self-avoiding walk on a lattice, and its dynamics consists of single-monomer jumps from one lattice site to another neighboring one. Since we have a very efficient program to simulate such polymer dynamics, which we decribe in Chapter 2, we can perform long

  1. A new approach to polymer translocation

    NASA Astrophysics Data System (ADS)

    Dubbeldam, Johan; Rostiashvili, Vakhtang; Milchev, Andrey; Vilgis, Thomas

    2011-03-01

    Polymer translocation is ubiquitous in nature. It plays a role in phenomena like virus infections and in trafficking of proteins through pores in a cell membrane. Many theoretical models have been developed to explain scaling properties of simple polymer chains through tiny nanopores. This has not resolved the controversies in this field, however. In this paper we employ novel methods to shed light on the results that were obtained using the different models that are in use today. We use, for example fractional Brownian motion to explain the scaling of the variance in the translocation length with time and find good agreement between simulation results and theoretical predictions. An extension of the theory to nanopores with more complex geometries are discussed.

  2. The role of phospholipase D in Glut-4 translocation.

    PubMed

    Huang, Ping; Frohman, Michael A

    2003-01-01

    Insulin-stimulated Glut-4 translocation is regulated through a complex pathway. Increasing attention is being paid to the role undertaken in this process by Phospholipase D, a signal transduction-activated enzyme that generates the lipid second-messenger phosphatidic acid. Phospholipase D facilitates Glut-4 translocation at potentially multiple steps in its outward movement. Current investigation is centered on Phospholipase D promotion of Glut-4-containing membrane vesicle trafficking and vesicle fusion into the plasma membrane, in part through activation of atypical protein kinase C isoforms. PMID:14648804

  3. The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis.

    PubMed

    Wood, M W; Jones, M A; Watson, P R; Siber, A M; McCormick, B A; Hedges, S; Rosqvist, R; Wallis, T S; Galyov, E E

    2000-08-01

    Salmonella-induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip-dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin. We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of enteritis.

  4. The mechanics of translocation: a molecular "spring-and-ratchet" system.

    PubMed

    Moran, Stephen J; Flanagan, John F; Namy, Olivier; Stuart, David I; Brierley, Ian; Gilbert, Robert J C

    2008-05-01

    The translation of genetic information into proteins is a fundamental process of life. Stepwise addition of amino acids to the growing polypeptide chain requires the coordinated movement of mRNA and tRNAs through the ribosome, a process known as translocation. Here, we review current understanding of the kinetics and mechanics of translocation, with particular emphasis on the structure of a functional mammalian ribosome stalled during translocation by an mRNA pseudoknot. In the context of a pseudoknot-stalled complex, the translocase EF-2 is seen to compress a hybrid-state tRNA into a strained conformation. We propose that this strain energy helps overcome the kinetic barrier to translocation and drives tRNA into the P-site, with EF-2 biasing this relaxation in one direction. The tRNA can thus be considered a molecular spring and EF-2 a Brownian ratchet in a "spring-and-ratchet" system within the translocation process.

  5. A complex-centric view of protein network evolution.

    PubMed

    Yosef, Nir; Kupiec, Martin; Ruppin, Eytan; Sharan, Roded

    2009-07-01

    The recent availability of protein-protein interaction networks for several species makes it possible to study protein complexes in an evolutionary context. In this article, we present a novel network-based framework for reconstructing the evolutionary history of protein complexes. Our analysis is based on generalizing evolutionary measures for single proteins to the level of whole subnetworks, comprehensively considering a broad set of computationally derived complexes and accounting for both sequence and interaction changes. Specifically, we compute sets of orthologous complexes across species, and use these to derive evolutionary rate and age measures for protein complexes. We observe significant correlations between the evolutionary properties of a complex and those of its member proteins, suggesting that protein complexes form early in evolution and evolve as coherent units. Additionally, our approach enables us to directly quantify the extent to which gene duplication has played a role in the evolution of complexes. We find that about one quarter of the sets of orthologous complexes have originated from evolutionary cores of homodimers that underwent duplication and divergence, testifying to the important role of gene duplication in protein complex evolution. PMID:19465379

  6. Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

    SciTech Connect

    Inadomi, Chiaki; Murata, Hiroaki; Ihara, Yoshito; Goto, Shinji; Urata, Yoshishige; Yodoi, Junji; Kondo, Takahito; Sumikawa, Koji

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. Black-Right-Pointing-Pointer NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. Black-Right-Pointing-Pointer Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

  7. Affinity Purification of Protein Complexes Using TAP Tags

    PubMed Central

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities. PMID:26096502

  8. Strandwise translocation of a DNA glycosylase on undamaged DNA.

    PubMed

    Qi, Yan; Nam, Kwangho; Spong, Marie C; Banerjee, Anirban; Sung, Rou-Jia; Zhang, Michael; Karplus, Martin; Verdine, Gregory L

    2012-01-24

    Base excision repair of genotoxic nucleobase lesions in the genome is critically dependent upon the ability of DNA glycosylases to locate rare sites of damage embedded in a vast excess of undamaged DNA, using only thermal energy to fuel the search process. Considerable interest surrounds the question of how DNA glycosylases translocate efficiently along DNA while maintaining their vigilance for target damaged sites. Here, we report the observation of strandwise translocation of 8-oxoguanine DNA glycosylase, MutM, along undamaged DNA. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. We further report that alterations of single base-pairs or a single amino acid substitution (R112A) can induce strandwise translocation. Molecular dynamics simulations confirm that MutM can translocate along DNA in a strandwise fashion. These observations reveal a previously unobserved mode of movement for a DNA-binding protein along the surface of DNA. PMID:22219368

  9. Complex translocation t(1;12;14)(q42;q14;q32) and HMGA2 deletion in a fetus presenting growth delay and bilateral cataracts.

    PubMed

    Raymond, Laure; Francou, Bruno; Petit, François; Tosca, Lucie; Briand-Suleau, Audrey; Metay, Corinne; Martinovic, Jelena; Cordier, Anne-Gaël; Benachi, Alexandra; Pineau, Dominique; Guiochon-Mantel, Anne; Goossens, Michel; Tachdjian, Gérard; Brisset, Sophie

    2015-11-01

    We report the prenatal detection of a de novo unbalanced complex chromosomal rearrangement (CCR), in a fetus with growth delay and bilateral cataracts. Standard karyotype and FISH analyses on amniotic fluid revealed a complex de novo translocation, resulting in a 46,XY,t(1;12;14)(q42;q14;q32) karyotype. CGH-array showed a significant deletion of 387  kb at 12q14.3, at a distance of only 200-700 kb from the breakpoint at 12q14, which encompassed the HMGA2 gene and occurred de novo. Although 12q14 microdeletions are associated with growth delay in several reports in the literature, we present here the smallest deletion prenatally detected, and we detail the clinical description of the fetus. The correlation between cataracts and this complex genotype is puzzling. Among the genes disrupted by the breakpoint in 12q14, GRIP1 has been associated with abnormal eye development in mice, including lens degeneration. Interestingly, HMGA2 is expressed in the mouse's developing lens, and its expression is decreased in lens of elderly humans, correlated with the severity of lens opacity. In this report, we refine the link between HMGA2 loss of function and growth delay during prenatal development. We also discuss the correlation between cataracts and genotype in this unbalanced CCR case of unexpected complexity.

  10. Polymer translocation through a cylindrical channel

    PubMed Central

    Wong, Chiu TaiAndrew; Muthukumar, M.

    2008-01-01

    A formalism of polymer translocation through a cylindrical channel of finite diameter and length between two spherical compartments is developed. Unlike previous simplified systems, the finite diameter of the channel allows the number of polymer segments inside the channel to be adjusted during translocation according to the free energy of possible conformations. The translocation process of a Gaussian chain without excluded volume and hydrodynamic interactions is studied using exact formulas of confinement free energy under this formalism. The free energy landscape for the translocation process, the distribution of the translocation time, and the average translocation time are presented. The complex dependencies of the average translocation time on the length and diameter of the channel, the sizes of the donor and receptor compartments, and the chain length are illustrated. PMID:18433273

  11. Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane

    NASA Astrophysics Data System (ADS)

    Rajapaksha, Suneth P.; Pal, Nibedita; Zheng, Desheng; Lu, H. Peter

    2015-11-01

    We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ˜15 Å diameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a

  12. Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane.

    PubMed

    Rajapaksha, Suneth P; Pal, Nibedita; Zheng, Desheng; Lu, H Peter

    2015-01-01

    We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ∼15 Å diameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a

  13. Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane.

    PubMed

    Rajapaksha, Suneth P; Pal, Nibedita; Zheng, Desheng; Lu, H Peter

    2015-01-01

    We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ∼15 Å diameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a

  14. The Rieske Fe/S protein of the cytochrome b6/f complex in chloroplasts: missing link in the evolution of protein transport pathways in chloroplasts?

    PubMed

    Molik, S; Karnauchov, I; Weidlich, C; Herrmann, R G; Klösgen, R B

    2001-11-16

    The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochrome b(6)/f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the DeltapH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH(2)-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of DeltapH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways. PMID:11526115

  15. The role of Hsp90 in protein complex assembly.

    PubMed

    Makhnevych, Taras; Houry, Walid A

    2012-03-01

    Hsp90 is a ubiquitous and essential molecular chaperone that plays central roles in many signaling and other cellular pathways. The in vivo and in vitro activity of Hsp90 depends on its association with a wide variety of cochaperones and cofactors, which form large multi-protein complexes involved in folding client proteins. Based on our proteomic work mapping the molecular chaperone interaction networks in yeast, especially that of Hsp90, as well as, on experiments and results presented in the published literature, one major role of Hsp90 appears to be the promotion and maintenance of proper assembly of protein complexes. To highlight this role of Hsp90, the effect of the chaperone on the assembly of the following seven complexes is discussed in this review: snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase-related protein kinase (PIKK), telomere complex, kinetochore, RNA induced silencing complexes (RISC), and 26S proteasome. For some complexes, it is observed that Hsp90 mediates complex assembly by stabilizing an unstable protein subunit and facilitating its incorporation into the complex; for other complexes, Hsp90 promotes change in the composition of that complex. In all cases, Hsp90 does not appear to be part of the final assembled complex. This article is part of a Special Issue entitled:Heat Shock Protein 90 (HSP90). PMID:21945180

  16. Systematic Analysis of Translocator Protein 18 kDa (TSPO) Ligands on Toll-like Receptors-mediated Pro-inflammatory Responses in Microglia and Astrocytes

    PubMed Central

    Lee, Ji-Won; Nam, Hyeri

    2016-01-01

    Translocator protein 18 kDa (TSPO) is a mitochondrial protein highly expressed on reactive microglia and astrocytes, and is considered as a biomarker for neurodegeneration and brain damage, especially neuroinflammation. Toll-like receptors (TLRs) are closely related with inflammatory responses of microglia and astrocytes and these signaling pathways regulate neuroinflammation. Previous reports have identified the anti-inflammatory effects of TSPO ligands, however study of their effects in relation to the TLR signaling was limited. Here, we investigated the effects of five representative TSPO ligands on microglia and astrocytes following activation by various TLR ligands. Our results show that TSPO ligands reduce the pro-inflammatory response elicited by the TLR ligands with more profound effects on microglia than astrocytes. PMID:27790060

  17. In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system.

    PubMed

    Kim, Jeong Hee; Lee, Min Jun; Hwang, Inhwan; Hwang, Hyun Jin

    2013-01-01

    This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s). The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.

  18. Design and characterization of complex protein films

    NASA Astrophysics Data System (ADS)

    Bui, Holt P.

    Once a biomaterial is implanted into biological system, a layer of protein is immediately deposited on the surface of that material. The newly formed protein film will dictate how the implanted material will interact with the surrounding biological environment and lead to either the acceptance or rejection of the biomaterial. One method to enhance performance involves the activation the surface of the biomaterial with one or more proteins to direct specific interactions with the host environment. The focus of my dissertation was to develop and characterize model biomaterials surfaces that are activated with one or more proteins to help understand how the protein films may affect biological processes and a biomaterial's performance. One model system consisted of a patterned film of two proteins on a gold surface. Characterization of this protein pattern indicated that patterning protein films with a focused ion beam produced protein patterns with high biological contrast and high spatial control. The second model protein film involved the adsorption of fibronectin on surfaces with different surface energies. The characterization of the adsorbed fibronectin films suggest that fibronectin adsorbed on a hydrophilic surface is in an orientation that projects hydrophilic amino acid residues towards surface of the protein and dehydration causes reorientation to project hydrophobic amino acids towards the surface. In contrast, fibronectin is adsorbed onto a hydrophobic surface in a manner that resulted in dehydration and denaturation during the adsorption process. The last model protein film studied in this work consisted of fibronectin patterned in a manner so that the film consisted of spatially controlled domains of fibronectin adsorbed onto a hydrophilic surface as well as a hydrophobic surface. Lateral characterization of this pattern demonstrated a difference in secondary structure of fibronectin adsorbed on the two domains with varying surface energies.

  19. Heavy Metal-induced Metallothionein Expression Is Regulated by Specific Protein Phosphatase 2A Complexes*

    PubMed Central

    Chen, Liping; Ma, Lu; Bai, Qing; Zhu, Xiaonian; Zhang, Jinmiao; Wei, Qing; Li, Daochuan; Gao, Chen; Li, Jie; Zhang, Zhengbao; Liu, Caixia; He, Zhini; Zeng, Xiaowen; Zhang, Aihua; Qu, Weidong; Zhuang, Zhixiong; Chen, Wen; Xiao, Yongmei

    2014-01-01

    Induction of metallothionein (MT) expression is involved in metal homeostasis and detoxification. To identify the key pathways that regulate metal-induced cytotoxicity, we investigate how phosphorylated metal-responsive transcription factor-1 (MTF-1) contributed to induction of MT expression. Immortal human embryonic kidney cells (HEK cells) were treated with seven kinds of metals including cadmium chloride (CdCl2), zinc sulfate (ZnSO4), copper sulfate(CuSO4), lead acetate (PbAc), nickel sulfate (NiSO4), sodium arsenite (NaAsO2), and potassium bichromate (K2Cr2O7). The MT expression was induced in a dose-response and time-dependent manner upon various metal treatments. A cycle of phosphorylation and dephosphorylation was required for translocation of MTF-1 from cytoplasm to nucleus, leading to the up-regulation of MTs expression. Protein phosphatase 2A (PP2A) participated in regulating MT expression through dephosphorylation of MTF-1. A loss-of-function screen revealed that the specific PP2A complexes containing PR110 were involved in metal-induced MT expression. Suppression of PP2A PR110 in HEK cells resulted in the persistent MTF-1 phosphorylation and the disturbance of MTF-1 nuclear translocation, which was concomitant with a significant decrease of MT expression and enhanced cytotoxicity in HEK cells. Notably, MTF-1 was found in complex with specific PP2A complexes containing the PR110 subunit upon metal exposure. Furthermore, we identify that the dephosphorylation of MTF-1 at residue Thr-254 is directly regulated by PP2A PR110 complexes and responsible for MTF-1 activation. Taken together, these findings delineate a novel pathway that determines cytotoxicity in response to metal treatments and provide new insight into the role of PP2A in cellular stress response. PMID:24962574

  20. High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli.

    PubMed

    Albiniak, Anna M; Matos, Cristina F R O; Branston, Steven D; Freedman, Robert B; Keshavarz-Moore, Eli; Robinson, Colin

    2013-08-01

    The twin-arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat-type signal peptide is present at the N-terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA-GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16-h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large-scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L⁻¹ culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing. PMID:23745597

  1. Recording information on protein complexes in an information management system.

    PubMed

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  2. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. PMID:26659058

  3. Recording information on protein complexes in an information management system.

    PubMed

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.

  4. Multi-LZerD: Multiple protein docking for asymmetric complexes

    PubMed Central

    Esquivel-Rodríguez, Juan; Yang, Yifeng David; Kihara, Daisuke

    2012-01-01

    The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Here, we have developed a novel computational multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on eleven hetero-multimeric complexes resulted in near native conformations for all but one of them (a root mean square deviation smaller than 2.5Å). We also show that our method copes with unbound docking cases well, outperforming the methodology that can be directly compared to our approach. Multi-LZerD was able to predict near native structures for multimeric complexes of various topologies. PMID:22488467

  5. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering.

  6. Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

    PubMed Central

    Gangopadhyay, Jaya Pal; Ikemoto, Noriaki

    2010-01-01

    We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca2+ leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes [Hamada et al., 2009]. The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca2+/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca2+ transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca2+ transients results in the appearance of trains of Ca2+ spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca2+ events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program. PMID

  7. Intracellular translocation of calmodulin and Ca{sup 2+}/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

    SciTech Connect

    Gangopadhyay, Jaya Pal; Ikemoto, Noriaki

    2010-05-28

    We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca{sup 2+} leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 ). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca{sup 2+}/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca{sup 2+} transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca{sup 2+} transients results in the appearance of trains of Ca{sup 2+} spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca{sup 2+} events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional

  8. Pseudomonas syringae lytic transglycosylases coregulated with the type III secretion system contribute to the translocation of effector proteins into plant cells.

    PubMed

    Oh, Hye-Sook; Kvitko, Brian H; Morello, Joanne E; Collmer, Alan

    2007-11-01

    Pseudomonas syringae translocates virulence effector proteins into plant cells via a type III secretion system (T3SS) encoded by hrp (for hypersensitive response and pathogenicity) genes. Three genes coregulated with the Hrp T3SS system in P. syringae pv. tomato DC3000 have predicted lytic transglycosylase domains: PSPTO1378 (here designated hrpH), PSPTO2678 (hopP1), and PSPTO852 (hopAJ1). hrpH is located between hrpR and avrE1 in the Hrp pathogenicity island and is carried in the functional cluster of P. syringae pv. syringae 61 hrp genes cloned in cosmid pHIR11. Strong expression of DC3000 hrpH in Escherichia coli inhibits bacterial growth unless the predicted catalytic glutamate at position 148 is mutated. Translocation tests involving C-terminal fusions with a Cya (Bordetella pertussis adenylate cyclase) reporter indicate that HrpH and HopP1, but not HopAJ1, are T3SS substrates. Pseudomonas fluorescens carrying a pHIR11 derivative lacking hrpH is poorly able to translocate effector HopA1, and this deficiency can be restored by HopP1 and HopAJ1, but not by HrpH(E148A) or HrpH(1-241). DC3000 mutants lacking hrpH or hrpH, hopP1, and hopAJ1 combined are variously reduced in effector translocation, elicitation of the hypersensitive response, and virulence. However, the mutants are not reduced in secretion of T3SS substrates in culture. When produced in wild-type DC3000, the HrpH(E148A) and HrpH(1-241) variants have a dominant-negative effect on the ability of DC3000 to elicit the hypersensitive response in nonhost tobacco and to grow and cause disease in host tomato. The three Hrp-associated lytic transglycosylases in DC3000 appear to have overlapping functions in contributing to T3SS functions during infection.

  9. Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

    SciTech Connect

    Yamagishi, Nobuyuki; Ishihara, Keiichi; Saito, Youhei; Hatayama, Takumi . E-mail: hatayama@mb.kyoto-phu.ac.jp

    2006-10-15

    Hsp105 (Hsp105{alpha} and Hsp105{beta}), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105{alpha} has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105{alpha} regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105{alpha} or Hsp105{beta} by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105{alpha} or Hsp105{beta}. In addition, we found that overexpression of Hsp105{alpha} or Hsp105{beta} suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105{alpha} or Hsp105{beta}. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells.

  10. Psychiatric disorder in a familial 15;18 translocation and sublocalization of myelin basic protein to 18q22.3

    SciTech Connect

    Calzolari, E.; Aiello, V.; Palazzi, P.; Sensi, A.

    1996-04-09

    Two related patients with similar clinical features consisting of a few dysmorphic signs and psychiatric disturbance were reported to have a partial trisomy of chromosomes 15(pter-q13.3) and 18(q23-qter) deriving from a familial translocation t(15;18). One patient is affected by bipolar disorder and the other by schizoaffective disorder. Both cases have a predominantly affective course; nevertheless, a clear diagnosis is difficult in the first patient, who is 15 years of age, and only a longitudinal course will allow us to establish a definite diagnosis. The possibility that these two pathologies belong to a single category is discussed, and the presence of a susceptibility locus on chromosome 18 is hypothesized. Cytogenetic data, FISH, and DNA studies indicate that the myelin basic protein (MPB) gene is not involved in the translocation, and localize it centromeric to the breakpoint on chromosome 18(q22.3). Thus, it is unlikely to be involved in the disease. 58 refs., 8 figs.

  11. Promoter swapping between the genes for a novel zinc finger protein and beta-catenin in pleiomorphic adenomas with t(3;8)(p21;q12) translocations.

    PubMed

    Kas, K; Voz, M L; Röijer, E; Aström, A K; Meyen, E; Stenman, G; Van de Ven, W J

    1997-02-01

    Pleiomorphic adenoma of the salivary glands is a benign epithelial tumour occurring primarily in the major and minor salivary glands. It is by far the most common type of salivary gland tumour. Microscopically, pleiomorphic adenomas show a marked histological diversity with epithelial, myoepithelial and mesenchymal components in a variety of patterns. In addition to a cytogenetic subgroup with normal karyotypes, pleiomorphic adenomas are characterized by recurrent chromosome rearrangements, particularly reciprocal translocations, with breakpoints at 8q12, 3p21, and 12q13-15, in that order of frequency. The most common abnormality is a reciprocal t(3;8)(p21;q12). We here demonstrate that the t(3;8)(p21;q12) results in promoter swapping between PLAG1, a novel, developmentally regulated zinc finger gene at 8q12, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein interface functioning in the WG/WNT signalling pathway and specification of cell fate during embryogenesis. Fusions occur in the 5'-non-coding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. Due to the t(3;8)(p21;q12), PLAG1 is activated and expression levels of CTNNB1 are reduced. Activation of PLAG1 was also observed in an adenoma with a variant translocation t(8;15)(q12;q14). Our results indicate that PLAG1 activation due to promoter swapping is a crucial event in salivary gland tumourigenesis.

  12. BZLF1, an Epstein-Barr virus immediate-early protein, induces p65 nuclear translocation while inhibiting p65 transcriptional function

    SciTech Connect

    Morrison, Thomas E.; Kenney, Shannon C. . E-mail: shann@med.unc.edu

    2004-10-25

    We have previously demonstrated that the Epstein-Barr virus immediate-early BZLF1 protein interacts with, and is inhibited by, the NF-{kappa}B family member p65. However, the effects of BZLF1 on NF-{kappa}B activity have not been intensively studied. Here we show that BZLF1 inhibits p65-dependent gene expression. BZLF1 inhibited the ability of IL-1, as well as transfected p65, to activate the expression of two different NF-{kappa}B-responsive genes, ICAM-1 and I{kappa}B-{alpha}. BZLF1 also reduced the constitutive level of I{kappa}B-{alpha} protein in HeLa and A549 cells, and increased the amount of nuclear NF-{kappa}B to a similar extent as tumor necrosis factor-alpha (TNF-{alpha}) treatment. In spite of this BZLF1-associated increase in the nuclear form of NF-{kappa}B, BZLF1 did not induce binding of NF-{kappa}B to NF-{kappa}B responsive promoters (as determined by chromatin immunoprecipitation assay) in vivo, although TNF-{alpha} treatment induced NF-{kappa}B binding as expected. Overexpression of p65 dramatically inhibited the lytic replication cycle of EBV in 293-EBV cells, confirming that NF-{kappa}B also inhibits BZLF1 transcriptional function. Our results are consistent with a model in which BZLF1 inhibits the transcriptional function of p65, resulting in decreased transcription of I{kappa}B-{alpha}, decreased expression of I{kappa}B-{alpha} protein, and subsequent translocation of NF-{kappa}B to the nucleus. This nuclear translocation of NF-{kappa}B may promote viral latency by negatively regulating BZLF1 transcriptional activity. In situations where p65 activity is limiting in comparison to BZLF1, the ability of BZLF1 to inhibit p65 transcriptional function may protect the virus from the host immune system during the lytic form of infection.

  13. Studying protein complexes by the yeast two-hybrid system.

    PubMed

    Rajagopala, Seesandra V; Sikorski, Patricia; Caufield, J Harry; Tovchigrechko, Andrey; Uetz, Peter

    2012-12-01

    Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

  14. Reversible cAMP-induced translocation of cytoskeleton-associated 300- to 350-kDa proteins from nucleus to cytoplasm

    SciTech Connect

    Nakayama, Tokiko; Nishizawa, Kimiko; Sato, Chicako )

    1988-08-01

    The authors previously reported that treatment of SV-3Y1 cells in an exponential growth state with db-cAMP plus theophylline induced reversible disappearance of nuclear dots stained by monoclonal anti-microtubule-associated protein (MAP)-1 antibody. In the present study, the authors examined the relation between the intracellular localization and phosphorylation of 300- to 350-kDa proteins that are intracellular antigens for our anti-Map-1 and -2 antibodies. Treatment with db-cAMP plus theophylline was found to result in a reversible decrease in immunofluorescent staining of the nucleus with polyclonal MAP-1 or -2 antibody, and a reversible increase in that of the cytoplasm. Simultaneous treatment with colchicine, colcemid, putrescine, or {alpha}-naphthyl phosphate in the presence of db-cAMP plus theophylline almost prevented this effect of db-cAMP plus theophylline. They examined the cytoplasmic and nuclear fractions by immunoperoxidase staining, immunoprecipitation, and {sup 125}I-protein A with anti-MAP-1 and -2 antibodies. The present research indicated that treatment with db-cAMP plus theophylline resulted in the reversible translocation of 300- to 350-kDa proteins from the nucleus to the cytoplasm accompanied by the dephosphorylation of these proteins.

  15. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Voigtländer, J.; Niedermeier, D.; Wex, H.; Stratmann, F.

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

  16. In the absence of cellular poly (A) binding protein, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53.

    PubMed

    Thangima Zannat, Mst; Bhattacharjee, Rumpa B; Bag, Jnanankur

    2011-06-01

    The cytoplasmic poly (A) binding protein (PABP) interacts with 3' poly (A) tract of eukaryotic mRNA and is important for both translation and stability of mRNA. Previously, we have shown that depletion of PABP by siRNA prevents protein synthesis and consequently leads to cell death through apoptosis. In the present investigation, we studied the mechanism of cell apoptosis. We show that in the absence of PABP, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53. As a result, p53 translocated to the mitochondria to initiate Bax mediated apoptosis.

  17. Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa*

    PubMed Central

    Discola, Karen F.; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

    2014-01-01

    The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

  18. Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors.

    PubMed

    Kurata, Shinya; Shen, Ben; Liu, Jun O; Takeuchi, Nono; Kaji, Akira; Kaji, Hideko

    2013-01-01

    Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3's known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.

  19. Immunogold evidence suggests that endoplasmic reticulum is the site of protamine-type protein synthesis and participates in translocation of these proteins into the nucleus during Chara vulgaris spermiogenesis.

    PubMed

    Popłonska, Katarzyna; Kwiatkowska, Maria; Wojtczak, Agnieszka; Polit, Justyna

    2009-03-01

    During spermiogenesis of an alga Chara vulgaris, which in many aspects resembles that of animals, histones are replaced by protamine-type proteins. Our earlier immunocytochemical studies showed that this replacement started during the short stage V of spermiogenesis, when electronograms revealed an extensive system of cisternae and vesicles of endoplasmic reticulum (ER). The present studies revealed at stage V intensive incorporation of labeled (3)H-arginine and (3)H-lysine quickly translocating into a nucleus visualized with pulse-chase autoradiography of semithin sections. The immunogold technique with the use of the antibodies to protamine-type proteins isolated from Chara tomentosa show that both ER cisternae and vesicles are labeled with gold grains, which are absent from the spermatids not treated with the antibodies; thus, the ER is probably the site of the protamine-type protein synthesis. These proteins then are translocated to a nucleus through ER channels connected with the nuclear envelope, as suggested by gold labeling of an inner membrane of the nuclear envelope adjacent to condensed chromatin. The above results correspond with those of other authors showing that in animals, protamines bind with lamin B receptors localized in the inner membrane of the nuclear envelope. A hypothesis has been put forward that during Chara spermiogenesis the inner membrane of the nuclear envelope invaginates into a nucleus together with protamine-type proteins, which become separated from the membrane and penetrate into chromatin.

  20. Disrupted in Schizophrenia 1 and Nudel form a neurodevelopmentally regulated protein complex: implications for schizophrenia and other major neurological disorders.

    PubMed

    Brandon, N J; Handford, E J; Schurov, I; Rain, J-C; Pelling, M; Duran-Jimeniz, B; Camargo, L M; Oliver, K R; Beher, D; Shearman, M S; Whiting, P J

    2004-01-01

    Disrupted In Schizophrenia 1 (DISC1) was identified as a potential susceptibility gene for schizophrenia due to its disruption by a balanced t(1;11) (q42;q14) translocation, which has been shown to cosegregate with major psychiatric disease in a large Scottish family. We have demonstrated that DISC1 exists in a neurodevelopmentally regulated protein complex with Nudel. The complex is abundant at E17 and in early postnatal life but is greatly reduced in the adult. Nudel has previously been shown to bind Lis1, a gene underlying lissencephaly in humans. Critically, we show that the predicted peptide product resulting from the Scottish translocation removes the interaction domain for Nudel. DISC1 interacts with Nudel through a leucine zipper domain and binds to a novel DISC1-interaction domain on Nudel, which is independent from the Lis1 binding site. We show that Nudel is able to act as a bridge between DISC1 and Lis1 to allow formation of a trimolecular complex. Nudel has been implicated to play a role in neuronal migration, together with the developmental variation in the abundance of the DISC1-Nudel complex, may implicate a defective DISC1-Nudel complex as a neurodevelopmental cause of schizophrenia.

  1. Accumulation of small protein molecules in a macroscopic complex coacervate.

    PubMed

    Lindhoud, Saskia; Claessens, Mireille M A E

    2016-01-14

    To obtain insight into the accumulation of proteins into macroscopic complex coacervate phases, the lysozyme concentration in complex coacervates containing the cationic polyelectrolyte poly-(N,N dimethylaminoethyl methacrylate) and the anionic polyelectrolyte polyacrylic acid was investigated as a function of the mixing ratio, protein concentration and ionic strength. Maximal protein enrichment of the complex coacervate phase was observed to require the presence of all three macromolecules. Under optimized conditions the protein concentrations in the complex coacervate were as high as 200 g L(-1). Such high concentrations are comparable to the protein concentration in the cytosol, suggesting that these interesting liquid phases may serve a suitable model system for the phase behavior of the cytosol and genesis and function of membrane-less organelles. The high stability of the complexes and the salt dependent uptake of protein suggest that complex coacervates may provide a way to store hydrated proteins at high concentrations and might therefore be of interest in the formulation of high protein foods.

  2. Recording information on protein complexes in an information management system

    PubMed Central

    Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

    2011-01-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  3. Mass Spectrometry of Protein Complexes: From Origins to Applications

    NASA Astrophysics Data System (ADS)

    Mehmood, Shahid; Allison, Timothy M.; Robinson, Carol V.

    2015-04-01

    Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase.

  4. Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1.

    PubMed Central

    Bogdan, J A; Adams-Burton, C; Pedicord, D L; Sukovich, D A; Benfield, P A; Corjay, M H; Stoltenborg, J K; Dicker, I B

    1998-01-01

    The human BTG1 protein is thought to be a potential tumour suppressor because its overexpression inhibits NIH 3T3 cell proliferation. However, little is known about how BTG1 exerts its anti-proliferative activity. In this study, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-associative factor 1 (hCAF-1), a homologue of mouse CAF-1 (mCAF-1) and Saccharomyces cerevisiae yCAF-1/POP2. In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34cdc2 kinase site on BTG1 (Ser-159). In yeast, the Ala-159 mutant did not interact with hCAF-1. In addition, phosphorylation of Ser-159 in vitro showed specificity for the cell cycle kinases p34CDK2/cyclin E and p34CDK2/cyclin A, but not for p34CDK4/cyclin D1 or p34cdc2/cyclin B. Cell synchrony experiments with primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated that message and protein levels of rat CAF-1 (rCAF-1) were up-regulated under conditions of cell contact, as previously reported for BTG1 [Wilcox, Scott, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulation 92, I34-I35]. Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was physically associated with BTG1, as determined by co-immunoprecipitation with anti-hCAF-1 antisera. Overexpression of hCAF-1 in NIH 3T3 and osteosarcoma (U-2-OS) cells was itself anti-proliferative with colony formation reduced by 67% and 90% respectively. Taken together, these results indicate that formation of the hCAF-1/BTG1 complex is driven by phosphorylation at BTG1 (Ser-159) and implicates this complex in the signalling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact. PMID:9820826

  5. Protein camouflage in cytochrome c-calixarene complexes

    NASA Astrophysics Data System (ADS)

    McGovern, Róise E.; Fernandes, Humberto; Khan, Amir R.; Power, Nicholas P.; Crowley, Peter B.

    2012-07-01

    Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host-guest interactions have been published. Although there is growing interest in protein-calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein-calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein-protein interactions, with potential applications in generating assemblies and promoting crystallization.

  6. Embracing proteins: structural themes in aptamer-protein complexes.

    PubMed

    Gelinas, Amy D; Davies, Douglas R; Janjic, Nebojsa

    2016-02-01

    Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer-protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer-protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity. PMID:26919170

  7. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.

    PubMed

    Banks, Charles A S; Kong, Stephanie E; Washburn, Michael P

    2012-12-01

    Characterizing protein complexes and identifying their subunits promote our understanding of the machinery involved in many in vivo processes. Proteomic studies can identify a protein's binding partners, and this can provide insight into how protein complexes function and how they are regulated. In addition, the composition of a protein complex within an organism can be investigated as a function of time, as a function of location, or during the response of an organism to a change in environment. There are many ways to isolate a complex and identify its constituents. This review will focus on complex isolation using affinity purification and will address issues that biochemists should bear in mind as they isolate protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT)(1). Protein complex analysis by mass spectrometry frequently involves the collaborative efforts of biochemists or biologists who purify protein complexes and proteomic specialists who analyze the samples - for fruitful collaborations it can be helpful for these specialized groups to be acquainted with basic principles of their collaborator's discipline. With this in mind, we first review the variety of affinity purification methods which might be considered for preparing complexes for analysis, and then provide brief primers on the principles of MudPIT mass spectrometry and data analysis. From this foundation, we then discuss how these techniques are integrated and optimized and suggest salient points to consider when preparing purified samples for protein identification, performing mass spectrometry runs, and analyzing the resulting data.

  8. Protein Connectivity in Chemotaxis Receptor Complexes

    PubMed Central

    Eismann, Stephan; Endres, Robert G.

    2015-01-01

    The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET) measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures. PMID:26646441

  9. Structural study of coacervation in protein-polyelectrolyte complexes

    NASA Astrophysics Data System (ADS)

    Chodankar, S.; Aswal, V. K.; Kohlbrecher, J.; Vavrin, R.; Wagh, A. G.

    2008-09-01

    Coacervation is a dense liquid-liquid phase separation and herein we report coacervation of protein bovine serum albumin (BSA) in the presence of polyelectrolyte sodium polystyrene sulfonate (NaPSS) under varying solution conditions. Small-angle neutron scattering (SANS) measurements have been performed on above protein-polyelectrolyte complexes to study the structural evolution of the process that leads to coacervation and the phase separated coacervate as a function of solution pH , protein-polyelectrolyte ratio and ionic strength. SANS study prior to phase separation on the BSA-NaPSS complex shows a fractal structure representing a necklace model of protein macromolecules randomly distributed along the polystyrene sulfonate chain. The fractal dimension of the complex decreases as pH is shifted away from the isoelectric point (˜4.7) of BSA protein, which indicates the decrease in the compactness of the complex structure due to increase in the charge repulsion between the protein macromolecules bound to the polyelectrolyte. Concentration-dependence studies of the polyelectrolyte in the complex suggest coexistence of two populations of polyelectrolytes, first one fully saturated with proteins and another one free from proteins. Coacervation phase has been obtained through the turbidity measurement by varying pH of the aqueous solution containing protein and polyelectrolyte from neutral to acidic regime to get them to where the two components are oppositely charged. The spontaneous formation of coacervates is observed for pH values less than 4. SANS study on coacervates shows two length scales related to complex aggregations (mesh size and overall extent of the complex) hierarchically branched to form a larger network. The mesh size represents the distance between cross-linked points in the primary complex, which decreases with increase in ionic strength and remains the same on varying the protein-polyelectrolyte ratio. On the other hand, the overall extent of the

  10. Blotting protein complexes from native gels to electron microscopy grids.

    PubMed

    Knispel, Roland Wilhelm; Kofler, Christine; Boicu, Marius; Baumeister, Wolfgang; Nickell, Stephan

    2012-01-08

    We report a simple and generic method for the direct transfer of protein complexes separated by native gel electrophoresis to electron microscopy grids. After transfer, sufficient material remains in the gel for identification and characterization by mass spectrometry. The method should facilitate higher-throughput single-particle analysis by substantially reducing the time needed for protein purification, as demonstrated for three complexes from Thermoplasma acidophilum.

  11. Identification of Post-translational Modifications of Plant Protein Complexes

    PubMed Central

    Piquerez, Sophie J. M.; Balmuth, Alexi L.; Sklenář, Jan; Jones, Alexandra M.E.; Rathjen, John P.; Ntoukakis, Vardis

    2014-01-01

    Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein. PMID:24637539

  12. Coupling protein complex analysis to peptide based proteomics.

    PubMed

    Gao, Qiang; Madian, Ashraf G; Liu, Xiuping; Adamec, Jiri; Regnier, Fred E

    2010-12-01

    Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.

  13. Visualizing active membrane protein complexes by electron cryotomography

    PubMed Central

    Gold, Vicki A.M.; Ieva, Raffaele; Walter, Andreas; Pfanner, Nikolaus; van der Laan, Martin; Kühlbrandt, Werner

    2014-01-01

    Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future. PMID:24942077

  14. Biochemical isolation of Argonaute protein complexes by Ago-APP

    PubMed Central

    Hauptmann, Judith; Schraivogel, Daniel; Bruckmann, Astrid; Manickavel, Sudhir; Jakob, Leonhard; Eichner, Norbert; Pfaff, Janina; Urban, Marc; Sprunck, Stefanie; Hafner, Markus; Tuschl, Thomas; Deutzmann, Rainer; Meister, Gunter

    2015-01-01

    During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells. PMID:26351695

  15. Replication forks blocked by protein-DNA complexes have limited stability in vitro.

    PubMed

    McGlynn, Peter; Guy, Colin P

    2008-08-29

    There are many barriers that replication forks must overcome in order to duplicate a genome in vivo. These barriers include damage to the template DNA and proteins bound to this template. If replication is halted by such a block, then the block must be either removed or bypassed for replication to continue. If continuation of replication employs the original fork, avoiding the need to reload the replication apparatus, then the blocked replisome must retain functionality. In vivo studies of Escherichia coli replication forks suggest that replication forks blocked by protein-DNA complexes retain the ability to resume replication upon removal of the block for several hours. Here we tested the functional stability of replication forks reconstituted in vitro and blocked by lac repressor-operator complexes. Once a fork comes to a halt at such a block, it cannot continue subsequently to translocate through the block until addition of IPTG induces repressor dissociation. However, the ability to resume replication is retained only for 4-6 min regardless of the topological state of the template DNA. Comparison of our in vitro data with previous in vivo data suggests that either accessory factors that stabilise blocked forks are present in vivo or the apparent stability of blocked forks in vivo is due to continual reloading of the replication apparatus at the site of the block.

  16. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  17. A Diatom Light-Harvesting Pigment-Protein Complex 1

    PubMed Central

    Friedman, Alan L.; Alberte, Randall S.

    1984-01-01

    A light-harvesting pigment-protein complex was isolated from the diatom Phaeodactylum tricornutum using the zwitterionic detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Detergent-solubilized membranes were fractionated by sucrose density gradient centrifugation into three components. The medium density fraction contained chlorophyll a, chlorophyll c, and fucoxanthin. This fraction was purified by DEAE-ion exchange chromatography, and contained chlorophyll a, chlorophyll c, and fucoxanthin in a molar ratio of 2.4:1.0:4.8. Fluorescence emission and excitation spectra of the isolated complex demonstrated that light energy absorbed by chlorophyll c and fucoxanthin was coupled to chlorophyll a fluorescence. Upon denaturation, the apoprotein yielded a polypeptide doublet at 17.5 to 18.0 kilodaltons which accounted for 30 to 40% of the toal membrane protein. These findings indicate that this pigment-protein complex is a major component of the diatom photosynthetic lammellae. The quantitative amino acid composition of the apoprotein was very similar to those reported for other membrane-bound pigment-protein complexes. Based on the protein to chlorophyll a ratio of 7700 grams protein per mole chlorophyll a for the complex, each apoprotein molecule contains, to the nearest integer, two chlorophyll a, one chlorophyll c, and five fucoxanthin molecules. Polyclonal antibodies raised against the 17.5 to 18.0 kilodaltons apoprotein showed a monospecific reaction with only the 17.5 to 18.0 protein zone from denatured P. tricornutum membranes as well as to the nondenatured pigment-protein complex. It appears that this complex is common to other diatom species. Images Fig. 2 Fig. 3 PMID:16663869

  18. Complexation between dodecyl sulfate surfactant and zein protein in solution.

    PubMed

    Ruso, Juan M; Deo, Namita; Somasundaran, P

    2004-10-12

    Interactions between sodium dodecyl sulfate and zein protein, a model system for the understanding of the effect of surfactants on skin, were investigated using a range of techniques involving UV-vis spectroscopy, TOC (total organic carbon analysis), electrophoresis, and static and dynamic light scattering. Zein protein was solubilized by SDS. The adsorption of SDS onto insoluble protein fraction caused the zeta potential of the complex to become more negative. From these values, we calculated the Gibbs energy of absorption, which decreases when the SDS concentration is raised. Finally the structure of the complex, based on the analysis by static and dynamic light scattering, is proposed to be rod like.

  19. Protein Complex Production from the Drug Discovery Standpoint.

    PubMed

    Moarefi, Ismail

    2016-01-01

    Small molecule drug discovery critically depends on the availability of meaningful in vitro assays to guide medicinal chemistry programs that are aimed at optimizing drug potency and selectivity. As it becomes increasingly evident, most disease relevant drug targets do not act as a single protein. In the body, they are instead generally found in complex with protein cofactors that are highly relevant for their correct function and regulation. This review highlights selected examples of the increasing trend to use biologically relevant protein complexes for rational drug discovery to reduce costly late phase attritions due to lack of efficacy or toxicity.

  20. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes

    PubMed Central

    Luo, Jiawei; Qi, Yi

    2015-01-01

    Background Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins. Method In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC), based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID), of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification. Results Experimental results based on three different PPI(protein-protein interaction) networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC). Conclusions LIDC is more effective for the prediction of essential proteins than other recently developed methods. PMID:26125187

  1. High-affinity lead binding proteins in rat kidney cytosol mediate cell-free nuclear translocation of lead

    SciTech Connect

    Mistry, P.; Lucier, G.W.; Fowler, B.A.

    1985-02-01

    The PbII binding characteristics of the previously reported PbII binding proteins of rat kidney cytosol were investigated further. Saturation and Scatchard analysis of /sup 203/Pb binding in whole cytosol and in 40% saturated ammonium sulfate precipitated fractions disclosed a class of relatively high-affinity sites with an apparent Kd of approximately 50 nM and binding capacities of approximately 41 and 9 pmol/mg of protein, respectively. Two /sup 203/Pb binding proteins with approximate molecular masses of 63K and 11.5K daltons and a high molecular weight component (greater than 200K) were isolated by Sepharose-6B column chromatography. The time course of association of /sup 203/Pb with cytosol and the 63K protein showed maximum binding at 18 hr which was stable up to 25 hr at 4 degrees C. The approximate half-time dissociation rate (T 1/2) of specifically bound /sup 203/Pb to the 63K protein was 100 min at 4 degrees C whereas the 11.5K protein showed little dissociation of specifically bound ligand at this temperature. Saturation analysis of the three isolated proteins disclosed low capacity, high-affinity sites with similar apparent Kd values to the cytosol assay. Sucrose density gradient analysis of kidney cytosol showed approximate sedimentation coefficients of 2S, 4.6S and 7S for the 11.5K, 63K and the high molecular weight proteins, respectively. Competitive binding studies with cytosol demonstrated displacement of /sup 203/Pb by PbII, CdII and ZnII ions but not CaII ions.

  2. Transcriptional regulation of protein complexes within and across species.

    PubMed

    Tan, Kai; Shlomi, Tomer; Feizi, Hoda; Ideker, Trey; Sharan, Roded

    2007-01-23

    Yeast two-hybrid and coimmunoprecipitation experiments have defined large-scale protein-protein interaction networks for many model species. Separately, systematic chromatin immunoprecipitation experiments have enabled the assembly of large networks of transcriptional regulatory interactions. To investigate the functional interplay between these two interaction types, we combined both within a probabilistic framework that models the cell as a network of transcription factors regulating protein complexes. This framework identified 72 putative coregulated complexes in yeast and allowed the prediction of 120 previously uncharacterized transcriptional interactions. Several predictions were tested by new microarray profiles, yielding a confirmation rate (58%) comparable with that of direct immunoprecipitation experiments. Furthermore, we extended our framework to a cross-species setting, identifying 24 coregulated complexes that were conserved between yeast and fly. Analyses of these conserved complexes revealed different conservation levels of their regulators and provided suggestive evidence that protein-protein interaction networks may evolve more slowly than transcriptional interaction networks. Our results demonstrate how multiple molecular interaction types can be integrated toward a global wiring diagram of the cell, and they provide insights into the evolutionary dynamics of protein complex regulation.

  3. Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

    PubMed Central

    Miczak, A; Kaberdin, V R; Wei, C L; Lin-Chao, S

    1996-01-01

    The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. Images Fig. 1 Fig. 2 PMID:8632981

  4. Border control: selectivity of chloroplast protein import and regulation at the TOC-complex

    PubMed Central

    Demarsy, Emilie; Lakshmanan, Ashok M.; Kessler, Felix

    2014-01-01

    Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC–TIC (translocon of outer membrane complex–translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms. PMID:25278954

  5. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  6. The human erythrocyte anion transport protein, band 3. Characterization of exofacial alkaline titratable groups involved in anion binding/translocation

    PubMed Central

    1992-01-01

    Chloride self-exchange across the human erythrocyte membrane at alkaline extracellular pH (pHO) and constant neutral intracellular pH (pH(i)) can be described by an exofacial deprotonatable reciprocating anion binding site model. The conversion of the transport system from the neutral to the alkaline state is related to deprotonation of a positively charged ionic strength- and substrate-sensitive group. In the absence of substrate ions ([ClO] = 0) the group has a pK of approximately 9.4 at constant high ionic strength (equivalent to approximately 150 mM KCl) and a pK of approximately 8.7 at approximately zero ionic strength. The alkaline ping-pong system (examined at constant high ionic strength) demonstrates outward recruitment of the binding sites with an asymmetry factor of approximately 0.2, as compared with the inward recruitment of the transport system at neutral pHO with an asymmetry factor of approximately 10. The intrinsic half-saturation constant for chloride binding, with [Cli] = [Clo], increased from approximately 30 mM at neutral to approximately 110 mM at alkaline pHO. The maximal transport rate was a factor of approximately 1.7 higher at alkaline pHO. This increase explains the stimulation of anion transport, the "modifier hump," observed at alkaline pHO. The translocation of anions at alkaline pHO was inhibited by deprotonation of another substrate- sensitive group with an intrinsic pK of approximately 11.3. This group together with the group with a pK of approximately 9.4 appear to form the essential part of the exofacial anion binding site. The effect of extracellular iodide inhibition on chloride transport as a function of pHO could, moreover, be simulated if three extracellular iodide binding constants were included in the model: namely, a competitive intrinsic iodide binding constant of approximately 1 mM in the neutral state, a self-inhibitor binding constant of approximately 120 mM in the neutral state, and a competitive intrinsic binding

  7. Morphological changes and nuclear translocation of DLC1 tumor suppressor protein precede apoptosis in human non-small cell lung carcinoma cells

    SciTech Connect

    Yuan Baozhu Jefferson, Amy M.; Millecchia, Lyndell; Popescu, Nicholas C.; Reynolds, Steven H.

    2007-11-01

    We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer.

  8. Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (Translocator Protein of 18 kDa (TSPO)).

    PubMed

    Šileikytė, Justina; Blachly-Dyson, Elizabeth; Sewell, Randall; Carpi, Andrea; Menabò, Roberta; Di Lisa, Fabio; Ricchelli, Fernanda; Bernardi, Paolo; Forte, Michael

    2014-05-16

    Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.

  9. Morphological changes and nuclear translocation of DLC1 tumor suppressor protein precede apoptosis in human non-small cell lung carcinoma cells.

    PubMed

    Yuan, Bao-Zhu; Jefferson, Amy M; Millecchia, Lyndell; Popescu, Nicholas C; Reynolds, Steven H

    2007-11-01

    We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer. PMID:17888903

  10. Identification and characterization of OSTL (RNF217) encoding a RING-IBR-RING protein adjacent to a translocation breakpoint involving ETV6 in childhood ALL

    PubMed Central

    Fontanari Krause, Luciana M.; Japp, Anna Sophia; Krause, Alexandre; Mooster, Jana; Chopra, Martin; Müschen, Markus; Bohlander, Stefan K.

    2014-01-01

    Genomic aberrations involving ETV6 on band 12p13 are amongst the most common chromosomal abnormalities in human leukemia. The translocation t(6;12)(q23;13) in a childhood B-cell acute lymphoblastic leukemia (ALL) cell line fuses ETV6 with the putative long non-coding RNA gene STL. Linking STL properties to leukemia has so far been difficult. Here, we describe a novel gene, OSTL (annotated as RNF217 in Genbank), which shares the first exon and a CpG island with STL but is transcribed in the opposite direction. Human RNF217 codes for a highly conserved RING finger protein and is mainly expressed in testis and skeletal muscle with different splice variants. RNF217 shows regulated splicing in B cell development, and is expressed in a number of human B cell leukemia cell lines, primary human chronic myeloid leukemia, acute myeloid leukemia with normal karyotype and acute T-ALL samples. Using a yeast two-hybrid screen, we identified the anti-apoptotic protein HAX1 to interact with RNF217. This interaction could be mapped to the C-terminal RING finger motif of RNF217. We propose that some of the recurring aberrations involving 6q might deregulate the expression of RNF217 and result in imbalanced apoptosis signalling via HAX1, promoting leukemia development. PMID:25298122

  11. Translocation of protein tyrosine phosphatase Pez/PTPD2/PTP36 to the nucleus is associated with induction of cell proliferation.

    PubMed

    Wadham, C; Gamble, J R; Vadas, M A; Khew-Goodall, Y

    2000-09-01

    Pez is a non-transmembrane tyrosine phosphatase with homology to the FERM (4.1, ezrin, radixin, moesin) family of proteins. The subcellular localisation of Pez in endothelial cells was found to be regulated by cell density and serum concentration. In confluent monolayers Pez was cytoplasmic, but in cells cultured at low density Pez was nuclear, suggesting that it is a nuclear protein in proliferating cells. This notion is supported by the loss of nuclear Pez when cells are serum-starved to induce quiescence, and the rapid return of Pez to the nucleus upon refeeding with serum to induce proliferation. Vascular endothelial cells normally exist as a quiescent confluent monolayer but become proliferative during angiogenesis or upon vascular injury. Using a 'wound' assay to mimic these events in vitro, Pez was found to be nuclear in the cells that had migrated and were proliferative at the 'wound' edge. TGFbeta, which inhibits cell proliferation but not migration, inhibited the translocation of Pez to the nucleus in the cells at the 'wound' edge, further strengthening the argument that Pez plays a role in the nucleus during cell proliferation. Together, the data presented indicate that Pez is a nuclear tyrosine phosphatase that may play a role in cell proliferation. PMID:10934049

  12. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters

    PubMed Central

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present “DyCluster”, a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

  13. Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

    PubMed Central

    Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

    2013-01-01

    Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID

  14. Emergence of Complexity in Protein Functions and Metabolic Networks

    NASA Technical Reports Server (NTRS)

    Pohorille, Andzej

    2009-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis of very large libraries of random amino acid sequences and subsequently subjecting them to in vitro evolution. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions, important clues have been uncovered. Considerable progress has been also achieved in understanding the origins of membrane proteins. We will address this issue in the example of ion channels - proteins that mediate transport of ions across cell walls. Remarkably, despite overall complexity of these proteins in contemporary cells, their structural motifs are quite simple, with -helices being most common. By combining results of experimental and computer simulation studies on synthetic models and simple, natural channels, I will show that, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during

  15. Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

    PubMed Central

    Hyung, Suk-Joon; Ruotolo, Brandon T.

    2013-01-01

    Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

  16. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    PubMed

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  17. ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB.

    PubMed

    Koronakis, V; Hughes, C; Koronakis, E

    1993-06-01

    The haemolysin exporter HlyB and its homologues are central to the unconventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP-binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-resistance P-glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytoplasmic domain as a C-terminal peptide fused to glutathione S-transferase allowed the direct affinity purification and concentration of 30-50 mg ml-1 of soluble protein (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST-Bctp bound to ADP-agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full-length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg(2+)-dependent ATPase activity of GST-Bctp (Vmax 1 mumol min-1 mg-1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P-glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F- and V-type ATPases, non-hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phospholipid phosphatidyl glycerol. PMID:8361361

  18. Protein Kinase C (PKC)ζ Pseudosubstrate Inhibitor Peptide Promiscuously Binds PKC Family Isoforms and Disrupts Conventional PKC Targeting and Translocation

    PubMed Central

    Bogard, Amy S.

    2015-01-01

    PKMζ is generated via an alternative transcriptional start site in the atypical protein kinase C (PKC)ζ isoform, which removes N-terminal regulatory elements, including the inhibitory pseudosubstrate domain, consequently rendering the kinase constitutively active. Persistent PKMζ activity has been proposed as a molecular mechanism for the long-term maintenance of synaptic plasticity underlying some forms of memory. Many studies supporting a role for PKMζ in synaptic plasticity and memory have relied on the PKCζ pseudosubstrate-derived ζ-inhibitory peptide (ZIP). However, recent studies have demonstrated that ZIP-induced impairments to synaptic plasticity and memory occur even in the absence of PKCζ, suggesting that ZIP exerts its actions via additional cellular targets. In this study, we demonstrated that ZIP interacts with conventional and novel PKC, in addition to atypical PKC isoforms. Moreover, when brain abundance of each PKC isoform and affinity for ZIP are taken into account, the signaling capacity of ZIP-responsive pools of conventional and novel PKCs may match or exceed that for atypical PKCs. Pseudosubstrate-derived peptides, like ZIP, are thought to exert their cellular action primarily by inhibiting PKC catalytic activity; however, the ZIP-sensitive catalytic core of PKC is known to participate in the enzyme’s subcellular targeting, suggesting an additional mode of ZIP action. Indeed, we have demonstrated that ZIP potently disrupts PKCα interaction with the PKC-targeting protein A-kinase anchoring protein (AKAP) 79 and interferes with ionomycin-induced translocation of conventional PKC to the plasma membrane. Thus, ZIP exhibits broad-spectrum action toward the PKC family of enzymes, and this action may contribute to its unique ability to impair memory. PMID:26199377

  19. DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma.

    PubMed Central

    Bailly, R A; Bosselut, R; Zucman, J; Cormier, F; Delattre, O; Roussel, M; Thomas, G; Ghysdael, J

    1994-01-01

    The 5' half of the EWS gene has recently been described to be fused to the 3' regions of genes encoding the DNA-binding domain of several transcriptional regulators, including ATF1, FLI-1, and ERG, in several human tumors. The most frequent occurrence of this situation results from the t(11;22)(q24;q12) chromosome translocation specific for Ewing sarcoma (ES) and related tumors which joins EWS sequences to the 3' half of FLI-1, which encodes a member of the Ets family of transcriptional regulators. We show here that this chimeric gene encodes an EWS-FLI-1 nuclear protein which binds DNA with the same sequence specificity as the wild-type parental FLI-1 protein. We further show that EWS-FLI-1 is an efficient sequence-specific transcriptional activator of model promoters containing FLI-1 (Ets)-binding sites, a property which is strictly dependent on the presence of its EWS domain. Comparison of the properties of the N-terminal activation domain of FLI-1 to those of the EWS domain of the fusion protein indicates that EWS-FLI-1 has altered transcriptional activation properties compared with FLI-1. These results suggest that EWS-FLI-1 contributes to the transformed phenotype of ES tumor cells by inducing the deregulated and/or unscheduled activation of genes normally responsive to FLI-1 or to other close members of the Ets family. ES and related tumors are characterized by an elevated level of c-myc expression. We show that EWS-FLI-1 is a transactivator of the c-myc promoter, suggesting that upregulation of c-myc expression is under control of EWS-FLI-1. Images PMID:8164678

  20. In the absence of cellular poly (A) binding protein, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53

    SciTech Connect

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-06-03

    Highlights: {yields} PABP knock down and cell apoptosis. {yields} Nuclear translocation of GAPDH in PABP depleted cells. {yields} Role of p53 in apoptosis of PABP depleted cells. {yields} Bax translocation and cytochrome C release and caspase 3 activation following PABP depletion. {yields} Association of p53 with Bcl2 and Bax. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) interacts with 3' poly (A) tract of eukaryotic mRNA and is important for both translation and stability of mRNA. Previously, we have shown that depletion of PABP by siRNA prevents protein synthesis and consequently leads to cell death through apoptosis. In the present investigation, we studied the mechanism of cell apoptosis. We show that in the absence of PABP, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53. As a result, p53 translocated to the mitochondria to initiate Bax mediated apoptosis.

  1. The 3;21 translocation in myelodysplasia results in a fusion transcript between the AML1 gene and the gene for EAP, a highly conserved protein associated with the Epstein-Barr virus small RNA EBER 1.

    PubMed Central

    Nucifora, G; Begy, C R; Erickson, P; Drabkin, H A; Rowley, J D

    1993-01-01

    In the 8;21 translocation, the AML1 gene, located at chromosome band 21q22, is translocated to chromosome 8 (q22), where it is fused to the ETO gene and transcribed as a chimeric gene. AML1 is the human homolog of the recently cloned mouse gene pebp2 alpha B, homologous to the DNA binding alpha subunit of the polyoma enhancer factor pebp2. AML1 is also involved in a translocation with chromosome 3 that is seen in patients with therapy-related acute myeloid leukemia and myelodysplastic syndrome and in chronic myelogenous leukemia in blast crisis. We have isolated a fusion cDNA clone from a t(3;21) library derived from a patient with therapy-related myelodysplastic syndrome; this clone contains sequences from AML1 and from EAP, which we have now localized to band 3q26. EAP has previously been characterized as a highly expressed small nuclear protein of 128 residues (EBER 1) associated with Epstein-Barr virus small RNA. The fusion clone contains the DNA binding 5' part of AML1 that is fused to ETO in the t(8;21) and, in addition, at least one other exon. The translocation replaces the last nine codons of AML1 with the last 96 codons of EAP. The fusion does not maintain the correct reading frame of EAP and may not lead to a functional chimeric protein. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8395054

  2. Synthetic RNA-protein complex shaped like an equilateral triangle

    NASA Astrophysics Data System (ADS)

    Ohno, Hirohisa; Kobayashi, Tetsuhiro; Kabata, Rinko; Endo, Kei; Iwasa, Takuma; Yoshimura, Shige H.; Takeyasu, Kunio; Inoue, Tan; Saito, Hirohide

    2011-02-01

    Synthetic nanostructures consisting of biomacromolecules such as nucleic acids have been constructed using bottom-up approaches. In particular, Watson-Crick base pairing has been used to construct a variety of two- and three-dimensional DNA nanostructures. Here, we show that RNA and the ribosomal protein L7Ae can form a nanostructure shaped like an equilateral triangle that consists of three proteins bound to an RNA scaffold. The construction of the complex relies on the proteins binding to kink-turn (K-turn) motifs in the RNA, which allows the RNA to bend by ~60° at three positions to form a triangle. Functional RNA-protein complexes constructed with this approach could have applications in nanomedicine and synthetic biology.

  3. Solid-State NMR Spectroscopy of Protein Complexes

    PubMed Central

    Sun, Shangjin; Han, Yun; Paramasivam, Sivakumar; Yan, Si; Siglin, Amanda E.; Williams, John C.; Byeon, In-Ja L.; Ahn, Jinwoo; Gronenborn, Angela M.; Polenova, Tatyana

    2016-01-01

    Protein-protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility or lack of long-range order. In the past several years, we have applied magic angle spinning SSNMR based methods to study several protein complexes. In this chapter, we discuss the general solid-state NMR methodologies employed for structural and dynamics analyses of protein complexes with specific examples from our work on thioredoxin reassemblies, HIV-1 capsid protein assemblies and microtubule-associated protein assemblies. We present protocols for sample preparation and characterization, pulse sequences, SSNMR spectra collection and data analysis. PMID:22167681

  4. A rare case of a three way complex variant positive Philadelphia translocation involving chromosome (9;11;22)(q34;p15;q11) in chronic myeloid leukemia: A case report

    PubMed Central

    Asif, Muhammad; Hussain, Abrar; Rasool, Mahmood

    2016-01-01

    The t(9;22)(q34;q11) translocation is present in 90–95% of patients with chronic myeloid leukemia (CML). Variant complex translocations have been observed in 5–8% of CML patients, in which a third chromosome other than (9;22) is involved. Imatinib mesylate is the first line breakpoint cluster region-Abelson gene (BCR/ABL)-targeted oral therapy for CML, and may produce a complete response in 70–80% of CML patients in the chronic phase. In the present study, a bone marrow sample was used for conventional cytogenetic analysis, and the fluorescence in situ hybridization (FISH) test was used for BCR/ABL gene detection. A hematological analysis was also performed to determine the white blood cell (WBC) count, red blood cell count, hemoglobin levels, packed and mean cell volumes, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and platelet values of the patient. The hematological analysis of the patient indicated the increased WBC of 186.5×103 cells/µl, and decreased hemoglobin levels of 11.1 g/dl. The FISH test revealed that 67% cells demonstrated BCR/ABL gene translocation. The patient was treated with 400 mg imatinib mesylate daily, and was monitored at various intervals over a 6-month period. The present study reports the rare case of a patient that demonstrates a three-way Philadelphia chromosome-positive translocation involving 46XY,t(9;11;22)(q34;p15;q11)[10], alongside CML in the chronic phase. The translocation was analyzed using cytogenetic and FISH tests.

  5. A rare case of a three way complex variant positive Philadelphia translocation involving chromosome (9;11;22)(q34;p15;q11) in chronic myeloid leukemia: A case report

    PubMed Central

    Asif, Muhammad; Hussain, Abrar; Rasool, Mahmood

    2016-01-01

    The t(9;22)(q34;q11) translocation is present in 90–95% of patients with chronic myeloid leukemia (CML). Variant complex translocations have been observed in 5–8% of CML patients, in which a third chromosome other than (9;22) is involved. Imatinib mesylate is the first line breakpoint cluster region-Abelson gene (BCR/ABL)-targeted oral therapy for CML, and may produce a complete response in 70–80% of CML patients in the chronic phase. In the present study, a bone marrow sample was used for conventional cytogenetic analysis, and the fluorescence in situ hybridization (FISH) test was used for BCR/ABL gene detection. A hematological analysis was also performed to determine the white blood cell (WBC) count, red blood cell count, hemoglobin levels, packed and mean cell volumes, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and platelet values of the patient. The hematological analysis of the patient indicated the increased WBC of 186.5×103 cells/µl, and decreased hemoglobin levels of 11.1 g/dl. The FISH test revealed that 67% cells demonstrated BCR/ABL gene translocation. The patient was treated with 400 mg imatinib mesylate daily, and was monitored at various intervals over a 6-month period. The present study reports the rare case of a patient that demonstrates a three-way Philadelphia chromosome-positive translocation involving 46XY,t(9;11;22)(q34;p15;q11)[10], alongside CML in the chronic phase. The translocation was analyzed using cytogenetic and FISH tests. PMID:27602125

  6. Structural and evolutionary versatility in protein complexes with uneven stoichiometry.

    PubMed

    Marsh, Joseph A; Rees, Holly A; Ahnert, Sebastian E; Teichmann, Sarah A

    2015-03-16

    Proteins assemble into complexes with diverse quaternary structures. Although most heteromeric complexes of known structure have even stoichiometry, a significant minority have uneven stoichiometry--that is, differing numbers of each subunit type. To adopt this uneven stoichiometry, sequence-identical subunits must be asymmetric with respect to each other, forming different interactions within the complex. Here we first investigate the occurrence of uneven stoichiometry, demonstrating that it is common in vitro and is likely to be common in vivo. Next, we elucidate the structural determinants of uneven stoichiometry, identifying six different mechanisms by which it can be achieved. Finally, we study the frequency of uneven stoichiometry across evolution, observing a significant enrichment in bacteria compared with eukaryotes. We show that this arises due to a general increased tendency for bacterial proteins to self-assemble and form homomeric interactions, even within the context of a heteromeric complex.

  7. Conformal Nanopatterning of Extracellular Matrix Proteins onto Topographically Complex Surfaces

    PubMed Central

    Sun, Yan; Jallerat, Quentin; Szymanski, John M.

    2015-01-01

    We report a method for conformal nanopatterning of extracellular matrix proteins onto engineered surfaces independent of underlying microtopography. This enables fibronectin, laminin, and other proteins to be applied to biomaterial surfaces in complex geometries inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface, used here to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment. PMID:25506720

  8. Biclustering Protein Complex Interactions with a Biclique FindingAlgorithm

    SciTech Connect

    Ding, Chris; Zhang, Anne Ya; Holbrook, Stephen

    2006-12-01

    Biclustering has many applications in text mining, web clickstream mining, and bioinformatics. When data entries are binary, the tightest biclusters become bicliques. We propose a flexible and highly efficient algorithm to compute bicliques. We first generalize the Motzkin-Straus formalism for computing the maximal clique from L{sub 1} constraint to L{sub p} constraint, which enables us to provide a generalized Motzkin-Straus formalism for computing maximal-edge bicliques. By adjusting parameters, the algorithm can favor biclusters with more rows less columns, or vice verse, thus increasing the flexibility of the targeted biclusters. We then propose an algorithm to solve the generalized Motzkin-Straus optimization problem. The algorithm is provably convergent and has a computational complexity of O(|E|) where |E| is the number of edges. It relies on a matrix vector multiplication and runs efficiently on most current computer architectures. Using this algorithm, we bicluster the yeast protein complex interaction network. We find that biclustering protein complexes at the protein level does not clearly reflect the functional linkage among protein complexes in many cases, while biclustering at protein domain level can reveal many underlying linkages. We show several new biologically significant results.

  9. Architecture and function of IFT complex proteins in ciliogenesis.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2012-02-01

    Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT. PMID:22118932

  10. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  11. Discovery of host-viral protein complexes during infection

    PubMed Central

    Rowles, Daniell L.; Terhune, Scott S.; Cristea, Ileana M.

    2014-01-01

    Summary Viruses have co-evolved with their hosts, developing effective approaches for hijacking and manipulating host cellular processes. Therefore, for their efficient replication and spread, viruses depend on dynamic and temporally-regulated interactions with host proteins. The rapid identification of host proteins targeted by viral proteins during infection provides significant insights into mechanisms of viral protein function. The resulting discoveries often lead to unique and innovative hypotheses on viral protein function. Here, we describe a robust method for identifying virus-host protein interactions and protein complexes, which we have successfully utilized to characterize spatial-temporal protein interactions during infections with either DNA or RNA viruses, including human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), human immunodeficiency virus (HIV-1), Sindbis, and West Nile virus (WNV). This approach involves cryogenic cell lysis, rapid immunoaffinity purification targeting a virus or host protein, followed by identification of associated proteins using mass spectrometry. Like most proteomic approaches, this methodology has evolved over the past few years and continues to evolve. We are presenting here the updated approaches for each step, and discuss alternative strategies allowing for the protocol to be optimized for different biological systems. PMID:23996249

  12. Multiscale Dynamics in Soft-Matter Systems: Enzyme Catalysis, Sec-Facilitated Protein Translocation, and Ion-Conduction in Polymers

    NASA Astrophysics Data System (ADS)

    Miller, Thomas

    Nature exhibits dynamics that span extraordinary ranges of space and time. In some cases, these dynamical hierarchies are well separated, simplifying their understanding and description. But chemistry and biology are replete with examples of dynamically coupled scales. In this talk, we will discuss the use of high-performance computing and new simulation methods that enable the inclusion of nuclear quantum effects, such as zero point energy and tunneling, in the reaction dynamics of enzymes, as well as coarse-graining strategies to enable minute-timescale simulations of protein targeting to cell membranes and ion-conduction in polymer electrolytes for lithium-ion battery applications.

  13. Antigen translocation machineries in adaptive immunity and viral immune evasion.

    PubMed

    Mayerhofer, Peter U; Tampé, Robert

    2015-03-13

    Protein homeostasis results in a steady supply of peptides, which are further degraded to fuel protein synthesis or metabolic needs of the cell. In higher vertebrates, a small fraction of the resulting peptidome, however, is translocated into the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Antigenic peptides are guided to major histocompatibility complex class I (MHC I) molecules and are finally displayed on the cell surface, where they mount an adaptive immune response against viral infected or malignantly transformed cells. Here, we review the structural organization and the molecular mechanism of this specialized antigen translocon. We discuss how the ATP-binding cassette (ABC) transporter TAP communicates and cooperates within the multi-component peptide loading machinery, mediating the proper assembly and editing of kinetically stable peptide/MHC I complexes. In light of its important role within the MHC I antigen processing pathway, TAP is a prime target for viral immune evasion strategies, and we summarize how this antigen translocation machinery is sabotaged by viral factors. Finally, we compare TAP with other ABC systems that facilitate peptide translocation.

  14. Utility of Translocator Protein (18 kDa) as a Molecular Imaging Biomarker to Monitor the Progression of Liver Fibrosis

    PubMed Central

    Hatori, Akiko; Yui, Joji; Xie, Lin; Kumata, Katsushi; Yamasaki, Tomoteru; Fujinaga, Masayuki; Wakizaka, Hidekatsu; Ogawa, Masanao; Nengaki, Nobuki; Kawamura, Kazunori; Wang, Feng; Zhang, Ming-Rong

    2015-01-01

    Hepatic fibrosis is the wound healing response to chronic hepatic injury caused by various factors. In this study, we aimed to evaluate the utility of translocator protein (18 kDa) (TSPO) as a molecular imaging biomarker for monitoring the progression of hepatic fibrosis to cirrhosis. Model rats were induced by carbon tetrachloride (CCl4), and liver fibrosis was assessed. Positron emission tomography (PET) with N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[18F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]-acetamide ([18F]FEDAC), a radioprobe specific for TSPO, was used for noninvasive visualisation in vivo. PET scanning, immunohistochemical staining, ex vivo autoradiography, and quantitative reverse-transcription polymerase chain reaction were performed to elucidate the relationships among radioactivity uptake, TSPO levels, and cellular sources enriching TSPO expression in damaged livers. PET showed that uptake of radioactivity in livers increased significantly after 2, 4, 6, and 8 weeks of CCl4 treatment. Immunohistochemistry demonstrated that TSPO was mainly expressed in macrophages and hepatic stellate cells (HSCs). TSPO-expressing macrophages and HSCs increased with the progression of liver fibrosis. Interestingly, the distribution of radioactivity from [18F]FEDAC was well correlated with TSPO expression, and TSPO mRNA levels increased with the severity of liver damage. TSPO was a useful molecular imaging biomarker and could be used to track the progression of hepatic fibrosis to cirrhosis with PET. PMID:26612465

  15. Radiosynthesis, In Vivo Biological Evaluation, and Imaging of Brain Lesions with [123I]-CLINME, a New SPECT Tracer for the Translocator Protein

    PubMed Central

    Mattner, F.; Quinlivan, M.; Greguric, I.; Pham, T.; Liu, X.; Jackson, T.; Berghofer, P.; Fookes, C. J. R.; Dikic, B.; Gregoire, M.-C.; Dolle, F.; Katsifis, A.

    2015-01-01

    The high affinity translocator protein (TSPO) ligand 6-chloro-2-(4′-iodophenyl)-3-(N,N-methylethyl)imidazo[1,2-a]pyridine-3-acetamide (CLINME) was radiolabelled with iodine-123 and assessed for its sensitivity for the TSPO in rodents. Moreover neuroinflammatory changes on a unilateral excitotoxic lesion rat model were detected using SPECT imaging. [123I]-CLINME was prepared in 70–80% radiochemical yield. The uptake of [123I]-CLINME was evaluated in rats by biodistribution, competition, and metabolite studies. The unilateral excitotoxic lesion was performed by injection of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid unilaterally into the striatum. The striatum lesion was confirmed and correlated with TSPO expression in astrocytes and activated microglia by immunohistochemistry and autoradiography. In vivo studies with [123I]-CLINME indicated a biodistribution pattern consistent with TPSO distribution and the competition studies with PK11195 and Ro 5-4864 showed that [123I]-CLINME is selective for this site. The metabolite study showed that the extractable radioactivity was unchanged [123I]-CLINME in organs which expresses TSPO. SPECT/CT imaging on the unilateral excitotoxic lesion indicated that the mean ratio uptake in striatum (lesion : nonlesion) was 2.2. Moreover, TSPO changes observed by SPECT imaging were confirmed by immunofluorescence, immunochemistry, and autoradiography. These results indicated that [123I]-CLINME is a promising candidate for the quantification and visualization of TPSO expression in activated astroglia using SPECT. PMID:26199457

  16. Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants.

    PubMed

    Mudgett, M B; Chesnokova, O; Dahlbeck, D; Clark, E T; Rossier, O; Bonas, U; Staskawicz, B J

    2000-11-21

    Strains of Xanthomonas campestris pv. vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance. Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent. N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants. By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv. Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants. The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal. PMID:11078519

  17. Repeated administration of AC-5216, a ligand for the 18 kDa translocator protein, improves behavioral deficits in a mouse model of post-traumatic stress disorder.

    PubMed

    Qiu, Zhi-Kun; Zhang, Li-Ming; Zhao, Nan; Chen, Hong-Xia; Zhang, You-Zhi; Liu, Yan-Qin; Mi, Tian-Yue; Zhou, Wen-Wen; Li, Yang; Yang, Ri-Fang; Xu, Jiang-Ping; Li, Yun-Feng

    2013-08-01

    Post-traumatic stress disorder (PTSD) is a severely disabling anxiety disorder that may occur following exposure to a serious traumatic event. It is a psychiatric condition that can afflict anyone who has experienced a life-threatening or violent event. Previous studies have shown that changes in 18 kDa translocator protein (TSPO) expression (or function), a promising target for treating neurological disorders without benzodiazepine-like side effects, may correlate with PTSD. However, few studies have investigated the anti-PTSD effects of TSPO ligands. AC-5216, a ligand for TSPO, induces anxiolytic- and anti-depressant-like effects in animal models. The present study aimed to determine whether AC-5216 ameliorates PTSD behavior in mice. Following the training session consisting of exposure to inescapable electric foot shocks, animals were administered AC-5216 daily during the behavioral assessments, i.e., situational reminders (SRs), the open field (OF) test, the elevated plus-maze (EPM) test, and the staircase test (ST). The results indicated that exposure to foot shocks induced long-term behavioral deficiencies in the mice, including freezing and anxiety-like behavior, which were significantly ameliorated by repeated treatment with AC-5216 but without any effect on spontaneous locomotor activity or body weight. In summary, this study demonstrated the anti-PTSD effects of AC-5216 treatment, suggesting that TSPO may represent a therapeutic target for anti-PTSD drug discovery and that TSPO ligands may be a promising new class of drugs for the future treatment of PTSD.

  18. In vivo (R)-[11C]PK11195 PET imaging of 18kDa translocator protein in recent onset psychosis

    PubMed Central

    van der Doef, Thalia F; de Witte, Lot D; Sutterland, Arjen L; Jobse, Ellen; Yaqub, Maqsood; Boellaard, Ronald; de Haan, Lieuwe; Eriksson, Jonas; Lammertsma, Adriaan A; Kahn, René S; van Berckel, Bart N M

    2016-01-01

    Evidence is accumulating that immune dysfunction is involved in the pathophysiology of schizophrenia. It has been hypothesized that microglia activation is present in patients with schizophrenia. Various in vivo and post-mortem studies have investigated this hypothesis, but as yet with inconclusive results. Microglia activation is associated with elevations in 18 kDa translocator protein (TSPO) levels, which can be measured with the positron emission tomography (PET) tracer (R)-[11C]PK11195. The purpose of the present study was to investigate microglia activation in psychosis in vivo at an early stage of the disease. (R)-[11C]PK11195 binding potential (BPND) was measured in 19 patients with recent onset psychosis and 17 age and gender-matched healthy controls. Total gray matter, as well as five gray matter regions of interest (frontal cortex, temporal cortex, parietal cortex, striatum, and thalamus) were defined a priori. PET data were analysed using a reference tissue approach and a supervised cluster analysis algorithm to identify the reference region. No significant difference in (R)-[11C]PK11195 BPND between patients and controls was found in total gray matter, nor one of the regions of interest. These findings suggest that microglia activation is not present in recent onset psychosis or that it is a subtle phenomenon that could not be detected using the design of the present study.

  19. Lentiviral-Mediated Overexpression of the 18 kDa Translocator Protein (TSPO) in the Hippocampal Dentate Gyrus Ameliorates LPS-Induced Cognitive Impairment in Mice

    PubMed Central

    Wang, Wei; Zhang, Liming; Zhang, Xiaoying; Xue, Rui; Li, Lei; Zhao, Weixing; Fu, Qiang; Mi, Weidong; Li, Yunfeng

    2016-01-01

    The 18 kDa translocator protein (TSPO) is involved in the immune/inflammatory response. However, the exact role that TSPO plays in neuroinflammation-induced cognitive impairment is still elusive. The purpose of our present study was to investigate the effects of lentiviral-mediated hippocampal overexpression of the TSPO in a mouse model of LPS-induced cognitive impairment. We established a mouse cognitive impairment model using systematic daily administration of lipopolysaccharide (LPS) (0.5 mg/kg). Microinjection of the dentate gyrus of the mouse with lentiviral vectors, which contained a cDNA targeting TSPO (Lv-TSPO), resulted in a significant increase in TSPO expression and allopregnanolone production. Mice treated with LPS showed cognitive deficits in the novel object recognition test and the Morris water maze test that could be ameliorated by TSPO overexpression. In addition, TSPO overexpression reversed LPS-induced microglial activation and accumulation of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Moreover, TSPO overexpression attenuated the LPS-induced impairment of hippocampal neurogenesis. Our results suggest that local overexpression of TSPO in the hippocampal dentate gyrus alleviated LPS-induced cognitive deficits, and its effects might be mediated by the attenuation of inflammatory cytokines, inhibition of microglial activation, and promotion of neurogenesis. PMID:27803668

  20. In vivo (R)-[(11)C]PK11195 PET imaging of 18kDa translocator protein in recent onset psychosis.

    PubMed

    van der Doef, Thalia F; de Witte, Lot D; Sutterland, Arjen L; Jobse, Ellen; Yaqub, Maqsood; Boellaard, Ronald; de Haan, Lieuwe; Eriksson, Jonas; Lammertsma, Adriaan A; Kahn, René S; van Berckel, Bart N M

    2016-01-01

    Evidence is accumulating that immune dysfunction is involved in the pathophysiology of schizophrenia. It has been hypothesized that microglia activation is present in patients with schizophrenia. Various in vivo and post-mortem studies have investigated this hypothesis, but as yet with inconclusive results. Microglia activation is associated with elevations in 18 kDa translocator protein (TSPO) levels, which can be measured with the positron emission tomography (PET) tracer (R)-[(11)C]PK11195. The purpose of the present study was to investigate microglia activation in psychosis in vivo at an early stage of the disease. (R)-[(11)C]PK11195 binding potential (BPND) was measured in 19 patients with recent onset psychosis and 17 age and gender-matched healthy controls. Total gray matter, as well as five gray matter regions of interest (frontal cortex, temporal cortex, parietal cortex, striatum, and thalamus) were defined a priori. PET data were analysed using a reference tissue approach and a supervised cluster analysis algorithm to identify the reference region. No significant difference in (R)-[(11)C]PK11195 BPND between patients and controls was found in total gray matter, nor one of the regions of interest. These findings suggest that microglia activation is not present in recent onset psychosis or that it is a subtle phenomenon that could not be detected using the design of the present study. PMID:27602389

  1. Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation.

    PubMed Central

    Weyrich, A S; McIntyre, T M; McEver, R P; Prescott, S M; Zimmerman, G A

    1995-01-01

    Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues. Images PMID:7537762

  2. Early repeated administration of progesterone improves the recovery of neuropathic pain and modulates spinal 18kDa-translocator protein (TSPO) expression.

    PubMed

    Liu, Xiaoming; Li, Weiyan; Dai, Lihua; Zhang, Tingting; Xia, Weiliang; Liu, Hongjun; Ma, Ke; Xu, Jianguo; Jin, Yi

    2014-09-01

    Although progesterone was reported to be a neuroprotective agent against injuries to the nervous system, including the peripheral neuropathy, the mechanisms of its dose or timing-related effects remain unclear. Translocator protein (TSPO) is predominantly located in the mitochondrial outer membrane and has been recently implicated in modulation of several brain injuries and nociception. This experiment was conducted using a rat model of L5 spinal nerve ligation (SNL) to observe the effects of progesterone against allodynia development in an 84-day period and to explore the spinal TSPO expression after treatment. Our results demonstrated that a 10-day progesterone treatment started right after injury at a dose of 15 mg/kg/d or more could significantly increase the mechanical thresholds within the 84-day observation period. Moreover, increased TSPO expression was observed in the ipsilateral spinal dorsal horn after SNL surgery and reached its peak on Day 14. A treatment regimen of pharmacological progesterone augmented this spinal TSPO activation and expression before Day 28 and after Day 56. Both the anti-nociception and TSPO activation augment effect of progesterone were completely abolished by 5α-reductase inhibitor finasteride but not progesterone receptor antagonist mifepristone. These results indicate that early repeated administration of progesterone could improve the recovery of neuropathic pain and modulate spinal TSPO activation which were dependent on its 5α-reductase metabolites.

  3. Biodistribution and Radiation Dosimetry in Humans of a New PET Ligand, 18F-PBR06, to Image Translocator Protein (18 kDa)

    PubMed Central

    Fujimura, Yota; Kimura, Yasuyuki; Siméon, Fabrice G.; Dickstein, Leah P.; Pike, Victor W.; Innis, Robert B.; Fujita, Masahiro

    2010-01-01

    As a PET biomarker for inflammation, translocator protein (18 kDa) (TSPO) can be measured with an 18F-labeled aryloxyanilide, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06), in the human brain. The objective of this study was to estimate the radiation absorbed doses of 18F-PBR06 based on biodistribution data in humans. Methods After the injection of 18F-PBR06, images were acquired from head to thigh in 7 healthy humans. Urine was collected at various time points. Radiation absorbed doses were estimated by the MIRD scheme. Results Moderate to high levels of radioactivity were observed in organs with high densities of TSPO and in organs of metabolism and excretion. Bone had low levels of radioactivity. The effective dose was 18.5 µSv/MBq. Conclusion The effective dose of 18F-PBR06, compared with other 18F radioligands, was moderate. This radioligand had negligible defluorination, as indirectly assessed by bone radioactivity. Doses to the gallbladder wall and spleen may limit the amount of permissible injected radioactivity. PMID:20008980

  4. In vivo (R)-[11C]PK11195 PET imaging of 18kDa translocator protein in recent onset psychosis

    PubMed Central

    van der Doef, Thalia F; de Witte, Lot D; Sutterland, Arjen L; Jobse, Ellen; Yaqub, Maqsood; Boellaard, Ronald; de Haan, Lieuwe; Eriksson, Jonas; Lammertsma, Adriaan A; Kahn, René S; van Berckel, Bart N M

    2016-01-01

    Evidence is accumulating that immune dysfunction is involved in the pathophysiology of schizophrenia. It has been hypothesized that microglia activation is present in patients with schizophrenia. Various in vivo and post-mortem studies have investigated this hypothesis, but as yet with inconclusive results. Microglia activation is associated with elevations in 18 kDa translocator protein (TSPO) levels, which can be measured with the positron emission tomography (PET) tracer (R)-[11C]PK11195. The purpose of the present study was to investigate microglia activation in psychosis in vivo at an early stage of the disease. (R)-[11C]PK11195 binding potential (BPND) was measured in 19 patients with recent onset psychosis and 17 age and gender-matched healthy controls. Total gray matter, as well as five gray matter regions of interest (frontal cortex, temporal cortex, parietal cortex, striatum, and thalamus) were defined a priori. PET data were analysed using a reference tissue approach and a supervised cluster analysis algorithm to identify the reference region. No significant difference in (R)-[11C]PK11195 BPND between patients and controls was found in total gray matter, nor one of the regions of interest. These findings suggest that microglia activation is not present in recent onset psychosis or that it is a subtle phenomenon that could not be detected using the design of the present study. PMID:27602389

  5. The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein.

    PubMed

    Johnson, Christopher L; Ridley, Helen; Marchetti, Roberta; Silipo, Alba; Griffin, David C; Crawford, Lucy; Bonev, Boyan; Molinaro, Antonio; Lakey, Jeremy H

    2014-05-01

    Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell's outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low-copy-number, high-affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high-affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor-binding domain (ColN-R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid- and solid-state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPS(Δ) (LIPID) ) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.

  6. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export.

    PubMed

    Minamino, Tohru; Morimoto, Yusuke V; Hara, Noritaka; Aldridge, Phillip D; Namba, Keiichi

    2016-03-01

    The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+-protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration. PMID:26943926

  7. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export

    PubMed Central

    Minamino, Tohru; Morimoto, Yusuke V.; Hara, Noritaka; Aldridge, Phillip D.; Namba, Keiichi

    2016-01-01

    The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+–protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration. PMID:26943926

  8. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export.

    PubMed

    Minamino, Tohru; Morimoto, Yusuke V; Hara, Noritaka; Aldridge, Phillip D; Namba, Keiichi

    2016-03-01

    The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+-protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration.

  9. Plasmodium falciparum Bloom homologue, a nucleocytoplasmic protein, translocates in 3' to 5' direction and is essential for parasite growth.

    PubMed

    Rahman, Farhana; Tarique, Mohammed; Tuteja, Renu

    2016-05-01

    Malaria caused by Plasmodium, particularly Plasmodium falciparum, is the most serious and widespread parasitic disease of humans. RecQ helicase family members are essential in homologous recombination-based error-free DNA repair processes in all domains of life. RecQ helicases present in each organism differ and several homologues have been identified in various multicellular organisms. These proteins are involved in various pathways of DNA metabolism by providing duplex unwinding function. Five members of RecQ family are present in Homo sapiens but P. falciparum contains only two members of this family. Here we report the detailed biochemical and functional characterization of the Bloom (Blm) homologue (PfBlm) from P. falciparum 3D7 strain. Purified PfBlm exhibits ATPase and 3' to 5' direction specific DNA helicase activity. The calculated average reaction rate of ATPase was ~13 pmol of ATP hydrolyzed/min/pmol of enzyme. The immunofluorescence assay results show that PfBlm is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain. In some stages of development in addition to nucleus PfBlm also localizes in the cytoplasm. The gene disruption studies of PfBlm by dsRNA showed that it is required for the ex-vivo intraerythrocytic development of the parasite P. falciparum 3D7 strain. The dsRNA mediated inhibition of parasite growth suggests that a variety of pathways are affected resulting in curtailing of the parasite growth. This study will be helpful in unravelling the basic mechanism of DNA transaction in the malaria parasite and additionally it may provide leads to understand the parasite specific characteristics of this protein. PMID:26917473

  10. Protein corona - from molecular adsorption to physiological complexity.

    PubMed

    Treuel, Lennart; Docter, Dominic; Maskos, Michael; Stauber, Roland H

    2015-01-01

    In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP-protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs.

  11. Structure of a PE–PPE–EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    PubMed Central

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-01-01

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed “type VII.” How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE–PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways. PMID:25275011

  12. Expression of the translocator protein (TSPO) from Pseudomonas fluorescens Pf0-1 requires the stress regulatory sigma factors AlgU and RpoH.

    PubMed

    Leneveu-Jenvrin, Charlène; Bouffartigues, Emeline; Maillot, Olivier; Cornelis, Pierre; Feuilloley, Marc G J; Connil, Nathalie; Chevalier, Sylvie

    2015-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is an evolutionary conserved protein that is found in many Eukarya, Archae, and Bacteria, in which it plays several important functions including for example membrane biogenesis, signaling, and stress response. A tspo homolog gene has been identified in several members of the Pseudomonas genus, among which the soil bacterium P. fluorescens Pf0-1. In this bacterium, the tspo gene is located in the vicinity of a putative hybrid histidine kinase-encoding gene. Since tspo has been involved in water stress related response in plants, we explored the effects of hyperosmolarity and temperature on P. fluorescens Pf0-1 tspo expression using a strategy based on lux-reporter fusions. We show that the two genes Pfl01_2810 and tspo are co-transcribed forming a transcription unit. The expression of this operon is growth phase-dependent and is increased in response to high concentrations of NaCl, sucrose and to a D-cycloserine treatment, which are conditions leading to activity of the major cell wall stress responsive extracytoplasmic sigma factor AlgU. Interestingly, the promoter region activity is strongly lowered in a P. aeruginosa algU mutant, suggesting that AlgU may be involved at least partly in the molecular mechanism leading to Pfl01_2810-tspo expression. In silico analysis of this promoter region failed to detect an AlgU consensus binding site; however, a putative binding site for the heat shock response RpoH sigma factor was detected. Accordingly, the promoter activity of the region containing this sequence is increased in response to high growth temperature and slightly lowered in a P. aeruginosa rpoH mutant strain. Taken together, our data suggest that P. fluorescens tspo gene may belong at least partly to the cell wall stress response. PMID:26441945

  13. Expression of the translocator protein (TSPO) from Pseudomonas fluorescens Pf0-1 requires the stress regulatory sigma factors AlgU and RpoH.

    PubMed

    Leneveu-Jenvrin, Charlène; Bouffartigues, Emeline; Maillot, Olivier; Cornelis, Pierre; Feuilloley, Marc G J; Connil, Nathalie; Chevalier, Sylvie

    2015-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is an evolutionary conserved protein that is found in many Eukarya, Archae, and Bacteria, in which it plays several important functions including for example membrane biogenesis, signaling, and stress response. A tspo homolog gene has been identified in several members of the Pseudomonas genus, among which the soil bacterium P. fluorescens Pf0-1. In this bacterium, the tspo gene is located in the vicinity of a putative hybrid histidine kinase-encoding gene. Since tspo has been involved in water stress related response in plants, we explored the effects of hyperosmolarity and temperature on P. fluorescens Pf0-1 tspo expression using a strategy based on lux-reporter fusions. We show that the two genes Pfl01_2810 and tspo are co-transcribed forming a transcription unit. The expression of this operon is growth phase-dependent and is increased in response to high concentrations of NaCl, sucrose and to a D-cycloserine treatment, which are conditions leading to activity of the major cell wall stress responsive extracytoplasmic sigma factor AlgU. Interestingly, the promoter region activity is strongly lowered in a P. aeruginosa algU mutant, suggesting that AlgU may be involved at least partly in the molecular mechanism leading to Pfl01_2810-tspo expression. In silico analysis of this promoter region failed to detect an AlgU consensus binding site; however, a putative binding site for the heat shock response RpoH sigma factor was detected. Accordingly, the promoter activity of the region containing this sequence is increased in response to high growth temperature and slightly lowered in a P. aeruginosa rpoH mutant strain. Taken together, our data suggest that P. fluorescens tspo gene may belong at least partly to the cell wall stress response.

  14. Expression of the translocator protein (TSPO) from Pseudomonas fluorescens Pf0-1 requires the stress regulatory sigma factors AlgU and RpoH

    PubMed Central

    Leneveu-Jenvrin, Charlène; Bouffartigues, Emeline; Maillot, Olivier; Cornelis, Pierre; Feuilloley, Marc G. J.; Connil, Nathalie; Chevalier, Sylvie

    2015-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is an evolutionary conserved protein that is found in many Eukarya, Archae, and Bacteria, in which it plays several important functions including for example membrane biogenesis, signaling, and stress response. A tspo homolog gene has been identified in several members of the Pseudomonas genus, among which the soil bacterium P. fluorescens Pf0-1. In this bacterium, the tspo gene is located in the vicinity of a putative hybrid histidine kinase-encoding gene. Since tspo has been involved in water stress related response in plants, we explored the effects of hyperosmolarity and temperature on P. fluorescens Pf0-1 tspo expression using a strategy based on lux-reporter fusions. We show that the two genes Pfl01_2810 and tspo are co-transcribed forming a transcription unit. The expression of this operon is growth phase-dependent and is increased in response to high concentrations of NaCl, sucrose and to a D-cycloserine treatment, which are conditions leading to activity of the major cell wall stress responsive extracytoplasmic sigma factor AlgU. Interestingly, the promoter region activity is strongly lowered in a P. aeruginosa algU mutant, suggesting that AlgU may be involved at least partly in the molecular mechanism leading to Pfl01_2810-tspo expression. In silico analysis of this promoter region failed to detect an AlgU consensus binding site; however, a putative binding site for the heat shock response RpoH sigma factor was detected. Accordingly, the promoter activity of the region containing this sequence is increased in response to high growth temperature and slightly lowered in a P. aeruginosa rpoH mutant strain. Taken together, our data suggest that P. fluorescens tspo gene may belong at least partly to the cell wall stress response. PMID:26441945

  15. Peripheral Benzodiazepine Receptor/Translocator Protein Global Knock-out Mice Are Viable with No Effects on Steroid Hormone Biosynthesis*♦

    PubMed Central

    Tu, Lan N.; Morohaku, Kanako; Manna, Pulak R.; Pelton, Susanne H.; Butler, W. Ronald; Stocco, Douglas M.; Selvaraj, Vimal

    2014-01-01

    Translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein implicated as essential for cholesterol import to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Previous research on TSPO was based entirely on in vitro experiments, and its critical role was reinforced by an early report that claimed TSPO knock-out mice were embryonic lethal. In a previous publication, we examined Leydig cell-specific TSPO conditional knock-out mice that suggested TSPO was not required for testosterone production in vivo. This raised controversy and several questions regarding TSPO function. To examine the definitive role of TSPO in steroidogenesis and embryo development, we generated global TSPO null (Tspo−/−) mice. Contrary to the early report, Tspo−/− mice survived with no apparent phenotypic abnormalities and were fertile. Examination of adrenal and gonadal steroidogenesis showed no defects in Tspo−/− mice. Adrenal transcriptome comparison of gene expression profiles showed that genes involved in steroid hormone biosynthesis (Star, Cyp11a1, and Hsd3b1) were unchanged in Tspo−/− mice. Adrenocortical ultrastructure illustrated no morphological alterations in Tspo−/− mice. In an attempt to correlate our in vivo findings to previously used in vitro models, we also determined that siRNA knockdown or the absence of TSPO in different mouse and human steroidogenic cell lines had no effect on steroidogenesis. These findings directly refute the dogma that TSPO is indispensable for steroid hormone biosynthesis and viability. By amending the current model, this study advances our understanding of steroidogenesis with broad implications in biology and medicine. PMID:24936060

  16. Study of protein complexes via homology modeling, applied to cysteine proteases and their protein inhibitors.

    PubMed

    Tastan Bishop, Ozlem; Kroon, Matthys

    2011-12-01

    This paper develops and evaluates large-scale calculation of 3D structures of protein complexes by homology modeling as a promising new approach for protein docking. The complexes investigated were papain-like cysteine proteases and their protein inhibitors, which play numerous roles in human and parasitic metabolisms. The structural modeling was performed in two parts. For the first part (evaluation set), nine crystal structure complexes were selected, 1325 homology models of known complexes were rebuilt by various templates including hybrids, allowing an analysis of the factors influencing the accuracy of the models. The important considerations for modeling the interface were protease coverage and inhibitor sequence identity. In the second part (study set), the findings of the evaluation set were used to select appropriate templates to model novel cysteine protease-inhibitor complexes from human and malaria parasites Plasmodium falciparum and Plasmodium vivax. The energy scores, considering the evaluation set, indicate that the models are of high accuracy. PMID:21365221

  17. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

  18. How to Build a Complex, Functional Propeller Protein, From Parts.

    PubMed

    Clark, Patricia L

    2016-04-01

    By combining ancestral sequence reconstruction and in vitro evolution, Smock et al. identified single motifs that assemble into a functional five-bladed β-propeller, and a likely route for conversion into the more complex, extant single chain fusion. Interestingly, although sequence diversification destabilized five-motif fusions, it also destabilized aggregation-prone intermediates, increasing the level of functional protein in vivo.

  19. Translocation of DNA across bacterial membranes.

    PubMed Central

    Dreiseikelmann, B

    1994-01-01

    DNA translocation across bacterial membranes occurs during the biological processes of infection by bacteriophages, conjugative DNA transfer of plasmids, T-DNA transfer, and genetic transformation. The mechanism of DNA translocation in these systems is not fully understood, but during the last few years extensive data about genes and gene products involved in the translocation processes have accumulated. One reason for the increasing interest in this topic is the discussion about horizontal gene transfer and transkingdom sex. Analyses of genes and gene products involved in DNA transfer suggest that DNA is transferred through a protein channel spanning the bacterial envelope. No common model exists for DNA translocation during phage infection. Perhaps various mechanisms are necessary as a result of the different morphologies of bacteriophages. The DNA translocation processes during conjugation, T-DNA transfer, and transformation are more consistent and may even be compared to the excretion of some proteins. On the basis of analogies and homologies between the proteins involved in DNA translocation and protein secretion, a common basic model for these processes is presented. PMID:7968916

  20. DOCK/PIERR: web server for structure prediction of protein-protein complexes.

    PubMed

    Viswanath, Shruthi; Ravikant, D V S; Elber, Ron

    2014-01-01

    In protein docking we aim to find the structure of the complex formed when two proteins interact. Protein-protein interactions are crucial for cell function. Here we discuss the usage of DOCK/PIERR. In DOCK/PIERR, a uniformly discrete sampling of orientations of one protein with respect to the other, are scored, followed by clustering, refinement, and reranking of structures. The novelty of this method lies in the scoring functions used. These are obtained by examining hundreds of millions of correctly and incorrectly docked structures, using an algorithm based on mathematical programming, with provable convergence properties.

  1. Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle.

    PubMed

    Wu, Fu-Gen; Jiang, Yao-Wen; Chen, Zhan; Yu, Zhi-Wu

    2016-04-19

    The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxypoly(ethylene glycol)5K-block-poly(l-aspartic acid sodium salt)10 (mPEG(5K)-b-PLD10), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG(5K)-b-PLD10, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating-cooling-reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme-mPEG(5K)-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein

  2. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC.

  3. Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA.

    PubMed

    Doshi, Rupak; van Veen, Hendrik W

    2013-07-26

    ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP. PMID:23766512

  4. Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain.

    PubMed

    Waters, Brian M; Uauy, Cristobal; Dubcovsky, Jorge; Grusak, Michael A

    2009-01-01

    The NAM-B1 gene is a NAC transcription factor that affects grain nutrient concentrations in wheat (Triticum aestivum). An RNAi line with reduced expression of NAM genes has lower grain protein, iron (Fe), and zinc (Zn) concentrations. To determine whether decreased remobilization, lower plant uptake, or decreased partitioning to grain are responsible for this phenotype, mineral dynamics were quantified in wheat tissues throughout grain development. Control and RNAi wheat were grown in potting mix and hydroponics. Mineral (Ca, Cu, Fe, K, Mg, Mn, P, S, and Zn) and nitrogen (N) contents of organs were determined at regular intervals to quantify the net remobilization from vegetative tissues and the accumulation of nutrients in grain. Total nutrient accumulation was similar between lines, but grain Fe, Zn, and N were at lower concentrations in the NAM knockdown line. In potting mix, net remobilization of N, Fe, and Zn from vegetative tissues was impaired in the RNAi line. In hydroponics with ample nutrients, net remobilization was not observed, but grain Fe and Zn contents and concentrations remained lower in the RNAi line. When Fe or Zn was withheld post-anthesis, both lines demonstrated remobilization. These results suggest that a major effect of the NAM genes is an increased efflux of nutrients from the vegetative tissues and a higher partitioning of nutrients to grain. PMID:19858116