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Sample records for protein translocation complexes

  1. Two alternative binding mechanisms connect the protein translocation Sec71/Sec72 complex with heat shock proteins.

    PubMed

    Tripathi, Arati; Mandon, Elisabet C; Gilmore, Reid; Rapoport, Tom A

    2017-03-12

    The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum (ER) membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71/Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71/Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the ER membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome- associated nascent polypeptide chains also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71/72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.

  2. Human apo-SRP72 and SRP68/72 complex structures reveal the molecular basis of protein translocation.

    PubMed

    Gao, Yina; Zhang, Qi; Lang, Yue; Liu, Yang; Dong, Xiaofei; Chen, Zhenhang; Tian, Wenli; Tang, Jun; Wu, Wei; Tong, Yufeng; Chen, Zhongzhou

    2017-03-20

    The co-translational targeting or insertion of secretory and membrane proteins into the endoplasmic reticulum (ER) is a key biological process mediated by the signal recognition particle (SRP). In eukaryotes, the SRP68-SRP72 (SRP68/72) heterodimer plays an essential role in protein translocation. However, structural information on the two largest SRP proteins, SRP68 and SRP72, is limited, especially regarding their interaction. Herein, we report the first crystal structures of human apo-SRP72 and the SRP68/72 complex at 2.91Å and 1.7Å resolution, respectively. The SRP68-binding domain of SRP72 contains four atypical tetratricopeptide repeats (TPR) and a flexible C-terminal cap. Apo-SRP72 exists mainly as dimers in solution. To bind to SRP68, the SRP72 homodimer disassociates, and the indispensable C-terminal cap undergoes a pronounced conformational change to assist formation of the SRP68/72 heterodimer. A 23-residue polypeptide of SRP68 is sufficient for tight binding to SRP72 through its unusually hydrophobic and extended surface. Structural, biophysical, and mutagenesis analyses revealed that cancer-associated mutations disrupt the SRP68-SRP72 interaction and their co-localization with ER in mammalian cells. The results highlight the essential role of the SRP68-SRP72 interaction in SRP-mediated protein translocation and provide a structural basis for disease diagnosis, pathophysiology, and drug design.

  3. 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.

  4. Hey bHLH Proteins Interact with a FBXO45 Containing SCF Ubiquitin Ligase Complex and Induce Its Translocation into the Nucleus.

    PubMed

    Salat, Daniela; Winkler, Anja; Urlaub, Henning; Gessler, Manfred

    2015-01-01

    The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors.

  5. Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex*

    PubMed Central

    Romano, Fabian B.; Tang, Yuzhou; Rossi, Kyle C.; Monopoli, Kathryn R.; Ross, Jennifer L.; Heuck, Alejandro P.

    2016-01-01

    A type 3 secretion system is used by many bacterial pathogens to inject proteins into eukaryotic cells. Pathogens insert a translocon complex into the target eukaryotic membrane by secreting two proteins known as translocators. How these translocators form a translocon in the lipid bilayer and why both proteins are required remains elusive. Pseudomonas aeruginosa translocators PopB and PopD insert pores into membranes forming homo- or hetero-complexes of undetermined stoichiometry. Single-molecule fluorescence photobleaching experiments revealed that PopD formed mostly hexameric structures in membranes, whereas PopB displayed a bi-modal distribution with 6 and 12 subunits peaks. However, individually the proteins are not functional for effector translocation. We have found that when added together, the translocators formed distinct hetero-complexes containing 8 PopB and 8 PopD molecules. Thus, the interaction between PopB and PopD guide the assembly of a unique hetero-oligomer in membranes. PMID:26786106

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. Interaction of Tim23 with Tim50 Is essential for protein translocation by the mitochondrial TIM23 complex.

    PubMed

    Gevorkyan-Airapetov, Lada; Zohary, Keren; Popov-Celeketic, Dusan; Mapa, Koyeli; Hell, Kai; Neupert, Walter; Azem, Abdussalam; Mokranjac, Dejana

    2009-02-20

    The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23. We established two independent methods, chemical cross-linking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 that abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction between the two proteins in vivo, leading to defective import of preproteins via the TIM23 complex and to cell death at higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to identify specific residues of Tim23 that are vital for the interaction with Tim50.

  12. 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

  13. 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.

  14. Computational analysis of maltose binding protein translocation

    NASA Astrophysics Data System (ADS)

    Chinappi, Mauro; Cecconi, Fabio; Massimo Casciola, Carlo

    2011-05-01

    We propose a computational model for the study of maltose binding protein translocation across α-hemolysin nanopores. The phenomenological approach simplifies both the pore and the polypeptide chain; however it retains the basic structural protein-like properties of the maltose binding protein by promoting the correct formation of its native key interactions. By considering different observables characterising the channel blockade and molecule transport, we verified that MD simulations reproduce qualitatively the behaviour observed in a recent experiment. Simulations reveal that blockade events consist of a capture stage, to some extent related to the unfolding kinetics, and a single file translocation process in the channel. A threshold mechanics underlies the process activation with a critical force depending on the protein denaturation state. Finally, our results support the simple interpretation of translocation via first-passage statistics of a driven diffusion process of a single reaction coordinate.

  15. 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

  16. 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

  17. 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.

  18. Simulation of polymer translocation through protein channels

    NASA Astrophysics Data System (ADS)

    Muthukumar, M.; Kong, C. Y.

    2006-04-01

    A modeling algorithm is presented to compute simultaneously polymer conformations and ionic current, as single polymer molecules undergo translocation through protein channels. The method is based on a combination of Langevin dynamics for coarse-grained models of polymers and the Poisson-Nernst-Planck formalism for ionic current. For the illustrative example of ssDNA passing through the -hemolysin pore, vivid details of conformational fluctuations of the polymer inside the vestibule and -barrel compartments of the protein pore, and their consequent effects on the translocation time and extent of blocked ionic current are presented. In addition to yielding insights into several experimentally reported puzzles, our simulations offer experimental strategies to sequence polymers more efficiently.

  19. Control of protein function through optochemical translocation.

    PubMed

    Engelke, Hanna; Chou, Chungjung; Uprety, Rajendra; Jess, Phillip; Deiters, Alexander

    2014-10-17

    Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applications-light-triggered transcription and light-triggered protease cleavage-both based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology.

  20. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

    PubMed

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-12-29

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

  1. Translocator protein-mediated pharmacology of cholesterol transport and steroidogenesis.

    PubMed

    Papadopoulos, Vassilios; Aghazadeh, Yasaman; Fan, Jinjiang; Campioli, Enrico; Zirkin, Barry; Midzak, Andrew

    2015-06-15

    Steroidogenesis begins with cholesterol transfer into mitochondria through the transduceosome, a complex composed of cytosolic proteins that include steroidogenesis acute regulatory protein (STAR), 14-3-3 adaptor proteins, and the outer mitochondrial membrane proteins Translocator Protein (TSPO) and Voltage-Dependent Anion Channel (VDAC). TSPO is a drug- and cholesterol-binding protein found at particularly high levels in steroid synthesizing cells. Its aberrant expression has been linked to cancer, neurodegeneration, neuropsychiatric disorders and primary hypogonadism. Brain steroids serve as local regulators of neural development and excitability. Reduced levels of these steroids have been linked to depression, anxiety and neurodegeneration. Reduced serum testosterone is common among subfertile young men and aging men, and is associated with depression, metabolic syndrome and reduced sexual function. Although testosterone-replacement therapy is available, there are undesired side-effects. TSPO drug ligands have been proposed as therapeutic agents to regulate steroid levels in the brain and testis.

  2. 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

  3. 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.

  4. 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.

  5. Structural framework for DNA translocation via the viral portal protein

    PubMed Central

    Lebedev, Andrey A; Krause, Margret H; Isidro, Anabela L; Vagin, Alexei A; Orlova, Elena V; Turner, Joanne; Dodson, Eleanor J; Tavares, Paulo; Antson, Alfred A

    2007-01-01

    Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X-ray structure of the bacteriophage SPP1 portal protein in its isolated 13-subunit form and the pseudoatomic structure of a 12-subunit assembly. The first defines the DNA-interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal–DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a ‘mexican wave' of positional and conformational changes propagating sequentially along this belt. PMID:17363899

  6. DNA translocation through protein and synthetic nano pores

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Aniket

    2007-03-01

    DNA translocation through narrow protein channels is recognized as an important process in biology. Recently it has attracted lot of attention in the biophysical community following several experiments on DNA translocation through protein nano-pores, and more recently, through synthetic silicon nano-pores. A fundamental understanding is needed for various biological processes, e.g., entry and exit of a DNA in and out of a cell, efficient separation methods for macromolecules, and, possibly fast DNA sequencing. In this talk I will be presenting results for the DNA translocation using a coarse-grained model for an idealized DNA as well as the pore. I will consider several scenarios for the DNA translocation. First, I will show scaling of translocation time of a homopolymer as it escapes from the trans side to the cis side of an idealized thin membrane. Then I will consider DNA dynamics subject to a driving force inside the pore. Next, I will consider heteropolymer threading through a nano-pore. Specifically we will consider both highly ordered and completely random sequences of the chain and relate specific sequences to the distribution of the translocation time and the residence time inside the pore. These studies also will include effects due to different environment on either side of the pore, specific DNA-pore interactions located at selective sites, etc.. I will discuss relevance of these simulation results to recent experiments and theoretical models. A. Milchev, K. Binder, and Aniket Bhattacharya, J. Chem. Phys. 121, 6042 (2004).

  7. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein

    PubMed Central

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-01-01

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins. DOI: http://dx.doi.org/10.7554/eLife.11897.001 PMID:26714107

  8. Prediction of the translocation kinetics of a protein from its mechanical properties.

    PubMed

    West, Daniel K; Brockwell, David J; Paci, Emanuele

    2006-09-01

    Proteins are actively unfolded to pass through narrow channels in macromolecular complexes that catalyze protein translocation and degradation. Catalyzed unfolding shares many features that characterize the mechanical unfolding of proteins using the atomic force microscope (AFM). However, simulations of unfolding induced by the AFM and when a protein is translocated through a pore suggest that each process occurs by distinct pathways. The link, if any, between each type of unfolding, therefore, is not known. We show that the mechanical unfolding energy landscape of a protein, obtained using an atomistic molecular model, can be used to predict both the relative mechanical strength of proteins when unfolded using the AFM and when unfolded by translocation into a pore. We thus link the two processes and show that the import rate through a pore not only depends on the location of the initiation tag but also on the mechanical properties of the protein when averaged over all the possible geometries that are relevant for a given translocation initiation site.

  9. Transport and proofreading of proteins by the twin-arginine translocation (Tat) system in bacteria.

    PubMed

    Robinson, Colin; Matos, Cristina F R O; Beck, Daniel; Ren, Chao; Lawrence, Janna; Vasisht, Nishi; Mendel, Sharon

    2011-03-01

    The twin-arginine translocation (Tat) system operates in plant thylakoid membranes and the plasma membranes of most free-living bacteria. In bacteria, it is responsible for the export of a number of proteins to the periplasm, outer membrane or growth medium, selecting substrates by virtue of cleavable N-terminal signal peptides that contain a key twin-arginine motif together with other determinants. Its most notable attribute is its ability to transport large folded proteins (even oligomeric proteins) across the tightly sealed plasma membrane. In Gram-negative bacteria, TatABC subunits appear to carry out all of the essential translocation functions in the form of two distinct complexes at steady state: a TatABC substrate-binding complex and separate TatA complex. Several studies favour a model in which these complexes transiently coalesce to generate the full translocase. Most Gram-positive organisms possess an even simpler "minimalist" Tat system which lacks a TatB component and contains, instead, a bifunctional TatA component. These Tat systems may involve the operation of a TatAC complex together with a separate TatA complex, although a radically different model for TatAC-type systems has also been proposed. While bacterial Tat systems appear to require the presence of only a few proteins for the actual translocation event, there is increasing evidence for the operation of ancillary components that carry out sophisticated "proofreading" activities. These activities ensure that redox proteins are only exported after full assembly of the cofactor, thereby avoiding the futile export of apo-forms. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

  10. PARK7 protein translocating into spermatozoa mitochondria in Chinese asthenozoospermia.

    PubMed

    Sun, Yi; Zhang, Wen-Jia; Zhao, Xin; Yuan, Ren-Pei; Jiang, Hui; Pu, Xiao-Ping

    2014-09-01

    PARK7 (DJ1) is a multifunctional oxidative stress response protein that protects cells against reactive oxygen species (ROS) and mitochondrial damage. PARK7 defects are known to cause various physiological dysfunctions, including infertility. Asthenozoospermia (AS), i.e. low-motile spermatozoa in the ejaculate, is a common cause of human male infertility. In this study, we found that downregulation of PARK7 resulted in increased levels of lipid peroxide and ROS, decreased mitochondrial membrane potential, and reduced mitochondrial complex I enzyme activity in the spermatozoa from AS patients. Furthermore, it was observed that PARK7 was translocated into the mitochondria of damaged spermatozoa in AS. Finally, we examined the oxidative state of PARK7 and the results demonstrated the enhancement of oxidation, expressed by increased sulfonic acid residues, the highest form of oxidation, as the sperm motility decreased. Taken together, these results revealed that PARK7 deficiency may increase the oxidative stress damage to spermatozoa. Our present findings open new avenues of therapeutic intervention targeting PARK7 for the treatment of AS.

  11. Retro-translocation of mitochondrial intermembrane space proteins

    PubMed Central

    Bragoszewski, Piotr; Wasilewski, Michal; Sakowska, Paulina; Gornicka, Agnieszka; Böttinger, Lena; Qiu, Jian; Wiedemann, Nils; Chacinska, Agnieszka

    2015-01-01

    The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance. PMID:26056291

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

    PubMed

    Li, Long; Park, Eunyong; Ling, JingJing; Ingram, Jessica; Ploegh, Hidde; Rapoport, Tom A

    2016-03-17

    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 lipid. 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. How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (~10 Å) or are of insufficient quality. 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 carboxy (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.

  13. Decatransin, a new natural product inhibiting protein translocation at the Sec61/SecYEG translocon.

    PubMed

    Junne, Tina; Wong, Joanne; Studer, Christian; Aust, Thomas; Bauer, Benedikt W; Beibel, Martin; Bhullar, Bhupinder; Bruccoleri, Robert; Eichenberger, Jürg; Estoppey, David; Hartmann, Nicole; Knapp, Britta; Krastel, Philipp; Melin, Nicolas; Oakeley, Edward J; Oberer, Lukas; Riedl, Ralph; Roma, Guglielmo; Schuierer, Sven; Petersen, Frank; Tallarico, John A; Rapoport, Tom A; Spiess, Martin; Hoepfner, Dominic

    2015-03-15

    A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.

  14. Two distinct membrane potential-dependent steps drive mitochondrial matrix protein translocation.

    PubMed

    Schendzielorz, Alexander Benjamin; Schulz, Christian; Lytovchenko, Oleksandr; Clancy, Anne; Guiard, Bernard; Ieva, Raffaele; van der Laan, Martin; Rehling, Peter

    2017-01-02

    Two driving forces energize precursor translocation across the inner mitochondrial membrane. Although the membrane potential (Δψ) is considered to drive translocation of positively charged presequences through the TIM23 complex (presequence translocase), the activity of the Hsp70-powered import motor is crucial for the translocation of the mature protein portion into the matrix. In this study, we show that mitochondrial matrix proteins display surprisingly different dependencies on the Δψ. However, a precursor's hypersensitivity to a reduction of the Δψ is not linked to the respective presequence, but rather to the mature portion of the polypeptide chain. The presequence translocase constituent Pam17 is specifically recruited by the receptor Tim50 to promote the transport of hypersensitive precursors into the matrix. Our analyses show that two distinct Δψ-driven translocation steps energize precursor passage across the inner mitochondrial membrane. The Δψ- and Pam17-dependent import step identified in this study is positioned between the two known energy-dependent steps: Δψ-driven presequence translocation and adenosine triphosphate-driven import motor activity.

  15. Dityromycin and GE82832 bind protein S12 and block EF-G catalyzed translocation

    PubMed Central

    Bulkley, David; Brandi, Letizia; Polikanov, Yury S.; Fabbretti, Attilio; O’Connor, Michael; Gualerzi, Claudio O.; Steitz, Thomas A.

    2017-01-01

    Summary The translocation of messenger RNA and transfer RNA through the ribosome is catalyzed by EF-G, a universally conserved GTPase. The mechanism by which the closely related decapeptide antibiotics dityromycin and GE82832 inhibit EF-G-catalyzed translocation is elucidated in this study. Using crystallographic and biochemical experiments we demonstrate that these antibiotics bind to ribosomal protein S12 in solution as well as within the small ribosomal subunit, inducing long-range effects on the ribosomal head. The crystal structure of the antibiotic in complex with the 70S ribosome reveals that the binding involves conserved amino acid residues of S12 whose mutations result in in vitro and in vivo antibiotic resistance and loss of antibiotic binding. The data also suggest that GE82832/dityromycin inhibits EF-G-catalyzed translocation by disrupting a critical contact between EF-G and S12 that is required to stabilize the post-translocational conformation of EF-G, thereby preventing the ribosome-EF-G complex from entering a conformation productive for translocation. PMID:24412368

  16. Lost in translocation: the functions of the 18-kD translocator protein.

    PubMed

    Gut, Philipp; Zweckstetter, Markus; Banati, Richard B

    2015-07-01

    Research spanning nearly four decades has assigned to the translocator protein (18 kDa) (TSPO) a critical role, among others, in the mitochondrial import of cholesterol, the subsequent steps of (neuro)steroid production, and systemic endocrine regulation, with implications for the pathophysiology of immune, inflammatory, neurodegenerative, and psychiatric as well as neoplastic diseases. Recent knockout studies in mice unexpectedly report normal or latent phenotypes, raising doubts about the protein's role in steroidogenesis and other previously postulated functions and challenging the validity of earlier data on the selectivity of TSPO-binding drugs. Here we provide a synthesis of the current debate from a structural and molecular biology perspective, discuss the limits of inference in loss-of-function (gene knockout) studies, and suggest new functions of TSPO.

  17. Translocation of structural P proteins in the phloem.

    PubMed Central

    Golecki, B; Schulz, A; Thompson, G A

    1999-01-01

    Phloem-specific proteins (P proteins) are particularly useful markers to investigate long-distance trafficking of macromolecules in plants. In this study, genus-specific molecular probes were used in combination with intergeneric grafts to reveal the presence of a pool of translocatable P protein subunits. Immunoblot analyses demonstrated that Cucurbita spp P proteins PP1 and PP2 are translocated from Cucurbita maxima stocks and accumulate in Cucumis sativus scions. Cucurbita maxima or Cucurbita ficifolia PP1 and PP2 mRNAs were not detected in Cucumis sativus scions by either RNA gel blot analysis or reverse transcription-polymerase chain reaction, indicating that the proteins, rather than transcripts, are translocated. Tissue prints of the Cucumis sativus scion, using antibodies raised against Cucurbita maxima PP1 or PP2, detected both proteins in the fascicular phloem of the stem at points distal to the graft union and in the petiole of a developing leaf, suggesting that the proteins move within the assimilate stream toward sink tissues. Cucurbita maxima PP1 was immunolocalized by light microscopy in sieve elements of the extrafascicular phloem of Cucumis sativus scions, whereas Cucurbita maxima PP2 was detected in both sieve elements and companion cells. PMID:9878637

  18. Translocation of signalling proteins to the plasma membrane revealed by a new bioluminescent procedure

    PubMed Central

    2011-01-01

    Background Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors. High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive. Results Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca2+)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca2+ concentration [Ca2+] difference between bulk cytosolic and the sub-plasma membrane rim. Conclusion This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane. Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation. Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs

  19. 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.

  20. Analysis of Translocation-Competent Secretory Proteins by HDX-MS.

    PubMed

    Tsirigotaki, A; Papanastasiou, M; Trelle, M B; Jørgensen, T J D; Economou, A

    2017-01-01

    Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded. However, once they reach the appropriate cellular compartment, they can fold to their native states. The nonnative states of preproteins remain structurally poorly characterized since increased disorder, protein sizes, aggregation propensity, and the observation timescale are often limiting factors for typical structural approaches such as X-ray crystallography and NMR. Here, we present an alternative approach for the in vitro analysis of nonfolded translocation-competent protein states and their comparison with their native states. We make use of hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS), a method based on differentiated isotope exchange rates in structured vs unstructured protein states/regions, and highly dynamic vs more rigid regions. We present a complete structural characterization pipeline, starting from the preparation of the polypeptides to data analysis and interpretation. Proteolysis and mass spectrometric conditions for the analysis of the labeled proteins are discussed, followed by the analysis and interpretation of HDX-MS data. We highlight the suitability of HDX-MS for identifying short structured regions within otherwise highly flexible protein states, as illustrated by an exported protein example, experimentally tested in our lab. Finally, we discuss statistical analysis in comparative HDX-MS. The protocol is applicable to any protein and protein size, exhibiting slow or fast loss of translocation competence. It could be easily adapted to more complex assemblies, such as the interaction of chaperones with nonnative protein states.

  1. 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.

  2. 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

  3. The complex of tmRNA-SmpB and EF-G on translocating ribosomes.

    PubMed

    Ramrath, David J F; Yamamoto, Hiroshi; Rother, Kristian; Wittek, Daniela; Pech, Markus; Mielke, Thorsten; Loerke, Justus; Scheerer, Patrick; Ivanov, Pavel; Teraoka, Yoshika; Shpanchenko, Olga; Nierhaus, Knud H; Spahn, Christian M T

    2012-05-06

    Bacterial ribosomes stalled at the 3' end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD-SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome-tmRNA complexes at low to intermediate resolution, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S-tmRNA-SmpB-EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3 Å resolution. This post-translocational intermediate (TI(POST)) presents the TLD-SmpB module in an intrasubunit ap/P hybrid site and a tRNA(fMet) in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA-SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways.

  4. Requirements for a conservative protein translocation pathway in chloroplasts.

    PubMed

    Vojta, Lea; Soll, Jürgen; Bölter, Bettina

    2007-06-12

    The chloroplast inner envelope translocon subunit Tic110 is imported via a soluble stromal translocation intermediate. In this study an in-organellar import system is established which allows for an accumulation of this intermediate in order to analyze its requirements for reexport. All results demonstrate that the re-export of Tic110 from the soluble intermediate stage into the inner envelope requires ATP hydrolysis, which cannot be replaced by other NTPs. Furthermore, the molecular chaperone Hsp93 seems prominently involved in the reexport pathway of Tic110, because other stromal intermediates like that of the oxygen evolving complex subunit OE33 (iOE33) en route to the thylakoid lumen interacts preferentially with Hsp70.

  5. Autophosphorylation of the C2 domain inhibits translocation of the novel protein kinase C (nPKC) Apl II.

    PubMed

    Farah, Carole A; Lindeman, Amanda A; Siu, Vincent; Gupta, Micaela Das; Sossin, Wayne S

    2012-11-01

    Protein kinase Cs (PKCs) are critical signaling molecules controlled by complex regulatory pathways. Herein, we describe an important regulatory role for C2 domain phosphorylation. Novel PKCs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. Previously, we described an autophosphorylation site in the Aplysia novel PKC Apl II that increased the binding of the C2 domain to lipids. In this study, we show that the function of this phosphorylation is to inhibit PKC translocation. Indeed, a phosphomimetic serine-glutamic acid mutation reduced translocation of PKC Apl II while blocking phosphorylation with a serine-alanine mutation enhanced translocation and led to the persistence of the kinase at the membrane longer after the end of the stimulation. Consistent with a role for autophosphorylation in regulating kinase translocation, inhibiting PKC activity using bisindolymaleimide 1 increased physiological translocation of PKC Apl II, whereas inhibiting phosphatase activity using calyculin A inhibited physiological translocation of PKC Apl II in neurons. Our results suggest a major role for autophosphorylation-dependent regulation of translocation.

  6. Electrically facilitated translocations of proteins through silicon nitride nanopores: conjoint and competitive action of diffusion, electrophoresis, and electroosmosis.

    PubMed

    Firnkes, Matthias; Pedone, Daniel; Knezevic, Jelena; Döblinger, Markus; Rant, Ulrich

    2010-06-09

    Solid-state nanopores bear great potential to be used to probe single proteins; however, the passage of proteins through nanopores was found to be complex, and unexpected translocation behavior with respect to the passage direction, rate, and duration was observed. Here we study the translocation of a model protein (avidin) through silicon nitride nanopores focusing on the electrokinetic effects that facilitate protein transport across the pore. The nanopore zeta potential zeta(pore) and the protein zeta potential zeta(protein) are measured independently as a function of solution pH. Our results reveal that electroosmotic transport may enhance or dominate and reverse electrophoretic transport in nanopores. The translocation behavior is rationalized by accounting for the charging states of the protein and the pore, respectively; the resulting translocation direction can be predicted according to the difference in zeta potentials, zeta(protein) - zeta(pore). When electrophoresis and electroosmosis cancel each other out, diffusion becomes an effective (and bias-independent) mechanism which facilitates protein transport across the pore at a significant rate.

  7. 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.

  8. 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

  9. The Mitochondrial Translocator Protein and Arrhythmogenesis in Ischemic Heart Disease

    PubMed Central

    Akar, Fadi G.

    2015-01-01

    Mitochondrial dysfunction is a hallmark of multiple cardiovascular disorders, including ischemic heart disease. Although mitochondria are well recognized for their role in energy production and cell death, mechanisms by which they control excitation-contraction coupling, excitability, and arrhythmias are less clear. The translocator protein (TSPO) is an outer mitochondrial membrane protein that is expressed in multiple organ systems. The abundant expression of TSPO in macrophages has been leveraged to image the immune response of the heart to inflammatory processes. More recently, the recognition of TSPO as a regulator of energy-dissipating mitochondrial pathways has extended its utility from a diagnostic marker of inflammation to a therapeutic target influencing diverse pathophysiological processes. Here, we provide an overview of the emerging role of TSPO in ischemic heart disease. We highlight the importance of TSPO in the regenerative process of reactive oxygen species (ROS) induced ROS release through its effects on the inner membrane anion channel (IMAC) and the permeability transition pore (PTP). We discuss evidence implicating TSPO in arrhythmogenesis in the settings of acute ischemia-reperfusion injury and myocardial infarction. PMID:25918579

  10. A G-Protein Subunit Translocation Embedded Network Motif Underlies GPCR Regulation of Calcium Oscillations

    PubMed Central

    Giri, Lopamudra; Patel, Anilkumar K.; Karunarathne, W.K. Ajith; Kalyanaraman, Vani; Venkatesh, K.V.; Gautam, N.

    2014-01-01

    G-protein βγ subunits translocate reversibly from the plasma membrane to internal membranes on receptor activation. Translocation rates differ depending on the γ subunit type. There is limited understanding of the role of the differential rates of Gβγ translocation in modulating signaling dynamics in a cell. Bifurcation analysis of the calcium oscillatory network structure predicts that the translocation rate of a signaling protein can regulate the damping of system oscillation. Here, we examined whether the Gβγ translocation rate regulates calcium oscillations induced by G-protein-coupled receptor activation. Oscillations in HeLa cells expressing γ subunit types with different translocation rates were imaged and quantitated. The results show that differential Gβγ translocation rates can underlie the diversity in damping characteristics of calcium oscillations among cells. Mathematical modeling shows that a translocation embedded motif regulates damping of G-protein-mediated calcium oscillations consistent with experimental data. The current study indicates that such a motif may act as a tuning mechanism to design oscillations with varying damping patterns by using intracellular translocation of a signaling component. PMID:24988358

  11. 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

  12. 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.

  13. A Translocated Bacterial Protein Protects Vascular Endothelial Cells from Apoptosis

    PubMed Central

    Schmid, Michael C; Scheidegger, Florine; Dehio, Michaela; Balmelle-Devaux, Nadège; Schulein, Ralf; Guye, Patrick; Chennakesava, Cuddapah S; Biedermann, Barbara; Dehio, Christoph

    2006-01-01

    The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane–associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium. PMID:17121462

  14. ATM modulates the loading of recombination proteins onto a chromosomal translocation breakpoint hotspot.

    PubMed

    Sun, Jiying; Oma, Yukako; Harata, Masahiko; Kono, Kazuteru; Shima, Hiroki; Kinomura, Aiko; Ikura, Tsuyoshi; Suzuki, Hidekazu; Mizutani, Shuki; Kanaar, Roland; Tashiro, Satoshi

    2010-10-27

    Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots.

  15. 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.

  16. 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.

  17. A septal chromosome segregator protein evolved into a conjugative DNA-translocator protein

    PubMed Central

    Sepulveda, Edgardo; Vogelmann, Jutta

    2011-01-01

    Streptomycetes, Gram-positive soil bacteria well known for the production of antibiotics feature a unique conjugative DNA transfer system. In contrast to classical conjugation which is characterized by the secretion of a pilot protein covalently linked to a single-stranded DNA molecule, in Streptomyces a double-stranded DNA molecule is translocated during conjugative transfer. This transfer involves a single plasmid encoded protein, TraB. A detailed biochemical and biophysical characterization of TraB, revealed a close relationship to FtsK, mediating chromosome segregation during bacterial cell division. TraB translocates plasmid DNA by recognizing 8-bp direct repeats located in a specific plasmid region clt. Similar sequences accidentally also occur on chromosomes and have been shown to be bound by TraB. We suggest that TraB mobilizes chromosomal genes by the interaction with these chromosomal clt-like sequences not relying on the integration of the conjugative plasmid into the chromosome. PMID:22479692

  18. 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

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

    PubMed Central

    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. PMID:25988176

  20. Role of AIP and its homologue the blindness-associated protein AIPL1 in regulating client protein nuclear translocation.

    PubMed

    van der Spuy, J; Cheetham, M E

    2004-08-01

    Mutations in the AIPL1 (aryl hydrocarbon receptor interacting protein-like 1) cause the blinding disease Leber's congenital amaurosis. AIPL1 is a homologue of the AIP. AIP functions as part of a chaperone heterocomplex to facilitate signalling by the AhR and plays an important role in regulating the nuclear translocation of the receptor. We review the evidence for the role of AIP in protein translocation and compare the potential functions of AIPL1 in the translocation of its interacting partner the NEDD8 ultimate buster protein 1.

  1. A Role for Cytoskeletal Elements in the Light-Driven Translocation of Proteins in Rod Photoreceptors

    PubMed Central

    Peterson, James J.; Orisme, Wilda; Fellows, Jonathan; McDowell, J. Hugh; Shelamer, Charles L.; Dugger, Donald R.; Clay Smith, W.

    2006-01-01

    PURPOSE. Light-driven protein translocation is responsible for the dramatic redistribution of some proteins in vertebrate rod photoreceptors. In this study, the involvement of microtubules and microfilaments in the light-driven translocation of arrestin and transducin was investigated. METHODS. Pharmacologic reagents were applied to native and transgenic Xenopus tadpoles, to disrupt the microtubules (thiabendazole) and microfilaments (cytochalasin D and latrunculin B) of the rod photoreceptors. Quantitative confocal imaging was used to assess the impact of these treatments on arrestin and transducin translocation. A series of transgenic tadpoles expressing arrestin truncations were also created to identify portions of arrestin that enable arrestin to translocate. RESULTS. Application of cytochalasin D or latrunculin B to disrupt the microfilament organization selectively slowed only transducin movement from the inner to the outer segments. Perturbation of the microtubule cytoskeleton with thiabendazole slowed the translocation of both arrestin and transducin, but only in moving from the outer to the inner segments. Transgenic Xenopus expressing fusions of green fluorescent protein (GFP) with portions of arrestin implicates the C terminus of arrestin as an important portion of the molecule for promoting translocation. This C-terminal region can be used independently to promote translocation of GFP in response to light. CONCLUSIONS. The results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motor-driven mechanism for complete movement of arrestin and transducin. PMID:16249472

  2. 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

  3. The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

    PubMed Central

    Wunder, Tobias; Martin, Roman; Löffelhardt, Wolfgang; Schleiff, Enrico; Steiner, Jürgen M

    2007-01-01

    Background Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems. Results To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon. Conclusion The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a "primitive" translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface. The phenylalanine has

  4. PIAS proteins are involved in the SUMO-1 modification, intracellular translocation and transcriptional repressive activity of RET finger protein

    SciTech Connect

    Matsuura, Tetsuo; Shimono, Yohei; Kawai, Kumi; Murakami, Hideki; Urano, Takeshi; Niwa, Yasumasa; Goto, Hidemi; Takahashi, Masahide . E-mail: mtakaha@med.nagoya-u.ac.jp

    2005-08-01

    Ret finger protein (RFP) is a nuclear protein that is highly expressed in testis and in various tumor cell lines. RFP functions as a transcriptional repressor and associates with Enhancer of Polycomb 1 (EPC1), a member of the Polycomb group proteins, and Mi-2{beta}, a main component of the nucleosome remodeling and deacetylase (NuRD) complex. We show that RFP binds with PIAS (protein inhibitor of activated STAT) proteins, PIAS1, PIAS3, PIASx{alpha} and PIASy at their carboxyl-terminal region and is covalently modified by SUMO-1 (sumoylation). PIAS proteins enhance the sumoylation of RFP in a dose-dependent manner and induce the translocation of RFP into nuclear bodies reminiscent of the PML bodies. In addition, co-expression of PIAS proteins or SUMO-1 strengthened the transcriptional repressive activity of RFP. Finally, our immunohistochemical results show that RFP, SUMO-1 and PIASy localize in a characteristic nuclear structure juxtaposed with the inner nuclear membrane (XY body) of primary spermatocytes in mouse testis. These results demonstrate that the intracellular location and the transcriptional activity of RFP are modified by PIAS proteins which possess SUMO E3 ligase activities and suggest that they may play a co-operative role in spermatogenesis.

  5. A translocation signal for delivery of oomycete effector proteins into host plant cells.

    PubMed

    Whisson, Stephen C; Boevink, Petra C; Moleleki, Lucy; Avrova, Anna O; Morales, Juan G; Gilroy, Eleanor M; Armstrong, Miles R; Grouffaud, Severine; van West, Pieter; Chapman, Sean; Hein, Ingo; Toth, Ian K; Pritchard, Leighton; Birch, Paul R J

    2007-11-01

    Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK--representing a change that conserves physicochemical properties of the protein--P. infestans fails to deliver Avr3a or an Avr3a-GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

  6. Evidence that small proteins translocate through silicon nitride pores in a folded conformation

    NASA Astrophysics Data System (ADS)

    Stefureac, Radu I.; Trivedi, Dhruti; Marziali, Andre; Lee, Jeremy S.

    2010-11-01

    The interaction of three proteins (histidine-containing phosphocarrier protein, HPr, calmodulin, CaM, and maltose binding protein, MBP) with synthetic silicon nitride (SiNx) membranes has been studied. The proteins which have a net negative charge were electrophoretically driven into pores of 7 and 5 nm diameter with a nominal length of 15 nm. The % blockade current and event duration were measured at three different voltages. For a translocation event it was expected that the % block would be constant with voltage whilst the event duration would decrease with increasing voltage. On the basis of these criteria, we deduce that MBP whose largest dimension is 6.5 nm does not translocate whereas up to 40% of CaM molecules can translocate the 7 nm pore as can a majority of HPr molecules, with some translocations being observed for the 5 nm pore. For translocation events the magnitude of the % blockade current is consistent with a folded conformation of the proteins surrounded by a hydration shell of 0.5-1.0 nm.

  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

    2008-06-06

    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.

  8. Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria.

    PubMed

    Popov-Celeketić, Dusan; Mapa, Koyeli; Neupert, Walter; Mokranjac, Dejana

    2008-05-21

    The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments.

  9. 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.

  10. 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

  11. 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…

  12. Proteins : paradigms of complexity /

    SciTech Connect

    Frauenfelder, Hans,

    2001-01-01

    Proteins are the working machines of living systems. Directed by the DNA, of the order of a few hundred building blocks, selected from twenty different amino acids, are covalently linked into a linear polypeptide chain. In the proper environment, the chain folds into the working protein, often a globule of linear dimensions of a few nanometers. The biologist considers proteins units from which living systems are built. Many physical scientists look at them as systems in which the laws of complexity can be studied better than anywhere else. Some of the results of such studies will be sketched.

  13. S-Nitrosylation Regulates Nuclear Translocation of Chloride Intracellular Channel Protein CLIC4*

    PubMed Central

    Malik, Mariam; Shukla, Anjali; Amin, Palak; Niedelman, Wendy; Lee, Jessica; Jividen, Kasey; Phang, Juanita M.; Ding, Jinhui; Suh, Kwang S.; Curmi, Paul M. G.; Yuspa, Stuart H.

    2010-01-01

    Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca2+-induced differentiation, stress-induced apoptosis, and modulating TGF-β signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin α and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor α-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor α-induced nitrosylation and association with importin α and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution. PMID:20504765

  14. 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.

  15. Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons

    SciTech Connect

    Vaccarino, F.; Guidotti, A.; Costa, E.

    1987-12-01

    In primary cultures of cerebellar granule cells, protein kinase C (PKC) translocation and activation can be triggered by the stimulation of excitatory amino acid neurotransmitter receptors. Glutamate evokes a dose-related translocation of 4-..beta..-(/sup 3/H)phorbol 12,13-dibutyrate /(/sup 3/H)-P(BtO)/sub 2// binding sites from the cytosol to the neuronal membrane and stimulates the incorporation of /sup 32/P into a number of membrane proteins, particularly protein bands in the range of 80, 50, and 40 kDa. The glutamate-evoked PKC translocation is Mg/sup 2 +/ sensitive, is prevented by 2-amino-5-phosphonovalerate and phencyclidine, is not inhibited by nitrendipine (a voltage-dependent Ca/sup 2 +/-channel-blocker) but is abolished by the removal of Ca/sup 2 +/ from the incubation medium, suggesting that glutamate-mediated Ca/sup 2 +/ influx is operative in the redistribution of PKC. Exposure of granule cells to the gangliosides trisialosylgangliotetraglycosylceramide (GT1b) of monosialosylgangliotetraglycosylceramide (GM1) inhibits the translocation and activation of PKC evoked by glutamate. These glycosphingolipids fail to interfere with glutamate binding to its high-affinity recognition site of with the (/sup 3/H)P(BtO)/sub 2/ binding, nor do they affect the Ca/sup 2 +/ influx. These gangliosides may prevent PKC translocation by interfering with the PKC binding to the neuronal membrane phosphatidylserine.

  16. Carbohydrate-protein interactions that drive processive polysaccharide translocation in enzymes revealed from a computational study of cellobiohydrolase processivity.

    PubMed

    Knott, Brandon C; Crowley, Michael F; Himmel, Michael E; Ståhlberg, Jerry; Beckham, Gregg T

    2014-06-18

    Translocation of carbohydrate polymers through protein tunnels and clefts is a ubiquitous biochemical phenomenon in proteins such as polysaccharide synthases, glycoside hydrolases, and carbohydrate-binding modules. Although static snapshots of carbohydrate polymer binding in proteins have long been studied via crystallography and spectroscopy, the molecular details of polysaccharide chain processivity have not been elucidated. Here, we employ simulation to examine how a cellulose chain translocates by a disaccharide unit during the processive cycle of a glycoside hydrolase family 7 cellobiohydrolase. Our results demonstrate that these biologically and industrially important enzymes employ a two-step mechanism for chain threading to form a Michaelis complex and that the free energy barrier to chain threading is significantly lower than the hydrolysis barrier. Taken with previous studies, our findings suggest that the rate-limiting step in enzymatic cellulose degradation is the glycosylation reaction, not chain processivity. Based on the simulations, we find that strong electrostatic interactions with polar residues that are conserved in GH7 cellobiohydrolases, but not in GH7 endoglucanases, at the leading glucosyl ring provide the thermodynamic driving force for polysaccharide chain translocation. Also, we consider the role of aromatic-carbohydrate interactions, which are widespread in carbohydrate-active enzymes and have long been associated with processivity. Our analysis suggests that the primary role for these aromatic residues is to provide tunnel shape and guide the carbohydrate chain to the active site. More broadly, this work elucidates the role of common protein motifs found in carbohydrate-active enzymes that synthesize or depolymerize polysaccharides by chain translocation mechanisms coupled to catalysis.

  17. 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.

  18. Clustering of C-terminal stromal domains of Tha4 homo-oligomers during translocation by the Tat protein transport system.

    PubMed

    Dabney-Smith, Carole; Cline, Kenneth

    2009-04-01

    The chloroplast Twin arginine translocation (Tat) pathway uses three membrane proteins and the proton gradient to transport folded proteins across sealed membranes. Precursor proteins bind to the cpTatC-Hcf106 receptor complex, triggering Tha4 assembly and protein translocation. Tha4 is required only for the translocation step and is thought to be the protein-conducting component. The organization of Tha4 oligomers was examined by substituting pairs of cysteine residues into Tha4 and inducing disulfide cross-links under varying stages of protein translocation. Tha4 formed tetramers via its transmembrane domain in unstimulated membranes and octamers in membranes stimulated by precursor and the proton gradient. Tha4 formed larger oligomers of at least 16 protomers via its carboxy tail, but such C-tail clustering only occurred in stimulated membranes. Mutational studies showed that transmembrane domain directed octamers as well as C-tail clusters require Tha4's transmembrane glutamate residue and its amphipathic helix, both of which are necessary for Tha4 function. A novel double cross-linking strategy demonstrated that both transmembrane domain directed- and C-tail directed oligomerization occur in the translocase. These results support a model in which Tha4 oligomers dock with a precursor-receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 into a larger transport-active homo-oligomer.

  19. 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

  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. Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation

    PubMed Central

    Adams, Thorsten M.; Wentzel, Alexander; Kolmar, Harald

    2005-01-01

    Intimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells. Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor. We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REIv, ubiquitin, calmodulin, β-lactamase inhibitor protein, or β-lactamase. By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure. We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane. These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface. PMID:15629924

  2. High content screening for G protein-coupled receptors using cell-based protein translocation assays.

    PubMed

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne; Linde, Viggo; Pedersen, Hans-Christian; Krog-Jensen, Christian; Rosenkilde, Mette M; Pagliaro, Len

    2005-06-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.

  3. Tau proteins harboring neurodegeneration-linked mutations impair kinesin translocation in vitro.

    PubMed

    Yu, Dezhi; LaPointe, Nichole E; Guzman, Elmer; Pessino, Veronica; Wilson, Leslie; Feinstein, Stuart C; Valentine, Megan T

    2014-01-01

    We tested the hypothesis that mutant tau proteins that cause neurodegeneration and dementia differentially alter kinesin translocation along microtubules (MTs) relative to normal tau in vitro. We employed complementary in vitro motility assays using purified recombinant kinesin, purified recombinant tau, and purified bovine brain α:β tubulin to isolate interactions among these components without any contribution by cellular regulatory mechanisms. We found that kinesin translocates slower along MTs assembled by any of three independent tau mutants (4-repeat P301L tau, 4-repeat ΔN296 tau, and 4-repeat R406W tau) relative to its translocation rate along MTs assembled by normal, 4-repeat wild type (WT) tau. Moreover, the R406W mutation exhibited isoform specific effects; while kinesin translocation along 4-repeat R406W tau assembled MTs is slower than along MTs assembled by 4-repeat WT tau, the R406W mutation had no effect in the 3-repeat tau context. These data provide strong support for the notion that aberrant modulation of kinesin translocation is a component of tau-mediated neuronal cell death and dementia. Finally, we showed that assembling MTs with taxol before coating them with mutant tau obscured effects of the mutant tau that were readily apparent using more physiologically relevant MTs assembled with tau alone, raising important issues regarding the use of taxol as an experimental reagent and novel insights into therapeutic mechanisms of taxol action.

  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. 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

  6. 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.

  7. 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

  8. A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

    PubMed Central

    Ezure, Toru; Nanatani, Kei; Sato, Yoko; Suzuki, Satomi; Aizawa, Keishi; Souma, Satoshi; Ito, Masaaki; Hohsaka, Takahiro; von Heijine, Gunnar; Utsumi, Toshihiko; Abe, Keietsu; Ando, Eiji; Uozumi, Nobuyuki

    2014-01-01

    Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins. PMID:25486605

  9. 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.

  10. The first dipeptide ligand of translocator protein: Design and anxiolytic activity.

    PubMed

    Gudasheva, T A; Deeva, O A; Mokrov, G V; Yarkov, S A; Yarkova, M A; Seredenin, S B

    2015-01-01

    On the basis of the structure of Alpidem, a pyrazolopyrimidine ligand of the translocator protein (TSPO), a dipeptide TSPO ligand, N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23), was designed and synthesized using our own original peptide design strategy. This compound exhibited anxiolytic activity in BALB/cAnN mice in the "open-field" test and in outbred CD1 mice in the "elevated plus maze" test. The stereoselectivity of the anxiolytic effect of GD-23 is demonstrated. The results of this study suggest that GD-23 is a ligand of the translocator protein, and its structure can become the basis for creating anxiolytics with a fundamentally new mechanism of action.

  11. 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.

  12. Characterization of the mitochondrial inner membrane protein translocator Tim17 from Trypanosoma brucei

    PubMed Central

    Singha, Ujjal K.; Peprah, Emmanuel; Williams, Shuntae; Walker, Robert; Saha, Lipi; Chaudhuri, Minu

    2010-01-01

    Mitochondrial protein translocation machinery in the kinetoplastid parasites, like Trypanosoma brucei, has been characterized poorly. In T. brucei genome data base, one homolog for a protein translocator of mitochondrial inner membrane (Tim) has been found, which is closely related to Tim17 from other species. The T. brucei Tim17 (TbTim17) has a molecular mass 16.2 kDa and it possesses four characteristic transmembrane domains. The protein is localized in the mitochondrial inner membrane. The level of TbTim17 protein is 6–7 fold higher in the procyclic form that has a fully active mitochondrion, than in the mammalian bloodstream form of T. brucei, where many of the mitochondrial activities are suppressed. Knockdown of TbTim17 expression by RNAi caused a cessation of cell growth in the procyclic form and reduced growth rate in the bloodstream form. Depletion of TbTim17 decreased mitochondrial membrane potential more in the procyclic than bloodstream form. However, TbTim17 knockdown reduced the expression level of several nuclear encoded mitochondrial proteins in both the forms. Furthermore, import of presequence containing nuclear encoded mitochondrial proteins was significantly reduced in TbTim17 depleted mitochondria of the procyclic as well as the bloodstream form, confirming that TbTim17 is critical for mitochondrial protein import in both developmental forms. Together, these show that TbTim17 is the translocator of nuclear encoded mitochondrial proteins and its expression is regulated according to mitochondrial activities in T. brucei. PMID:18325611

  13. Cytoplasm-Translocated Ku70/80 Complex Sensing of HBV DNA Induces Hepatitis-Associated Chemokine Secretion

    PubMed Central

    Li, Young; Wu, Yang; Zheng, Xiaohu; Cong, Jingjing; Liu, Yanyan; Li, Jiabin; Sun, Rui; Tian, Zhigang G.; Wei, Haiming M.

    2016-01-01

    Chronic hepatitis B virus (HBV) infection remains a serious disease, mainly due to its severe pathological consequences, which are difficult to cure using current therapies. When the immune system responds to hepatocytes experiencing rapid HBV replication, effector cells (such as HBV-specific CD8+ T cells, NK cells, NKT cells, and other subtypes of immune cells) infiltrate the liver and cause hepatitis. However, the precise recruitment of these cells remains unclear. In the present study, we found that the cytoplasm-translocated Ku70/80 complex in liver-derived cells sensed cytosolic HBV DNA and promoted hepatitis-associated chemokine secretion. Upon sensing HBV DNA, DNA-dependent protein kinase catalytic subunit and PARP1 were assembled. Then, IRF1 was activated and translocated into the nucleus, which upregulated CCL3 and CCL5 expression. Because CCR5, a major chemokine receptor for CCL3 and CCL5, is known to be critical in hepatitis B, Ku70/80 sensing of HBV DNA likely plays a critical role in immune cell recruitment in response to HBV infection. PMID:27994596

  14. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine.

    PubMed

    Aubin-Tam, Marie-Eve; Olivares, Adrian O; Sauer, Robert T; Baker, Tania A; Lang, Matthew J

    2011-04-15

    All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5-8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.

  15. Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli.

    PubMed Central

    Chen, L; Rhoads, D; Tai, P C

    1985-01-01

    We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli. We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used. Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped. This was the case not only with the OmpA protein, which is synthesized by free polysomes and hence is presumably exported posttranslationally in the cell, but also with alkaline phosphatase, which is synthesized only by membrane-bound polysomes and has been shown to be secreted cotranslationally in the cells. Prolonged incubation rendered the precursors inactive for subsequent translocation. Posttranslational translocation was impaired, like cotranslational translocation, by inhibitors of the proton motive force and by treatment of the vesicles with protease. Since it appears that E. coli can translocate the same proteins either cotranslationally or posttranslationally, the cotranslational mode may perhaps be more efficient, but not obligatory, for the secretion of bacterial proteins. Images PMID:3882674

  16. 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.

  17. Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease.

    PubMed

    Yeeles, Joseph T P; van Aelst, Kara; Dillingham, Mark S; Moreno-Herrero, Fernando

    2011-06-24

    AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase assays and our understanding of helicase mechanisms in general.

  18. CD4 and BST-2/Tetherin Proteins Retro-translocate from Endoplasmic Reticulum to Cytosol as Partially Folded and Multimeric Molecules

    PubMed Central

    Petris, Gianluca; Casini, Antonio; Sasset, Linda; Cesaratto, Francesca; Bestagno, Marco; Cereseto, Anna; Burrone, Oscar R.

    2014-01-01

    CD4 and BST-2/Tetherin are cellular membrane proteins targeted to degradation by the HIV-1 protein Vpu. In both cases proteasomal degradation following recruitment into the ERAD pathway has been described. CD4 is a type I transmembrane glycoprotein, with four extracellular immunoglobulin-like domains containing three intrachain disulfide bridges. BST-2/Tetherin is an atypical type II transmembrane glycoprotein with an N-terminal transmembrane domain and a C-terminal glycophosphatidylinositol anchor, which dimerizes through three interchain bridges. We investigated spontaneous and Vpu-induced retro-translocation of CD4 and BST-2/Tetherin using our novel biotinylation technique in living cells to determine ER-to-cytosol retro-translocation of proteins. We found that CD4 retro-translocates with oxidized intrachain disulfide bridges, and only upon proteasomal inhibition does it accumulate in the cytosol as already reduced and deglycosylated molecules. Similarly, BST-2/Tetherin is first exposed to the cytosol as a dimeric oxidized complex and then becomes deglycosylated and reduced to monomers. These results raise questions on the required features of the putative retro-translocon, suggesting alternative retro-translocation mechanisms for membrane proteins in which complete cysteine reduction and unfolding are not always strictly required before ER to cytosol dislocation. PMID:24257748

  19. 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

  20. Essential regions in the membrane domain of bacterial complex I (NDH-1): the machinery for proton translocation.

    PubMed

    Sato, Motoaki; Torres-Bacete, Jesus; Sinha, Prem Kumar; Matsuno-Yagi, Akemi; Yagi, Takao

    2014-08-01

    The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is the first and largest enzyme of the respiratory chain which has a central role in cellular energy production and is implicated in many human neurodegenerative diseases and aging. It is believed that the peripheral domain of complex I/NDH-1 transfers the electron from NADH to Quinone (Q) and the redox energy couples the proton translocation in the membrane domain. To investigate the mechanism of the proton translocation, in a series of works we have systematically studied all membrane subunits in the Escherichia coli NDH-1 by site-directed mutagenesis. In this mini-review, we have summarized our strategy and results of the mutagenesis by depicting residues essential for proton translocation, along with those for subunit connection. It is suggested that clues to understanding the driving forces of proton translocation lie in the similarities and differences of the membrane subunits, highlighting the communication of essential charged residues among the subunits. A possible proton translocation mechanism with all membrane subunits operating in unison is described.

  1. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  2. 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

    2016-05-11

    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.

  3. Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure

    PubMed Central

    Jaipuria, Garima; Leonov, Andrei; Giller, Karin; Vasa, Suresh Kumar; Jaremko, Łukasz; Jaremko, Mariusz; Linser, Rasmus; Becker, Stefan; Zweckstetter, Markus

    2017-01-01

    Cholesterol is an important regulator of membrane protein function. However, the exact mechanisms involved in this process are still not fully understood. Here we study how the tertiary and quaternary structure of the mitochondrial translocator protein TSPO, which binds cholesterol with nanomolar affinity, is affected by this sterol. Residue-specific analysis of TSPO by solid-state NMR spectroscopy reveals a dynamic monomer–dimer equilibrium of TSPO in the membrane. Binding of cholesterol to TSPO's cholesterol-recognition motif leads to structural changes across the protein that shifts the dynamic equilibrium towards the translocator monomer. Consistent with an allosteric mechanism, a mutation within the oligomerization interface perturbs transmembrane regions located up to 35 Å away from the interface, reaching TSPO's cholesterol-binding motif. The lower structural stability of the intervening transmembrane regions provides a mechanistic basis for signal transmission. Our study thus reveals an allosteric signal pathway that connects membrane protein tertiary and quaternary structure with cholesterol binding. PMID:28358007

  4. The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains.

    PubMed

    Olsson, Jan; Edqvist, Petra J; Bröms, Jeanette E; Forsberg, Ake; Wolf-Watz, Hans; Francis, Matthew S

    2004-07-01

    To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III translocon to microinject several anti-host Yop effectors into the cytosol of target eukaryotic cells. YopD has been implicated in several key steps during Yop effector translocation, including maintenance of yop regulatory control and pore formation in the target cell membrane through which effectors traverse. These functions are mediated, in part, by an interaction with the cognate chaperone, LcrH. To gain insight into the complex molecular mechanisms of YopD function, we performed a systematic mutagenesis study to search for discrete functional domains. We highlighted amino acids beyond the first three N-terminal residues that are dispensable for YopD secretion and confirmed that an interaction between YopD and LcrH is essential for maintenance of yop regulatory control. In addition, discrete domains within YopD that are essential for both pore formation and translocation of Yop effectors were identified. Significantly, other domains were found to be important for effector microinjection but not for pore formation. Therefore, YopD is clearly essential for several discrete steps during efficient Yop effector translocation. Recognition of this modular YopD domain structure provides important insights into the function of YopD.

  5. Endoplasmic Reticulum Tubule Protein Reticulon 4 Associates with the Legionella pneumophila Vacuole and with Translocated Substrate Ceg9.

    PubMed

    Haenssler, Eva; Ramabhadran, Vinay; Murphy, Connor S; Heidtman, Matthew I; Isberg, Ralph R

    2015-09-01

    Intracellular growth of Legionella pneumophila occurs in a replication vacuole constructed by host proteins that regulate vesicular traffic from the host endoplasmic reticulum (ER). This process is promoted by a combination of approximately 300 Icm/Dot translocated substrates (IDTS). One of these proteins, Ceg9, was previously identified in a screen for L. pneumophila IDTS that manipulate secretory traffic when overexpressed in yeast. Using ectopic expression of Ceg9 in mammalian cells, we demonstrate that Ceg9 interacts with isoforms of host reticulon 4 (Rtn4), a protein that regulates ER tubule formation. Binding occurs under conditions that prevent association with other known reticulon binding proteins, arguing that Ceg9 binding is stable. A tripartite complex was demonstrated among Rtn4, Ceg9, and atlastin 1, a previously characterized reticulon interacting partner. The binding of Ceg9 to Rtn4 was not due to bridging by atlastin 1 but resulted from the two interacting partners binding independently to reticulon. When Ceg9 is ectopically expressed in mammalian cells, it shows a localization pattern that is indistinguishable from that of Rtn4, perhaps due to interactions between and similar structural features of the two proteins. Consistent with Rtn4 playing a role in the formation of the Legionella-containing vacuole, it was recruited to almost 50% of the vacuoles within 20 min postinfection. Our studies suggest that L. pneumophila proteins interact with ER tubules at an early stage of replication vacuole formation.

  6. Endoplasmic Reticulum Tubule Protein Reticulon 4 Associates with the Legionella pneumophila Vacuole and with Translocated Substrate Ceg9

    PubMed Central

    Haenssler, Eva; Ramabhadran, Vinay; Murphy, Connor S.; Heidtman, Matthew I.

    2015-01-01

    Intracellular growth of Legionella pneumophila occurs in a replication vacuole constructed by host proteins that regulate vesicular traffic from the host endoplasmic reticulum (ER). This process is promoted by a combination of approximately 300 Icm/Dot translocated substrates (IDTS). One of these proteins, Ceg9, was previously identified in a screen for L. pneumophila IDTS that manipulate secretory traffic when overexpressed in yeast. Using ectopic expression of Ceg9 in mammalian cells, we demonstrate that Ceg9 interacts with isoforms of host reticulon 4 (Rtn4), a protein that regulates ER tubule formation. Binding occurs under conditions that prevent association with other known reticulon binding proteins, arguing that Ceg9 binding is stable. A tripartite complex was demonstrated among Rtn4, Ceg9, and atlastin 1, a previously characterized reticulon interacting partner. The binding of Ceg9 to Rtn4 was not due to bridging by atlastin 1 but resulted from the two interacting partners binding independently to reticulon. When Ceg9 is ectopically expressed in mammalian cells, it shows a localization pattern that is indistinguishable from that of Rtn4, perhaps due to interactions between and similar structural features of the two proteins. Consistent with Rtn4 playing a role in the formation of the Legionella-containing vacuole, it was recruited to almost 50% of the vacuoles within 20 min postinfection. Our studies suggest that L. pneumophila proteins interact with ER tubules at an early stage of replication vacuole formation. PMID:26099580

  7. 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.

  8. 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.

  9. Hph1 and Hph2 are novel components of the Sec63/Sec62 posttranslational translocation complex that aid in vacuolar proton ATPase biogenesis.

    PubMed

    Piña, Francisco J; O'Donnell, Allyson F; Pagant, Silvere; Piao, Hai Lan; Miller, John P; Fields, Stanley; Miller, Elizabeth A; Cyert, Martha S

    2011-01-01

    Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1Δ hph2Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1Δ hph2Δ and hph1Δ hph2Δ sec71Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1Δ hph2Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

  10. The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation

    PubMed Central

    1996-01-01

    Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha- factor. Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay. The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase. Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s. They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed. PMID:8947547

  11. Identification of a 120-kDa protein associated with aromatic hydrocarbon receptor nuclear translocator.

    PubMed

    Hossain, A; Kikuchi, H; Ikawa, S; Sagami, I; Watanabe, M

    1995-07-06

    The aromatic hydrocarbon receptor nuclear translocator (ARNT) is a basic helix-loop-helix-PAS protein which forms a heterodimer with aromatic hydrocarbon receptor (AHR), this heterodimer mediating the signal transduction in response to the various xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and directly interacting with target genes by binding to xenobiotic responsive elements. An anti-ARNT antibody was raised in rabbits against the bacterially expressed ARNT of amino acids 21-328 from the N-terminal. Using this antibody, besides ARNT itself, we detected at least one protein, 120 kDa, in the immunoprecipitate of anti-ARNT antibodies in HepG2 cells as well as in Hepa-1 cells. However, this protein is not present in the immunoprecipitate of the anti-AHR antisera nor in that of the preimmune sera of the rabbits used for the immunization.

  12. 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

    2016-04-21

    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.

  13. A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

    PubMed

    Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

    2015-05-01

    Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening.

  14. Coarse-grained Brownian dynamics simulations of protein translocation through nanopores

    NASA Astrophysics Data System (ADS)

    Lee, Po-Hsien; Helms, Volkhard; Geyer, Tihamér

    2012-10-01

    A crucial process in biological cells is the translocation of newly synthesized proteins across cell membranes via integral membrane protein pores termed translocons. Recent improved techniques now allow producing artificial membranes with pores of similar dimensions of a few nm as the translocon system. For the translocon system, the protein has to be unfolded, whereas the artificial pores are wide enough so that small proteins can pass through even when folded. To study how proteins permeate through such membrane pores, we used coarse-grained Brownian dynamics simulations where the proteins were modeled as single beads or bead-spring polymers for both folded and unfolded states. The pores were modeled as cylindrical holes through the membrane with various radii and lengths. Diffusion was driven by a concentration gradient created across the porous membrane. Our results for both folded and unfolded configurations show the expected reciprocal relation between the flow rate and the pore length in agreement with an analytical solution derived by Brunn et al. [Q. J. Mech. Appl. Math. 37, 311 (1984)], 10.1093/qjmam/37.2.311. Furthermore, we find that the geometric constriction by the narrow pore leads to an accumulation of proteins at the pore entrance, which in turn compensates for the reduced diffusivity of the proteins inside the pore.

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

    PubMed

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

    2016-10-14

    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.

  16. Physiological Role for Phosphatidic Acid in the Translocation of the Novel Protein Kinase C Apl II in Aplysia Neurons▿

    PubMed Central

    Farah, Carole A.; Nagakura, Ikue; Weatherill, Daniel; Fan, Xiaotang; Sossin, Wayne S.

    2008-01-01

    In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKCɛ, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition. PMID:18505819

  17. 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

  18. Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli.

    PubMed

    Kang, Dong Gyun; Seo, Jeong Hyun; Jo, Byung Hoon; Kim, Chang Sup; Choi, Suk Soon; Cha, Hyung Joon

    2016-07-08

    Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016.

  19. 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

  20. 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.

  1. The Mitochondrial Translocator Protein, TSPO, Inhibits HIV-1 Envelope Glycoprotein Biosynthesis via the Endoplasmic Reticulum-Associated Protein Degradation Pathway

    PubMed Central

    Zhou, Tao; Dang, Ying

    2014-01-01

    ABSTRACT The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4+ T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. IMPORTANCE The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral

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

    PubMed

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

    2016-05-01

    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.

  3. Carborane-conjugated 2-quinolinecarboxamide ligands of the translocator protein for boron neutron capture therapy.

    PubMed

    Cappelli, Andrea; Valenti, Salvatore; Mancini, Alessandra; Giuliani, Germano; Anzini, Maurizio; Altieri, Saverio; Bortolussi, Silva; Ferrari, Cinzia; Clerici, Anna Maria; Zonta, Cecilia; Carraro, Fabio; Filippi, Irene; Giorgi, Gianluca; Donati, Alessandro; Ristori, Sandra; Vomero, Salvatore; Concas, Alessandra; Biggio, Giovanni

    2010-12-15

    Potential boron neutron capture therapy (BNCT) agents have been designed on the basis of the evidence about translocator protein (TSPO) overexpression on the outer mitochondrial membrane of tumor cells. The structure of the first TSPO ligand bearing a carborane cage (compound 2d) has been modified in order to find a suitable candidate for in vivo studies. The designed compounds were synthesized and evaluated for their potential interaction with TSPO and tumor cells. In vitro biological evaluation showed in the case of fluoromethyl derivative 4b a nanomolar TSPO affinity very similar to that of 2d, a significantly lower cytotoxicity, and a slightly superior performance as boron carrier toward breast cancer cells. Moreover, compound 4b could be used as a ¹⁹F magnetic resonance imaging (MRI) agent as well as labeled with ¹¹C or ¹⁸F to obtain positron emission tomography (PET) radiotracers in order to apply the "see and treat" strategy in BNCT.

  4. Enzymatic product formation impairs both the chloroplast receptor- binding function as well as translocation competence of the NADPH: protochlorophyllide oxidoreductase, a nuclear-encoded plastid precursor protein

    PubMed Central

    1995-01-01

    The key enzyme of chlorophyll biosynthesis in higher plants, the light- dependent NADPH:protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), is a nuclear-encoded plastid protein. Its posttranslational transport into plastids of barley depends on the intraplastidic availability of one of its substrates, protochlorophyllide (PChlide). The precursor of POR (pPOR), synthesized from a corresponding full-length barley cDNA clone by coupling in vitro transcription and translation, is enzymatically active and converts PChlide to chlorophyllide (Chlide) in a light- and NADPH-dependent manner. Chlorophyllide formed catalytically remains tightly but noncovalently bound to the precursor protein and stabilizes a transport-incompetent conformation of pPOR. As shown by in vitro processing experiments, the chloroplast transit peptide in the Chlide-pPOR complex appears to be masked and thus is unable to physically interact with the outer plastid envelope membrane. In contrast, the chloroplast transit peptide in the naked pPOR (without its substrates and its product attached to it) and in the pPOR- substrate complexes, such as pPOR-PChlide or pPOR-PChlide-NADPH, seems to react independently of the mature region of the polypeptide, and thus is able to bind to the plastid envelope. When envelope-bound pPOR- PChlide-NADPH complexes were exposed to light during a short preincubation, the enzymatically produced Chlide slowed down the actual translocation step, giving rise to the sequential appearance of two partially processed translocation intermediates. However, ongoing translocation induced by feeding the chloroplasts delta-aminolevulinic acid, a precursor of PChlide, was able to override these two early blocks in translocation, suggesting that the plastid import machinery has a substantial capacity to denature a tightly folded, envelope-bound precursor protein. Together, our results show that pPOR with Chlide attached to it is impaired both in the ATP-dependent step of binding to a

  5. Enterococcal surface protein Esp does not facilitate intestinal colonization or translocation of Enterococcus faecalis in clindamycin-treated mice.

    PubMed

    Pultz, Nicole J; Shankar, Nathan; Baghdayan, Arto S; Donskey, Curtis J

    2005-01-15

    Enterococcal surface protein (Esp) is a cell wall-associated protein of Enterococcus faecalis that has been identified as a potential virulence factor. We used a mouse model to examine whether Esp facilitates intestinal colonization or translocation of E. faecalis to mesenteric lymph nodes. After clindamycin treatment, similar levels of high-density colonization were established after orogastric inoculation of an E. faecalis isolate containing the esp gene within a large pathogenicity island and an isogenic mutant created by allelic replacement of the esp gene with a chloramphenicol resistance cassette (P=0.7); translocation to mesenteric lymph nodes was detected in 3 of 12 (25%) mice in both groups. Isogenic mutants of FA2-2 (a plasmid-free derivative of E. faecalis strain JH2) with or without the esp gene failed to establish colonization of clindamycin-treated mice. These results suggest that Esp does not facilitate intestinal colonization or translocation of E. faecalis.

  6. The pathogenic mechanism of the Mycobacterium ulcerans virulence factor, mycolactone, depends on blockade of protein translocation into the ER.

    PubMed

    Hall, Belinda S; Hill, Kirsti; McKenna, Michael; Ogbechi, Joy; High, Stephen; Willis, Anne E; Simmonds, Rachel E

    2014-04-01

    Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but

  7. 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

  8. Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation.

    PubMed

    Barrès, Romain; Grémeaux, Thierry; Gual, Philippe; Gonzalez, Teresa; Gugenheim, Jean; Tran, Albert; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2006-11-01

    APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.

  9. Cytoplasm-to-nucleus translocation of a herpesvirus tegument protein during cell division.

    PubMed

    Elliott, G; O'Hare, P

    2000-03-01

    We have previously shown that the herpes simplex virus tegument protein VP22 localizes predominantly to the cytoplasm of expressing cells. We have also shown that VP22 has the unusual property of intercellular spread, which involves the movement of VP22 from the cytoplasm of these expressing cells into the nuclei of nonexpressing cells. Thus, VP22 can localize in two distinct subcellular patterns. By utilizing time-lapse confocal microscopy of live cells expressing a green fluorescent protein-tagged protein, we now report in detail the intracellular trafficking properties of VP22 in expressing cells, as opposed to the intercellular trafficking of VP22 between expressing and nonexpressing cells. Our results show that during interphase VP22 appears to be targeted exclusively to the cytoplasm of the expressing cell. However, at the early stages of mitosis VP22 translocates from the cytoplasm to the nucleus, where it immediately binds to the condensing cellular chromatin and remains bound there through all stages of mitosis and chromatin decondensation into the G(1) stage of the next cycle. Hence, in VP22-expressing cells the subcellular localization of the protein is regulated by the cell cycle such that initially cytoplasmic protein becomes nuclear during cell division, resulting in a gradual increase over time in the number of nuclear VP22-expressing cells. Importantly, we demonstrate that this process is a feature not only of VP22 expressed in isolation but also of VP22 expressed during virus infection. Thus, VP22 utilizes an unusual pathway for nuclear targeting in cells expressing the protein which differs from the nuclear targeting pathway used during intercellular trafficking.

  10. High-Risk Microgranular Acute Promyelocytic Leukemia with a Five-Way Complex Translocation Involving PML-RARA.

    PubMed

    Powers, Benjamin; Persons, Diane; Rao, Deepthi; Woodroof, Janet; Lin, Tara L

    2015-01-01

    Acute promyelocytic leukemia (APL) is classically characterized by chromosomal translocation (15;17), resulting in the PML-RARA fusion protein leading to disease. Here, we present a case of a 50-year-old man who presented with signs and symptoms of acute leukemia with concern for APL. Therapy was immediately initiated with all-trans retinoic acid. The morphology of his leukemic blasts was consistent with the hypogranular variant of APL. Subsequent FISH and cytogenetic analysis revealed a unique translocation involving five chromosomal regions: 9q34, 17q21, 15q24, 12q13, and 15q26.1. Molecular testing demonstrated PML/RARA fusion transcripts. Treatment with conventional chemotherapy was added and he went into a complete remission. Given his elevated white blood cell count at presentation, intrathecal chemotherapy for central nervous system prophylaxis was also given. The patient remains on maintenance therapy and remains in remission. This is the first such report of a 5-way chromosomal translocation leading to APL. Similar to APL with chromosomal translocations other than classical t(15;17) which result in the typical PML-RARA fusion, our patient responded promptly to an ATRA-containing regimen and remains in complete remission.

  11. Targeting and translocation of proteins into the hydrogenosome of the protist Trichomonas: similarities with mitochondrial protein import.

    PubMed Central

    Bradley, P J; Lahti, C J; Plümper, E; Johnson, P J

    1997-01-01

    Trichomonads are early-diverging eukaryotes that lack both mitochondria and peroxisomes. They do contain a double membrane-bound organelle, called the hydrogenosome, that metabolizes pyruvate and produces ATP. To address the origin and biological nature of hydrogenosomes, we have established an in vitro protein import assay. Using purified hydrogenosomes and radiolabeled hydrogenosomal precursor ferredoxin (pFd), we demonstrate that protein import requires intact organelles, ATP and N-ethylmaleimide-sensitive cytosolic factors. Protein import is also affected by high concentrations of the protonophore, m-chlorophenylhydrazone (CCCP). Binding and translocation of pFd into hydrogenosomes requires the presence of an eight amino acid N-terminal presequence that is similar to presequences found on all examined hydrogenosomal proteins. Upon import, pFd is processed to a size consistent with cleavage of the presequence. Mutation of a conserved leucine at position 2 in the presequence to a glycine disrupts import of pFd into the organelle. Interestingly, a comparison of hydrogenosomal and mitochondrial protein presequences reveals striking similarities. These data indicate that mechanisms underlying protein targeting and biogenesis of hydrogenosomes and mitochondria are similar, consistent with the notion that these two organelles arose from a common endosymbiont. PMID:9218791

  12. 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

    2016-03-03

    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.

  13. 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

  14. 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.

  15. Expression of constitutively active Akt/protein kinase B signals GLUT4 translocation in the absence of an intact actin cytoskeleton.

    PubMed

    Eyster, Craig A; Duggins, Quwanza S; Olson, Ann Louise

    2005-05-06

    The actin cytoskeleton has been shown to be required for insulin-dependent GLUT4 translocation; however, the role that the actin network plays is unknown. Actin may play a role in formation of an active signaling complex, or actin may be required for movement of vesicles to the plasma membrane surface. To distinguish between these possibilities, we examined the ability of myr-Akt, a constitutively active form of Akt that signals GLUT4 translocation to the plasma membrane in the absence of insulin, to signal translocation of an HA-GLUT4-GFP reporter protein in the presence or absence of an intact cytoskeleton in 3T3-L1 adipocytes. Expression of myr-Akt signaled the redistribution of the GLUT4 reporter protein to the cell surface in the absence or presence of 10 microm latrunculin B, a concentration sufficient to completely inhibit insulin-dependent redistribution of the GLUT4 reporter to the cell surface. These data suggest that the actin network plays a primary role in organization of the insulin-signaling complex. To further support this conclusion, we measured the activation of known signaling proteins using a saturating concentration of insulin in cells pretreated without or with 10 microm latrunculin B. We found that latrunculin treatment did not affect insulin-dependent tyrosine phosphorylation of the insulin receptor beta-subunit and IRS-1 but completely inhibited activation of Akt/PKB enzymatic activity. Phosphorylation of Akt/PKB at Ser-473 and Thr-308 was inhibited by latrunculin B treatment, indicating that the defect in signaling lies prior to Akt/PKB activation. In summary, our data support the hypothesis that the actin network plays a role in organization of the insulin-signaling complex but is not required for vesicle trafficking and/or fusion.

  16. 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.

  17. The RNA-binding protein, TB-RBP, is the mouse homologue of translin, a recombination protein associated with chromosomal translocations.

    PubMed

    Wu, X Q; Gu, W; Meng, X; Hecht, N B

    1997-05-27

    The mouse RNA-binding protein, TB-RBP, suppresses translation in vitro and attaches mRNAs to microtubules by binding to conserved elements in the 3' untranslated regions of specific mRNAs. We have now purified TB-RBP from testicular and brain cytoplasmic extracts and cloned its cDNA. We find that the mouse TB-RBP cDNAs contain an open reading frame of 228 amino acids with a leucine zipper domain within its C terminus, a transmembrane helix, and a group of putative phosphorylation sites. TB-RBP shows 99% identity to the human protein, translin, a recombination hotspot-binding protein associated with chromosomal translocations [Aoki, K., Suzuki, K., Sugano, T., Tasaka, T., Nakahara, K., Kuge, O., Omori, A. & Kasai, M. (1995) Nat. Genet. 10, 167-174]. As shown for translin, TB-RBP also binds to single-stranded DNAs containing a broad range of consensus sequences, many of which are similar to the Y and H RNA-binding sequences. Recombinant TB-RBP was synthesized and an antiserum was prepared against the recombinant protein. The identity between translin and TB-RBP was confirmed by demonstrating that immunoprecipitation of TB-RBP from testicular extracts abolished formation of the RNA-TB-RBP complex. Based upon its DNA binding to target sequences in clustered breakpoint regions, we propose that TB-RBP may be involved in DNA recombination or DNA repair in male germ cells.

  18. 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

  19. 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

  20. The PPE2 protein of Mycobacterium tuberculosis translocates to host nucleus and inhibits nitric oxide production

    PubMed Central

    Bhat, Khalid Hussain; Srivastava, Shruti; Kotturu, Sandeep Kumar; Ghosh, Sudip; Mukhopadhyay, Sangita

    2017-01-01

    Mycobacterium tuberculosis, the bacterium that causes tuberculosis, is one of the most successful pathogens of humans. It has evolved several adaptive skills and evasion mechanisms to hijack the immunologically educated host to suit its intracellular lifestyle. Here, we show that one of the unique PPE family member proteins of M. tuberculosis, PPE2, can limit nitric oxide (NO) production by inhibiting inos gene transcription. PPE2 protein has a leucine zipper DNA-binding motif and a functional nuclear localization signal. PPE2 was translocated into the macrophage nucleus via the classical importin α/β pathway where it interacted with a GATA-binding site overlapping with the TATA box of inos promoter and inhibited NO production. PPE2 prolonged intracellular survival of a surrogate bacterium M. smegmatis in vitro as well as in vivo. This information are likely to improve our knowledge of host-pathogen interactions during M. tuberculosis infection which is crucial for designing effective anti-TB therapeutics. PMID:28071726

  1. 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

  2. Putting a break on protein translocation: metabolic regulation of mitochondrial protein import.

    PubMed

    Herrmann, Johannes M

    2009-04-01

    Sequence-inherent targeting information directs polypeptides synthesized in the cytosol to their respective cellular compartment. Some proteins use ambiguous sorting signals or specific folding properties to be dually distributed between the cytosol and mitochondria. A study published in this issue of Molecular Microbiology shows that in the case of fumarase this distribution is controlled by the metabolic state of yeast cells. The metabolite-dependent distribution of fumarase represents an exciting example of regulated protein import into mitochondria that shows that eukaryotes can adapt the intracellular protein distribution to their physiological conditions.

  3. The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex.

    PubMed

    Oates, Joanne; Barrett, Claire M L; Barnett, James P; Byrne, Katheryne G; Bolhuis, Albert; Robinson, Colin

    2005-02-11

    The Tat system transports folded proteins across bacterial plasma and plant thylakoid membranes. To date, three key Tat subunits have been identified and mechanistic studies indicate the presence of two types of complex: a TatBC-containing substrate-binding unit and a separate TatA complex. Here, we used blue-native gel electrophoresis and affinity purification to study the nature of these complexes in Escherichia coli. Analysis of solubilized membrane shows that the bulk of TatB and essentially all of the TatC is found in a single 370kDa TatABC complex. TatABC was purified to homogeneity using an affinity tag on TatC and this complex runs apparently as an identical band. We conclude that this is the primary core complex, predicted to contain six or seven copies of TatBC together with a similar number of TatA subunits. However, the data indicate the presence of an additional form of Tat complex containing TatA and TatB, but not TatC; we speculate that this may be an assembly or disassembly intermediate of the translocator. The vast majority of TatA is found in separate complexes that migrate in blue-native gels as a striking ladder of bands with sizes ranging from under 100 kDa to over 500 kDa. Further analysis shows that the bands differ by an average of 34 kDa, indicating that TatA complexes are built largely, but possibly not exclusively, from modules of three or four TatA molecules. The range and nature of these complexes are similar in a TatC mutant that is totally inactive, indicating that the ladder of bands does not stem from ongoing translocation activity, and we show that purified TatA can self-assemble in vitro to form similar complexes. This spectrum of TatA complexes may provide the flexibility required to generate a translocon capable of transporting substrates of varying sizes across the plasma membrane in a folded state.

  4. Intraglomerular basement membrane translocation of immune complex (IC) in the development of passive in situ IC nephritis of rats.

    PubMed Central

    Fujigaki, Y.; Nagase, M.; Honda, N.

    1993-01-01

    A study was performed to elucidate the mechanisms of charge-based immune complex nephritis. A chronological observation after induction of nephritis was made by immunoelectron microscopy to clarify whether antigen (Ag) remains in association with antibody (Ab) and C3 during the translocation through the glomerular basement membrane (GBM). Fifteen minutes after intrarenal perfusion with cationized ferritin (pI > 10.0) as Ag, followed by injection of rabbit anti-ferritin Ab, deposition of subendothelial Ag-Ab-C3 complexes was observed. Between 2 hours and 1 day, a large number of Ag in close association with Ab was noted in the lamina densa, but only a small amount of C3 was detectable. During this time Ag and Ab in the subendothelial region gradually decreased. However, C3 reappeared in the subepithelial region together with the Ag-Ab complex after 1 day, and the subendothelial C3 significantly decreased. At 2 hours and day 1, the distributions of Ag and Ab in the GBM were similar in immersion-fixed kidneys regardless of the preperfusion with phosphate-buffered saline. On the other hand, the passage of Ag across the lamina densa was delayed in the experimental rats as compared with the controls. Significant albuminuria also appeared on day 1. Despite the general concept that Ab binding to cationized Ag results in low avidity immune complex, cationized Ag translocated across the GBM in close association with Ab. The complement was activated biphasically in the subendothelial and in the subepithelial space. The subendothelial complement activation may have contributed to the translocation of immune complex. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8456943

  5. 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.

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

    PubMed

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

    2016-03-01

    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.

  7. 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

  8. Structural Basis of Protein Translocation by the Vps4-Vta1 AAA ATPase.

    PubMed

    Monroe, Nicole; Han, Han; Shen, Peter S; Sundquist, Wesley I; Hill, Christopher P

    2017-04-05

    Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP•BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP-binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 'walks' along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.

  9. Translocator Protein (TSPO) Expression in Platelets of Depressed Patients Decreases during Antidepressant Therapy.

    PubMed

    Sarubin, N; Baghai, T C; Lima-Ojeda, J M; Melchner, D; Hallof-Buestrich, H; Wolf, L; Hilbert, S; Milenkovic, V M; Wetzel, C H; Rupprecht, R; Nothdurfter, C

    2016-09-01

    Introduction: A promising candidate in the field of pharmacological treatment options regarding major depressive disorder (MDD) is the mitochondrial translocator protein (18 kDa) (TSPO). TSPO is crucial for neurosteroid synthesis, which is in turn important for the regulation of emotions. It has already been shown that TSPO expression in platelets of depressed patients is reduced compared to healthy subjects. Methods: We measured TSPO levels in platelets of 37 depressed patients before and after 6 weeks of pharmacological treatment to test the hypotheses that i) such treatment would increase TSPO expression and ii) that this increase would be correlated with therapeutic response. Results: Surprisingly, TSPO levels in platelets of all patients were significantly reduced after 6 weeks of treatment (p=0.044). Within the responder group, a non-significant trend towards greater TSPO level reduction could be observed. Discussion: These results challenge our hypotheses that TSPO levels might increase during antidepressant therapy along with a decrease in depressive symptoms. Thus, we assume that TSPO expression in platelets is not a suitable state marker for MDD.

  10. Nucleocytoplasmic protein translocation during mitosis in the social amoebozoan Dictyostelium discoideum.

    PubMed

    O'Day, Danton H; Budniak, Aldona

    2015-02-01

    Mitosis is a fundamental and essential life process. It underlies the duplication and survival of all cells and, as a result, all eukaryotic organisms. Since uncontrolled mitosis is a dreaded component of many cancers, a full understanding of the process is critical. Evolution has led to the existence of three types of mitosis: closed, open, and semi-open. The significance of these different mitotic species, how they can lead to a full understanding of the critical events that underlie the asexual duplication of all cells, and how they may generate new insights into controlling unregulated cell division remains to be determined. The eukaryotic microbe Dictyostelium discoideum has proved to be a valuable biomedical model organism. While it appears to utilize closed mitosis, a review of the literature suggests that it possesses a form of mitosis that lies in the middle between truly open and fully closed mitosis-it utilizes a form of semi-open mitosis. Here, the nucleocytoplasmic translocation patterns of the proteins that have been studied during mitosis in the social amoebozoan D. discoideum are detailed followed by a discussion of how some of them provide support for the hypothesis of semi-open mitosis.

  11. Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin.

    PubMed

    Pukatzki, Stefan; Ma, Amy T; Revel, Andrew T; Sturtevant, Derek; Mekalanos, John J

    2007-09-25

    Genes encoding type VI secretion systems (T6SS) are widely distributed in pathogenic Gram-negative bacterial species. In Vibrio cholerae, T6SS have been found to secrete three related proteins extracellularly, VgrG-1, VgrG-2, and VgrG-3. VgrG-1 can covalently cross-link actin in vitro, and this activity was used to demonstrate that V. cholerae can translocate VgrG-1 into macrophages by a T6SS-dependent mechanism. Protein structure search algorithms predict that VgrG-related proteins likely assemble into a trimeric complex that is analogous to that formed by the two trimeric proteins gp27 and gp5 that make up the baseplate "tail spike" of Escherichia coli bacteriophage T4. VgrG-1 was shown to interact with itself, VgrG-2, and VgrG-3, suggesting that such a complex does form. Because the phage tail spike protein complex acts as a membrane-penetrating structure as well as a conduit for the passage of DNA into phage-infected cells, we propose that the VgrG components of the T6SS apparatus may assemble a "cell-puncturing device" analogous to phage tail spikes to deliver effector protein domains through membranes of target host cells.

  12. A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells.

    PubMed Central

    Knutton, S; Rosenshine, I; Pallen, M J; Nisan, I; Neves, B C; Bain, C; Wolff, C; Dougan, G; Frankel, G

    1998-01-01

    Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell. In EPEC, four proteins are known to be exported by a type III secretion system_EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell to become a receptor for intimin-mediated intimate attachment and 'attaching and effacing' (A/E) lesion formation. The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system. Here, we describe a novel EspA-containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells. PMID:9545230

  13. SPINAL TRANSLOCATOR PROTEIN (TSPO) MODULATES PAIN BEHAVIOR IN RATS WITH CFA-INDUCED MONOARTHRITIS

    PubMed Central

    Hernstadt, Hayley; Wang, Shuxing; Lim, Grewo; Mao, Jianren

    2009-01-01

    Translocator protein 18kDa (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is predominantly located in the mitochondrial outer membrane and plays an important role in steroidogenesis, immunomodulation, cell survival and proliferation. Previous studies have shown an increased expression of TSPO centrally in neuropathology, as well as in injured nerves. TSPO has also been implicated in modulation of nociception. In the present study, we examined the hypothesis that TSPO is involved in the initiation and maintenance of inflammatory pain using a rat model of Complete Freund’s Adjuvant (CFA)-induced monoarthritis of the tibio-tarsal joint. Immunohistochemistry was performed using Iba-1 (microglia), NeuN (neurons), anti-Glial Fibrillary Acidic Protein, GFAP (astrocytes) and anti-PBR (TSPO) on day 1, 7 and 14 after CFA-induced arthritis. Rats with CFA-induced monoarthritis showed mechanical allodynia and thermal hyperalgesia on the ipsilateral hindpaw, which correlated with the increased TSPO expression in ipsilateral lamina I-II on all experimental days. Iba-1 expression in the ipsilateral dorsal horn was also increased on Day 7 and 14. Moreover, TSPO was co-localized with Iba-1, GFAP and NeuN within the spinal cord dorsal horn. The TSPO agonist Ro5-4864, given intrathecally, dose-dependently retarded or prevented the development of mechanical allodynia and thermal hyperalgesia in rats with CFA-induced monoarthritis. These findings provide evidence that spinal TSPO is involved in the development and maintenance of inflammatory pain behaviors in rats. Thus, spinal TSPO may present a central target as a complementary therapy to reduce inflammatory pain. PMID:19555675

  14. Obesity-Induced Down-Regulation of the Mitochondrial Translocator Protein (TSPO) Impairs Placental Steroid Production

    PubMed Central

    Lassance, Luciana; Haghiac, Maricela; Minium, Judi; Catalano, Patrick

    2015-01-01

    Context: Low concentrations of estradiol and progesterone are hallmarks of adverse pregnancy outcomes as is maternal obesity. During pregnancy, placental cholesterol is the sole source of sex steroids. Cholesterol trafficking is the limiting step in sex steroid biosynthesis and is mainly mediated by the translocator protein (TSPO), present in the mitochondrial outer membrane. Objective: The objective of the study was to investigate the effects of maternal obesity in placental sex steroid biosynthesis and TSPO regulation. Design/Participants: One hundred forty-four obese (body mass index 30–35 kg/m2) and 90 lean (body mass index 19–25 kg/m2) pregnant women (OP and LP, respectively) recruited at scheduled term cesarean delivery. Placenta and maternal blood were collected. Setting: This study was conducted at MetroHealth Medical Center (Cleveland, Ohio). Main Outcome Measures: Maternal metabolic components (fasting glucose, insulin, leptin, estradiol, progesterone, and total cholesterol) and placental weight were measured. Placenta (mitochondria and membranes separated) and cord blood cholesterol values were verified. The expression and regulation of TSPO and mitochondrial function were analyzed. Results: Plasma estradiol and progesterone concentrations were significantly lower (P < .04) in OP as compared with LP women. Maternal and cord plasma cholesterol were not different between groups. Placental citrate synthase activity and mitochondrial DNA, markers of mitochondrial density, were unchanged, but the mitochondrial cholesterol concentrations were 40% lower in the placenta of OP. TSPO gene and protein expressions were decreased 2-fold in the placenta of OP. In vitro trophoblast activation of the innate immune pathways with lipopolysaccharide and long-chain saturated fatty acids reduced TSPO expression by 2- to 3-fold (P < .05). Conclusion: These data indicate that obesity in pregnancy impairs mitochondrial steroidogenic function through the negative regulation

  15. 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 ...

  16. Determinants of the rate of mRNA translocation in bacterial protein synthesis.

    PubMed

    Borg, Anneli; Ehrenberg, Måns

    2015-05-08

    Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30 s(-1) at 1 mM free Mg2+ concentration to the detrimentally low value of 1 s(-1) at 6 mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0-6 kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account.

  17. 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.

  18. Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation*

    PubMed Central

    Chotewutmontri, Prakitchai; Bruce, Barry D.

    2015-01-01

    Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences. PMID:25645915

  19. Purifying protein complexes for mass spectrometry: applications to protein translation.

    PubMed

    Link, Andrew J; Fleischer, Tracey C; Weaver, Connie M; Gerbasi, Vincent R; Jennings, Jennifer L

    2005-03-01

    Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

  20. Efficient tritiation of the translocator protein (18 kDa) selective ligand DPA-714.

    PubMed

    Damont, Annelaure; Garcia-Argote, Sébastien; Buisson, David-Alexandre; Rousseau, Bernard; Dollé, Frédéric

    2015-01-01

    DPA-714 (N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide) is a recently discovered fluorinated ligand of the translocator protein 18 kDa (TSPO). Labelled with the short-lived positron emitter fluorine-18, this structure is today the radioligand of reference for in vivo imaging of microglia activation and neuroinflammatory processes with positron emission tomography. In the present work, an isotopically tritium-labelled version was developed ([(3) H]DPA-714), in order to access high resolution in vitro and ex vivo microscopic autoradiography studies, repeated and long-lasting receptor binding studies and in vivo pharmacokinetic determination at late time points. Briefly, DPA-714 as reference, and its 3,5-dibrominated derivative as precursor for labelling, were both prepared from DPA-713 in nonoptimized 32% (two steps) and 10% (three steps) yields, respectively. Reductive debromination using deuterium gas and Pd/C as catalyst in methanol, performed at the micromolar scale, confirmed the regioselective introduction of two deuterium atoms at the meta positions of the phenyl ring. Tritiodebromination was analogously performed using no-carrier tritium gas. HPLC purification provided >96% radiochemically pure [(3) H]DPA-714 (7 GBq) with a 2.1 TBq/mmol specific radioactivity. Interestingly, additional hydrogen-for-tritium exchanges were also observed at the 5-methyl and 7-methyl positions of the pyrazolo[1,5-a]pyrimidine, opening novel perspectives in the labelling of compounds featuring this heterocyclic core.

  1. Translocator protein (18 kDa) as a pharmacological target in adipocytes to regulate glucose homeostasis.

    PubMed

    Li, Jiehan; Papadopoulos, Vassilios

    2015-09-01

    As a major regulator in obesity and its associated metabolic complications, the proper functioning of adipocytes is crucial for health maintenance, thus serving as an important target for the development of anti-obese and anti-diabetic therapies. There is increasing evidence that mitochondrial malfunction is a pivotal event in disturbing adipocyte cell homeostasis. Among major mitochondrial structure components, the high-affinity drug- and cholesterol-binding outer mitochondrial membrane translocator protein (18 kDa; TSPO) has shown importance across a broad spectrum of mitochondrial functions. Recent studies demonstrated the presence of TSPO in white adipocyte mitochondria of mice, and administration of TSPO drug ligands to obese mice reduced weight gain and lowered glucose level. Therefore, it is of great interest to assess whether TSPO in adipocytes could serve as a drug target to regulate adipocyte activities with potential influence on weight control and glucose metabolism. Two structurally distinct TSPO drug ligands, PK 11195 and FGIN-1-27, improved the intracellular dynamics of 3T3-L1 adipocytes, such as the production and release of adipokines, glucose uptake, and adipogenesis. TSPO knockdown in either differentiated adipocytes or preadipocytes impaired these functions. Findings from 3T3-L1 cells were related to human primary cells, where TSPO expression was tightly associated with the metabolic state of primary adipocytes and the differentiation of primary preadipocytes. These results suggest that TSPO expression is essential to safeguard healthy adipocyte functions, and that TSPO activation in adipocytes improves their metabolic status in regulating glucose homeostasis. Adipocyte TSPO may serve as a pharmacologic target for the treatment of obesity and diabetes.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26627829

  3. CRTC1 Nuclear Translocation Following Learning Modulates Memory Strength via Exchange of Chromatin Remodeling Complexes on the Fgf1 Gene.

    PubMed

    Uchida, Shusaku; Teubner, Brett J W; Hevi, Charles; Hara, Kumiko; Kobayashi, Ayumi; Dave, Rutu M; Shintaku, Tatsushi; Jaikhan, Pattaporn; Yamagata, Hirotaka; Suzuki, Takayoshi; Watanabe, Yoshifumi; Zakharenko, Stanislav S; Shumyatsky, Gleb P

    2017-01-10

    Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets.

  4. 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.

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

    PubMed

    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-->7q21.3::3q27-->3qter;3pter-->3 q23::11q21-->11qter; 11pter-->11q21::3q23-->3q27::7q21.3-->7 qter). 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.

  6. 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.

  7. Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

    PubMed Central

    Amyot, Whitney M.; deJesus, Dennise

    2013-01-01

    Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system. PMID:23798536

  8. 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.

  9. Analysis of protein complexes using mass spectrometry.

    PubMed

    Gingras, Anne-Claude; Gstaiger, Matthias; Raught, Brian; Aebersold, Ruedi

    2007-08-01

    The versatile combination of affinity purification and mass spectrometry (AP-MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP-MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP-MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein-complex assembly.

  10. A lignan induces lysosomal dependent degradation of FoxM1 protein to suppress β-catenin nuclear translocation

    PubMed Central

    Dong, Guang-zhi; Jeong, Ji Hye; Lee, Yu-ih; Han, Yeong Eun; Shin, Jung Sook; Kim, Yoon-Jung; Jeon, Raok; Kim, Young Hwa; Park, Tae Jun; Kim, Keun Il; Ryu, Jae-Ha

    2017-01-01

    Colon cancer is one of the most common cancers. In this study, we isolated a lignan [(−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol, DFS] from Alnus japonica (Betulaceae) and investigated its biological activity and mechanism of action on colon cancer. DFS reduced the viability of colon cancer cells and induced cell cycle arrest. DFS also suppressed β-catenin nuclear translocation and β-catenin target gene expression through a reduction in FoxM1 protein. To assess the mechanism of the action of DFS, we investigated the effect of DFS on endogenous and exogenous FoxM1 protein degradation in colon cancer cells. DFS-induced FoxM1 protein degradation was suppressed by lysosomal inhibitors, chloroquine and bafilomycin A1, but not by knock-down of proteasomal proteins. The mechanism of DFS for FoxM1 degradation is lysosomal dependent, which was not reported before. Furthermore, we found that FoxM1 degradation was partially lysosomal-dependent under normal conditions. These observations indicate that DFS from A. japonica suppresses colon cancer cell proliferation by reducing β-catenin nuclear translocation. DFS induces lysosomal-dependent FoxM1 protein degradation. This is the first report on the lysosomal degradation of FoxM1 by a small molecule. DFS may be useful in treating cancers that feature the elevated expression of FoxM1. PMID:28378765

  11. A lignan induces lysosomal dependent degradation of FoxM1 protein to suppress β-catenin nuclear translocation.

    PubMed

    Dong, Guang-Zhi; Jeong, Ji Hye; Lee, Yu-Ih; Han, Yeong Eun; Shin, Jung Sook; Kim, Yoon-Jung; Jeon, Raok; Kim, Young Hwa; Park, Tae Jun; Kim, Keun Il; Ryu, Jae-Ha

    2017-04-05

    Colon cancer is one of the most common cancers. In this study, we isolated a lignan [(-)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol, DFS] from Alnus japonica (Betulaceae) and investigated its biological activity and mechanism of action on colon cancer. DFS reduced the viability of colon cancer cells and induced cell cycle arrest. DFS also suppressed β-catenin nuclear translocation and β-catenin target gene expression through a reduction in FoxM1 protein. To assess the mechanism of the action of DFS, we investigated the effect of DFS on endogenous and exogenous FoxM1 protein degradation in colon cancer cells. DFS-induced FoxM1 protein degradation was suppressed by lysosomal inhibitors, chloroquine and bafilomycin A1, but not by knock-down of proteasomal proteins. The mechanism of DFS for FoxM1 degradation is lysosomal dependent, which was not reported before. Furthermore, we found that FoxM1 degradation was partially lysosomal-dependent under normal conditions. These observations indicate that DFS from A. japonica suppresses colon cancer cell proliferation by reducing β-catenin nuclear translocation. DFS induces lysosomal-dependent FoxM1 protein degradation. This is the first report on the lysosomal degradation of FoxM1 by a small molecule. DFS may be useful in treating cancers that feature the elevated expression of FoxM1.

  12. Membrane translocation of t-SNARE protein syntaxin-4 abrogates ground-state pluripotency in mouse embryonic stem cells

    PubMed Central

    Hagiwara-Chatani, Natsumi; Shirai, Kota; Kido, Takumi; Horigome, Tomoatsu; Yasue, Akihiro; Adachi, Naoki; Hirai, Yohei

    2017-01-01

    Embryonic stem (ES) and induced pluripotent stem (iPS) cells are attractive tools for regenerative medicine therapies. However, aberrant cell populations that display flattened morphology and lose ground-state pluripotency often appear spontaneously, unless glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase kinase (MEK1/2) are inactivated. Here, we show that membrane translocation of the t-SNARE protein syntaxin-4 possibly is involved in this phenomenon. We found that mouse ES cells cultured without GSK3β/MEK1/2 inhibitors (2i) spontaneously extrude syntaxin-4 at the cell surface and that artificial expression of cell surface syntaxin-4 induces appreciable morphological changes and mesodermal differentiation through dephosphorylation of Akt. Transcriptome analyses revealed several candidate elements responsible for this, specifically, an E-to P-cadherin switch and a marked downregulation of Zscan4 proteins, which are DNA-binding proteins essential for ES cell pluripotency. Embryonic carcinoma cell lines F9 and P19CL6, which maintain undifferentiated states independently of Zscan4 proteins, exhibited similar cellular behaviors upon stimulation with cell surface syntaxin-4. The functional ablation of E-cadherin and overexpression of P-cadherin reproduced syntaxin-4-induced cell morphology, demonstrating that the E- to P-cadherin switch executes morphological signals from cell surface syntaxin-4. Thus, spontaneous membrane translocation of syntaxin-4 emerged as a critical element for maintenance of the stem-cell niche. PMID:28057922

  13. Replacement of glycine 232 by aspartic acid in the KdpA subunit broadens the ion specificity of the K(+)-translocating KdpFABC complex.

    PubMed Central

    Schrader, M; Fendler, K; Bamberg, E; Gassel, M; Epstein, W; Altendorf, K; Dröse, S

    2000-01-01

    Replacement of glycine residue 232 with aspartate in the KdpA subunit of the K(+)-translocating KdpFABC complex of Escherichia coli leads to a transport complex that has reduced affinity for K(+) and has lost the ability to discriminate Rb(+) ions (, J. Biol. Chem. 270:6678-6685). This glycine residue is the first in a highly conserved GGG motif that was aligned with the GYG sequence of the selectivity filter (P- or H5-loop) of K(+) channels (, Nature. 371:119-122). Investigations with the purified and reconstituted KdpFABC complex using the potential sensitive fluorescent dye DiSC(3)(5) and the "caged-ATP/planar bilayer method" confirm the altered ion specificity observed in uptake measurements with whole cells. In the absence of cations a transient current was observed in the planar bilayer measurements, a phenomenon that was previously observed with the wild-type enzyme and with another kdpA mutant (A:Q116R) and most likely represents the movement of a protein-fixed charge during a conformational transition. After addition of K(+) or Rb(+), a stationary current could be observed, representing the continuous pumping activity of the KdpFABC complex. In addition, DiSC(3)(5) and planar bilayer measurements indicate that the A:G232D Kdp-ATPase also transports Na(+), Li(+), and H(+) with a reduced rate. Similarities to mutations in the GYG motif of K(+) channels are discussed. PMID:10920013

  14. 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.

  15. In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation.

    PubMed Central

    Toyn, J; Hibbs, A R; Sanz, P; Crowe, J; Meyer, D I

    1988-01-01

    Mutants defective in the ability to translocate proteins across the membrane of the endoplasmic reticulum were selected in Trp- Saccharomyces cerevisiae on the basis of their ability to retain a fusion protein in the cytosol. The fusion comprised the prepro region of prepro-alpha-factor (MF alpha 1) N-terminal to phosphoribosyl anthranilate isomerase (TRP1). The first of the protein translocation mutations, called ptl1, results in temperature-sensitivity of growth and protein translocation. At the non-permissive temperature, precursors to several secretory proteins accumulate in the cytosol. Using this mutant, we demonstrate that the prepro-carboxypeptidase Y that had been accumulated in the cytosol at the non-permissive temperature could be post-translationally translocated into the endoplasmic reticulum when cells were returned to the permissive temperature. This result indicates that post-translational translocation of preproteins across endoplasmic reticulum membranes can occur in vivo. We have also determined that the temperature-sensitive component is membrane-associated in ptl1, and that the membranes derived from this strain show a reversible temperature-sensitive translocation phenotype in vitro. Images PMID:3072198

  16. Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen)

    PubMed Central

    2017-01-01

    Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Although the exact mechanism leading to relief of allodynia and hyperalgesia caused by neuropathy is not known, the blocking effect of gabapentin on voltage-dependent calcium channels has been proposed to be involved. In order to further evaluate its analgesic mechanisms, we tested the efficacy of gabapentin on protein kinase C epsilon (PKCε) translocation in cultured peripheral neurons isolated from rat dorsal root ganglia (DRGs). We found that gabapentin significantly reduced PKCε translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen), a very commonly used analgesic drug, also produces inhibition of PKCε. We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a mechanism of action for the combined use of paracetamol and gabapentin, which has recently been shown to be effective, with a cumulative behavior, in the control of postoperative pain in human patients. PMID:28299349

  17. Cytoplasmic Phospholipase A2 Modulation of Adolescent Rat Ethanol-Induced Protein Kinase C Translocation and Behavior

    PubMed Central

    Santerre, J. L.; Kolitz, E. B.; Pal, R.; Rogow, J. A.; Werner, D. F.

    2015-01-01

    Ethanol consumption typically begins during adolescence, a developmental period which exhibits many age-dependent differences in ethanol behavioral sensitivity. Protein kinase C (PKC) activity is largely implicated in ethanol-behaviors, and our previous work indicates that regulation of novel PKC isoforms likely contributes to decreased high-dose ethanol sensitivity during adolescence. The cytoplasmic Phospholipase A2 (cPLA2) signaling cascade selectivity modulates novel and atypical PKC isoform activity, as well as adolescent ethanol hypnotic sensitivity. Therefore, the current study was designed to ascertain adolescent cPLA2 activity both basally and in response to ethanol, as well as it's involvement in ethanol-induced PKC isoform translocation patterns. cPLA2 expression was elevated during adolescence, and activity was increased only in adolescents following high-dose ethanol administration. Novel, but not atypical PKC isoforms translocate to cytosolic regions following high-dose ethanol administration. Inhibiting cPLA2 with AACOCF3 blocked ethanol-induced PKC cytosolic translocation. Finally, inhibition of novel, but not atypical, PKC isoforms when cPLA2 activity was elevated, modulated adolescent high-dose ethanol-sensitivity. These data suggest that the cPLA2/PKC pathway contributes to the acute behavioral effects of ethanol during adolescence. PMID:25791059

  18. Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen).

    PubMed

    Vellani, Vittorio; Giacomoni, Chiara

    2017-01-01

    Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Although the exact mechanism leading to relief of allodynia and hyperalgesia caused by neuropathy is not known, the blocking effect of gabapentin on voltage-dependent calcium channels has been proposed to be involved. In order to further evaluate its analgesic mechanisms, we tested the efficacy of gabapentin on protein kinase C epsilon (PKCε) translocation in cultured peripheral neurons isolated from rat dorsal root ganglia (DRGs). We found that gabapentin significantly reduced PKCε translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen), a very commonly used analgesic drug, also produces inhibition of PKCε. We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a mechanism of action for the combined use of paracetamol and gabapentin, which has recently been shown to be effective, with a cumulative behavior, in the control of postoperative pain in human patients.

  19. 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.

  20. 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

  1. 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.

  2. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  3. Endoproteolytic cleavage of TUG protein regulates GLUT4 glucose transporter translocation.

    PubMed

    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

    2012-07-06

    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.

  4. 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

  5. A de nevo complex t(7;13;8) translocation with a deletion in the TRPS gene region.

    PubMed

    Brandt, C A; Lüdecke, H J; Hindkjaer, J; Strømkjaer, H; Pinkel, D; Herlin, T; Bolund, L; Friedrich, U

    1997-09-01

    Molecular cytogenetic analyses have resolved the pathogenetic aberration of an 8-year-old girl with tricho-rhino-phalangeal syndrome type I (TRPS I), normal intelligence, and a karyotype originally described as 46,XX,t(8;13)(q24;q21). R- and Q-banding and high resolution R-banding analyses have also disclosed a seemingly mosaic abnormality of the distal short arm of chromosome 7 but have not fully characterized this abnormality. Combined primed in situ labelling and chromosome painting, and three-colour chromosome painting have revealed a complex, apparently balanced translocation t(7;13;8). Fluorescence in situ hybridization with yeast artificial chromosome and cosmid clones from 8q24.1 has shown an interstitial deletion of at least 3 Mb covering most of the TRPS I critical region.

  6. 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

  7. 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.

  8. Translocation of single stranded DNA through the α-hemolysin protein nanopore in acidic solutions

    PubMed Central

    de Zoysa, Ranulu Samanthi S.; Krishantha, D.M. Milan; Zhao, Qitao; Gupta, Jyoti; Guan, Xiyun

    2012-01-01

    The effect of acidic pH on the translocation of single-stranded DNA through the α-hemolysin pore is investigated. Two significantly different types of events, i.e., deep blockades and shallow blockades, are observed at low pH. The residence times of the shallow blockades are not significantly different from those of the DNA translocation events obtained at or near physiological pH, while the deep blockades have much larger residence times and blockage amplitudes. With a decrease in the pH of the electrolyte solution, the percentage of the deep blockades in the total events increases. Furthermore, the mean residence time of these long-lived events is dependent on the length of DNA, and also varies with the nucleotide base, suggesting that they are appropriate for use in DNA analysis. In addition to be used as an effective approach to affect DNA translocation in the nanopore, manipulation of the pH of the electrolyte solution provides a potential means to greatly enhance the sensitivity of nanopore stochastic sensing. PMID:21997574

  9. 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.

  10. 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.

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

    PubMed Central

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

    2005-01-01

    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

  12. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  13. 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.

  14. 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.

  15. 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

  16. ERdj5 Reductase Cooperates with Protein Disulfide Isomerase To Promote Simian Virus 40 Endoplasmic Reticulum Membrane Translocation

    PubMed Central

    Inoue, Takamasa; Dosey, Annie; Herbstman, Jeffrey F.; Ravindran, Madhu Sudhan; Skiniotis, Georgios

    2015-01-01

    ABSTRACT The nonenveloped polyomavirus (PyV) simian virus 40 (SV40) traffics from the cell surface to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol before mobilizing into the nucleus to cause infection. Prior to ER membrane penetration, ER lumenal factors impart structural rearrangements to the virus, generating a translocation-competent virion capable of crossing the ER membrane. Here we identify ERdj5 as an ER enzyme that reduces SV40's disulfide bonds, a reaction important for its ER membrane transport and infection. ERdj5 also mediates human BK PyV infection. This enzyme cooperates with protein disulfide isomerase (PDI), a redox chaperone previously implicated in the unfolding of SV40, to fully stimulate membrane penetration. Negative-stain electron microscopy of ER-localized SV40 suggests that ERdj5 and PDI impart structural rearrangements to the virus. These conformational changes enable SV40 to engage BAP31, an ER membrane protein essential for supporting membrane penetration of the virus. Uncoupling of SV40 from BAP31 traps the virus in ER subdomains called foci, which likely serve as depots from where SV40 gains access to the cytosol. Our study thus pinpoints two ER lumenal factors that coordinately prime SV40 for ER membrane translocation and establishes a functional connection between lumenal and membrane events driving this process. IMPORTANCE PyVs are established etiologic agents of many debilitating human diseases, especially in immunocompromised individuals. To infect cells at the cellular level, this virus family must penetrate the host ER membrane to reach the cytosol, a critical entry step. In this report, we identify two ER lumenal factors that prepare the virus for ER membrane translocation and connect these lumenal events with events on the ER membrane. Pinpointing cellular components necessary for supporting PyV infection should lead to rational therapeutic strategies for preventing and treating Py

  17. Exploration of twin‐arginine translocation for expression and purification of correctly folded proteins in Escherichia coli

    PubMed Central

    Fisher, Adam C.; Kim, Jae‐Young; Perez‐Rodriguez, Ritsdeliz; Tullman‐Ercek, Danielle; Fish, Wallace R.; Henderson, Lee A.; DeLisa, Matthew P.

    2008-01-01

    Summary Historically, the general secretory (Sec) pathway of Gram‐negative bacteria has served as the primary route by which heterologous proteins are delivered to the periplasm in numerous expression and engineering applications. Here we have systematically examined the twin‐arginine translocation (Tat) pathway as an alternative, and possibly advantageous, secretion pathway for heterologous proteins. Overall, we found that: (i) export efficiency and periplasmic yield of a model substrate were affected by the composition of the Tat signal peptide, (ii) Tat substrates were correctly processed at their N‐termini upon reaching the periplasm and (iii) proteins fused to maltose‐binding protein (MBP) were reliably exported by the Tat system, but only when correctly folded; aberrantly folded MBP fusions were excluded by the Tat pathway's folding quality control feature. We also observed that Tat export yield was comparable to Sec for relatively small, well‐folded proteins, higher relative to Sec for proteins that required cytoplasmic folding, and lower relative to Sec for larger, soluble fusion proteins. Interestingly, the specific activity of material purified from the periplasm was higher for certain Tat substrates relative to their Sec counterparts, suggesting that Tat expression can give rise to relatively pure and highly active proteins in one step. PMID:21261860

  18. A Protein Complex Network of Drosophila melanogaster

    PubMed Central

    Guruharsha, K. G.; Rual, J. -F.; Zhai, B.; Mintseris, J.; Vaidya, P.; Vaidya, N.; Beekman, C.; Wong, C.; Rhee, D. Y.; Cenaj, O.; McKillip, E.; Shah, S.; Stapleton, M.; Wan, K. H.; Yu, C.; Parsa, B.; Carlson, J. W.; Chen, X.; Kapadia, B.; VijayRaghavan, K.; Gygi, S. P.; Celniker, S. E.; Obar, R. A.; Artavanis-Tsakonas, S.

    2011-01-01

    SUMMARY Determining the composition of protein complexes is an essential step towards understanding the cell as an integrated system. Using co-affinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly five thousand individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a novel statistical framework to define individual protein-protein interactions, led to the generation of a Drosophila Protein interaction Map (DPiM) encompassing 556 protein complexes. The high quality of DPiM and its usefulness as a paradigm for metazoan proteomes is apparent from the recovery of many known complexes, significant enrichment for shared functional attributes and validation in human cells. DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution. PMID:22036573

  19. Accumulation of the Drosophila Torso-like protein at the blastoderm plasma membrane suggests that it translocates from the eggshell.

    PubMed

    Mineo, Alessandro; Furriols, Marc; Casanova, Jordi

    2015-04-01

    The eggshell serves as a depository for proteins that play an important role in early embryonic development. In particular, the Drosophila eggshell is responsible for transferring asymmetries from the egg chamber to specify the regions at both ends of the embryo through the uneven activation of the Torso (Tor) receptor in its membrane. This process relies on the restricted expression of the gene torso-like (tsl) in subpopulations of follicle cells during oogenesis and its protein accumulation at both poles of the eggshell, but it is not known how this signal is transmitted to the embryo. Here, we show that Tsl accumulates at the embryonic plasma membrane, even in the absence of the Tor receptor. However, during oogenesis, we detected Tsl accumulation only at the eggshell. These results suggest that there is a two-step mechanism to transfer the asymmetric positional cues from the egg chamber into the early embryo: initial anchoring of Tsl at the eggshell as it is secreted, followed by its later translocation to the egg plasma membrane, where it enables Tor receptor activation. Translocation of anchored determinants from the eggshell might then regulate the spatial and temporal control of early embryonic developmental processes.

  20. A complex three way translocation resulting in two sibs with partial trisomy 3p23----3pter.

    PubMed Central

    Voss, R; Gross-Kieselstein, E; Hurvitz, H; Dagan, J; Kerem, E; Zlotogora, J

    1984-01-01

    A male infant with multiple congenital anomalies and psychomotor retardation was found to have a translocation resulting in partial trisomy for the distal part of chromosome 3p. An older sister with similar clinical findings had an identical karyotype. Chromosome studies in the phenotypically normal parents revealed a balanced translocation in the mother involving chromosomes 3, 11, and 18. An identical translocation was found in one of the normal children. Images PMID:6512835

  1. [Isolation of proteins with complex forming agents].

    PubMed

    Schwenke, K D; Raab, B; Ender, B

    1975-01-01

    Taking vegetable albumins for models, the authors report of the possibilities of isolating proteins (which cannot be precipitated isoelectrically) by using their property of forming complexes with tannin or poly-anions. The precipitation of proteins with dextran sulphate or polyphosphates, which is due to electrostatic interaction, depends on the pH value and the electrolyte content of the solution. Under appropriate experimental conditions, protein yields of 100% are achieved. By means of tannin, the proteins are completely precipitated in a wide range of pH. The protein component of the poly-anion-containing complexes is isolated by precipitation with salt or by thermal coagulation after dissolving of the complexes. The isolation of protein from the tannin complexes is preferably realized by reaction with coffeine.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. Doxorubicin attenuates CHIP-guarded HSF1 nuclear translocation and protein stability to trigger IGF-IIR-dependent cardiomyocyte death

    PubMed Central

    Huang, Chih-Yang; Kuo, Wei-Wen; Lo, Jeng-Fan; Ho, Tsung-Jung; Pai, Pei-ying; Chiang, Shu-Fen; Chen, Pei-Yu; Tsai, Fu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang

    2016-01-01

    Doxorubicin (DOX) is one of the most effective antitumor drugs, but its cardiotoxicity has been a major concern for its use in cancer therapy for decades. Although DOX-induced cardiotoxicity has been investigated, the underlying mechanisms responsible for this cardiotoxicity have not been completely elucidated. Here, we found that the insulin-like growth factor receptor II (IGF-IIR) apoptotic signaling pathway was responsible for DOX-induced cardiotoxicity via proteasome-mediated heat shock transcription factor 1 (HSF1) degradation. The carboxyl-terminus of Hsp70 interacting protein (CHIP) mediated HSF1 stability and nuclear translocation through direct interactions via its tetratricopeptide repeat domain to suppress IGF-IIR expression and membrane translocation under physiological conditions. However, DOX attenuated the HSF1 inhibition of IGF-IIR expression by diminishing the CHIP–HSF1 interaction, removing active nuclear HSF1 and triggering HSF1 proteasomal degradation. Overexpression of CHIP redistributed HSF1 into the nucleus, inhibiting IGF-IIR expression and preventing DOX-induced cardiomyocyte apoptosis. Moreover, HSF1A, a small molecular drug that enhances HSF1 activity, stabilized HSF1 expression and minimized DOX-induced cardiac damage in vitro and in vivo. Our results suggest that the cardiotoxic effects of DOX result from the prevention of CHIP-mediated HSF1 nuclear translocation and activation, which leads to an upregulation of the IGF-IIR apoptotic signaling pathway. We believe that the administration of an HSF1 activator or agonist may further protect against the DOX-induced cell death of cardiomyocytes. PMID:27809308

  7. 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.

  8. Complex lasso: new entangled motifs in proteins

    NASA Astrophysics Data System (ADS)

    Niemyska, Wanda; Dabrowski-Tumanski, Pawel; Kadlof, Michal; Haglund, Ellinor; Sułkowski, Piotr; Sulkowska, Joanna I.

    2016-11-01

    We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules.

  9. Complex lasso: new entangled motifs in proteins

    PubMed Central

    Niemyska, Wanda; Dabrowski-Tumanski, Pawel; Kadlof, Michal; Haglund, Ellinor; Sułkowski, Piotr; Sulkowska, Joanna I.

    2016-01-01

    We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules. PMID:27874096

  10. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells.

    PubMed

    Drastichova, Zdenka; Novotny, Jiri

    2012-08-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol. Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of G(q/11)α protein and to determine their localization in isolated fractions and stability in naïve and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G(11)α protein. We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the G(q/11) protein, which were found to be membrane-bound. Both of these complexes dissociated after prolonged treatment with TRH. Still other G(q/11)α protein complexes of lower molecular weight were determined in the cytosol. These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h) hormone treatment. These results support the notion that a portion of G(q/11)α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor. At the same time, these findings indicate that the redistribution of G(q/11)α is brought about by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the G(q/11)α protein.

  11. 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

  12. Distinct patterns of cleavage and translocation of cell cycle control proteins in CD95-induced and p53-induced apoptosis.

    PubMed Central

    Park, Weon Seo; Jung, Kyeong Cheon; Chung, Doo Hyun; Nam, Woo-Dong; Choi, Won Jin; Bae, Youngmee

    2003-01-01

    Apoptotic cell death induced by p53 occurs at a late G1 cell cycle checkpoint termed the restriction (R) point, and it has been proposed that p53-induced apoptosis causes upregulation of CD95. However, as cells with defective in CD95 signaling pathway are still sensitive to p53-induced apoptosis, CD95 cannot be the sole factor resulting in apoptosis. In addition, unlike p53-induced apoptosis, the relationship between CD95-mediated apoptosis and the cell cycle is not clearly understood. It would therefore be worth investigating whether CD95-mediated cell death is pertinent with p53-induced apoptosis in view of cell cycle related molecules. In this report, biochemical analysis showed that etoposide-induced apoptosis caused the induction and the nuclear translocation of effector molecules involved in G1 cell cycle checkpoint. However, there was no such translocation in the case of CD95-mediated death. Thus, although both types of apoptosis involved caspase activation, the cell cycle related proteins responded differently. This argues against the idea that p53-induced apoptosis occurs through the induction of CD95/CD95L expression. PMID:12923319

  13. Construction of a chimeric ArsA-ArsB protein for overexpression of the oxyanion-translocating ATPase.

    PubMed

    Dou, D; Owolabi, J B; Dey, S; Rosen, B P

    1992-12-25

    Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodes an ATP-driven anion extrusion pump. The ars operon is composed of three structural genes, arsA, arsB, and arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amount of ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integral membrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress the arsB gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (delta G = -17 kcal/mol) between the third and twelfth codons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3' in frame to the first 1458 base pairs of the arsA gene to encode a 914-residue chimeric protein (486 residues of the ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiating methionine. The ArsA-ArsB chimera has been overexpressed at approximately 15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO2- from cells, demonstrating that the chimera can function as a component of the oxyanion-translocating ATPase.

  14. A familial complex chromosome translocation resulting in duplication of 6p25.

    PubMed

    Vermeesch, J R; Thoelen, R; Fryns, Jean Pierre

    2004-01-01

    We report on a girl with psychomotor retardation, severe speech developmental delay and mild dysmorphic features. Molecular cytogenetic analysis showed that the patient was carrier of an insertion (6)(p22.5-->22.4) in chromosome 12. Analysis of the chromosomes of the mother revealed the presence of a complex chromosomal rearrangement. In addition to the insertion (6)(p22.5-->22.4) in chromosome 12 and a pericentric inversion in chromosome 12, the 6p subtelomeric region was absent in the mother. This is, to our knowledge, the smallest pure duplication of chromosome 6p as well as the smallest cryptic subtelomeric 6pter deletion thus far reported.

  15. Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases

    PubMed Central

    Capano, Michela; Crompton, Martin

    2005-01-01

    The cytosolic protein Bax plays a key role in apoptosis by migrating to mitochondria and releasing proapoptotic proteins from the mitochondrial intermembrane space. The present study investigates the movement of Bax in isolated rat neonatal cardiomyocytes subjected to simulated ischaemia (minus glucose, plus cyanide), using green fluorescent protein-tagged Bax as a means of imaging Bax movements. Simulated ischaemia induced Bax translocation from the cytosol to mitochondria, commencing within 20 min of simulated ischaemia and progressing for several hours. Under the same conditions, there was an increase in the active, phosphorylated forms of p38 MAPK (mitogen-activated protein kinase) and AMPK (AMP-activated protein kinase). The AMPK activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin also stimulated Bax translocation. Inhibition of p38 MAPK with SB203580 attenuated the phosphorylation of the downstream substrates, MAPK-activated protein kinases 2 and 3, but not that of the upstream MAPK kinase 3, nor of AMPK. Under all conditions (ischaemia, AICAR and metformin), SB203580 blocked Bax translocation completely. It is concluded that Bax translocation to mitochondria is an early step in ischaemia and that it occurs in response to activation of p38 MAPK downstream of AMPK. PMID:16321138

  16. 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.

  17. 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.

  18. 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

  19. The claudin Megatrachea protein complex.

    PubMed

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

    2012-10-26

    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.

  20. Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo.

    PubMed

    Min, Gyesik; Kemper, J Kim; Kemper, Byron

    2002-07-19

    Phenobarbital (PB) induction of CYP2B genes is mediated by translocation of the constitutively active androstane receptor (CAR) to the nucleus. Interaction of CAR with p160 coactivators and enhancement of CAR transactivation by the coactivators have been shown in cultured cells. In the present studies, the interaction of CAR with the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) was examined in vitro and in vivo. Binding of GRIP1 to CAR was shown by glutathione S-transferase (GST) pull-down and affinity DNA binding. N- or C-terminal fragments of GRIP1 that contained the central receptor-interacting domain bound to GST-CAR, but the presence of ligand increased the binding to GST-CAR of only the fragments containing the C-terminal region. In gel shift analysis, binding to CAR was observed only with GRIP1 fragments containing the C-terminal region, and the binding was increased by a CAR agonist and decreased by a CAR antagonist. Expression of GRIP1 enhanced CAR-mediated transactivation in cultured hepatic-derived cells 2-3-fold. In hepatocytes transfected in vivo, expression of exogenous GRIP1 alone induced transactivation of the CYP2B1 PB-dependent enhancer 15-fold, whereas CAR expression alone resulted in only a 3-fold enhancement in untreated mice. Remarkably, CAR and GRIP1 together synergistically transactivated the enhancer about 150-fold, which is approximately equal to activation by PB treatment. In PB-treated mice, expression of exogenous CAR alone had little effect, expression of GRIP1 increased transactivation about 2-fold, and with CAR and GRIP, a 4-fold activation was observed. In untreated mice, expression of GRIP resulted in nuclear translocation of green fluorescent protein-CAR. These results strongly suggest that a p160 coactivator functions in CAR-mediated transactivation in vivo in response to PB treatment and that the synergistic activation of CAR by GRIP in untreated animals results from both nuclear translocation and

  1. Protein Domain of Unknown Function 3233 is a Translocation Domain of Autotransporter Secretory Mechanism in Gamma proteobacteria

    PubMed Central

    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 1/3rd 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

  2. Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules

    PubMed Central

    1986-01-01

    Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [alpha 32P]8-azidoadenosine 5'-triphosphate ([alpha 32P]8-N3ATP), a photoaffinity analogue of ATP, was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass of 292 kD that bound [alpha 32P]8-N3ATP. The incorporation of label is ultraviolet light-dependent and ATP- sensitive. Moreover, the 292-kD polypeptide could be isolated in association with vesicles or microtubules, depending on the conditions used, and the data indicate that the 292-kD polypeptide is similar to mammalian brain microtubule-associated protein 2 (MAP 2) for the following reasons: The 292-kD polypeptide isolated from either squid axoplasm or optic lobe cross-reacts with antiserum to porcine brain MAP 2. Furthermore, it purifies with taxol-stabilized microtubules and is released with salt. Based on these characteristics, the 292-kD polypeptide is distinct from the known force-generating molecules myosin and flagellar dynein, as well as the 110-130-kD kinesin-like polypeptides that have recently been described (Brady, S. T., 1985, Nature (Lond.), 317:73-75; Vale, R. D., T. S. Reese, and M. P. Sheetz, 1985b, Cell, 42:39-50; Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh, 1985, Nature (Lond.), 318:483-486). Because the 292-kD polypeptide binds ATP and is associated with vesicles that translocate on purified MAP-free microtubules in an ATP-dependent fashion, it is therefore believed to be involved in vesicle-microtubule interactions that promote organelle motility. PMID:3091608

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

    PubMed Central

    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. PMID:24995125

  4. Stimulation of phosphatidylinositol hydrolysis, protein kinase C translocation, and mitogen-activated protein kinase activity by bradykinin in rat ventricular myocytes: dissociation from the hypertrophic response.

    PubMed Central

    Clerk, A; Gillespie-Brown, J; Fuller, S J; Sugden, P H

    1996-01-01

    In ventricular myocytes cultured from neonatal rat hearts, bradykinin (BK), kallidin or BK(1-8) [(Des-Arg9)BK] stimulated PtdinsP2 hydrolysis by 3-4-fold. EC50 values were 6 nM (BK), 2 nM (kallidin), and 14 microM [BK(1-8)]. BK or kallidin stimulated the rapid (less than 30 s) translocation of more than 80% of the novel protein kinase C (PKC) isoforms nPKC-delta and nPKC-epsilon from the soluble to the particulate fraction. EC50 values for nPKC-delta translocation by BK or kallidin were 10 and 2 nM respectively. EC50 values for nPKC-epsilon translocation by BK or kallidin were 2 and 0.6 nM respectively. EC50 values for the translocation of nPKC-delta and nPKC-epsilon by BK(1-8) were more than 5 microM. The classical PKC, cPKC-alpha, and the atypical PKC, nPKC-zeta, did not translocate. BK caused activation and phosphorylation of p42-mitogen-activated protein kinase (MAPK) (maximal at 3-5 min, 30-35% of p42-MAPK phosphorylated). p44-MAPK was similarly activated. EC50 values for p42/p44-MAPK activation by BK were less than 1 nM whereas values for BK(1-8) were more than 10 microM. The order of potency [BK approximately equal to kallidin >> BK (1-8)] for the stimulation of PtdInsP2 hydrolysis, nPKC-delta and nPKC-epsilon translocation, and p42/p44-MAPK activities suggests involvement of the B2 BK receptor subtype. In addition, stimulation of all three processes by BK was inhibited by the B2BK receptor-selective antagonist HOE140 but not by the B1-selective antagonist Leu8BK(1-8). Exposure of cells to phorbol 12-myristate 13-acetate for 24 h inhibited subsequent activation of p42/p44-MAPK by BK suggesting participation of nPKC (and possibly cPKC) isoforms in the activation process. Thus, like hypertrophic agents such as endothelin-1 (ET-1) and phenylephrine (PE), BK activates PtdInsP2 hydrolysis, translocates nPKC-delta, and nPKC-epsilon, and activates p42/p44-MAPK. However, in comparison with ET-1 and PE, BK was only weakly hypertrophic as assessed by cell morphology

  5. Electrophoretic protein analysis for the identification of doubled haploid 1A-1R, 1B-1R wheat-rye double translocation lines and for the assessment of their genetic stability.

    PubMed

    Vahl, U; Müller, G; Böhme, T

    1993-06-01

    Eighteen available doubled haploid wheat lines with a cytologically proven 1A-1R, 1B-1R double translocation, which where derived via anther culture from four crosses of the 1A-1R wheat-rye translocation cv "Amigo" with several 1B-1R wheat-rye translocation forms, were subjected to electrophoretic seed protein analysis. Besides, the five parents used in the crosses and some other wheat cultivars and doubled haploid lines (19 with a 1B-1R single translocation, 10 with a 1A-1R translocation and 7 without any 1R translocation) were also included in the investigation. It was found that the gliadin patterns visualized after SDS polyacrylamide gel electrophoresis of alcohol-soluble seed protein extracts can differentiate not only 1B-1R and 1A-1R translocation forms from wheats without any 1R-translocation chromosome, but also 1B-1R and 1A-1R wheats from each other. Moreover, 1A-1R, 1B-1R double translocation lines can be distinguished as well due to characteristic differences revealed between 1A-1R and 1B-1R translocation forms. Thus, all of tested dh1- and dh2-grains of the double translocation lines showed the expected doublet: the 1A-1R translocation ("Amigo")-typical rye band and the 1B-1R translocation ("Kawkas")-typical rye band. Consequently, gliadin patterns estimated after SDS electrophoresis may be used as markers for the fast detection of the desired 1A-1R, 1B-1R double translocation forms among 1A-1R single translocation lines, 1B-1R single translocation lines and lines without any 1R-translocation in the progenies of appropriate crosses. Furthermore, by means of gliadin tests on the dh2-generation the excellent stability of the double translocation 1A-1R, 1B-1R during more than one propagation phase has been proven. Estimations of high-molecular weight (HMW) glutenin subunits coded by 1A and 1B chromosomes are compatible with the double translocation constitution. A few deviating results can be explained by crossing-over events. Seed protein analysis

  6. Subcellular distribution of the 18kDa translocator protein and transcript variant PBR-S in human cells.

    PubMed

    Liu, Guo-Jun; Middleton, Ryan J; Banati, Richard B

    2017-05-20

    Despite continued interest in the 18kDa translocator protein (PBR/TSPO) as a biomarker and a therapeutic target for a range of diseases, its functional role, such as in the steroid synthesis pathway and energy metabolism has either become contentious or remains to be described more precisely. The PBR/TSPO gene consists of four exons, while a shorter isoform termed PBR-S lacks exon 2. The PBR-S 102-codon open reading frame differs to that of PBR/TSPO, resulting in a protein that is completely unrelated to PBR/TSPO. To our knowledge, PBR-S protein has never been described and has no known or proposed function. To obtain possible clues on the role of this uncharacterised protein, we compared the subcellular distribution of PBR-S to that of PBR/TSPO. By expressing fluorescently tagged PBR/TSPO, we confirmed that full-length PBR/TSPO co-localises with mitochondria in HeLa, HEK-293, MDA-MB-231, BJ and U87-MG human cell lines. Unlike the strictly mitochondrial localisation of PBR/TSPO, PBR-S has a punctate distribution throughout the cytosol that co-localises with lysosomes in HeLa, HEK-293, MDA-MB-231, BJ and U87-MG cells. In summary, within the cell lines examined we confirm mitochondria rather than occasionally reported other localisations, such as the cell nucleus, to be the only site where PBR/TSPO resides. Due to the lack of any shared protein sequences and the different subcellular locations, we suggest that the previously uncharacterised PBR-S protein variant of the PBR/TSPO gene is likely to serve a different yet to be discovered function compared to PBR/TSPO.

  7. Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways†

    PubMed Central

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A.; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-01-01

    Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome. PMID:16973564

  8. Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.

    PubMed

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-10-01

    Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

  9. Pathogenesis of hemorrhage-induced bacteria/endotoxin translocation in rats. Effects of recombinant bactericidal/permeability-increasing protein.

    PubMed Central

    Yao, Y M; Bahrami, S; Leichtfried, G; Redl, H; Schlag, G

    1995-01-01

    OBJECTIVES: This study was conducted to determine the role of gut-derived bacteria/endotoxin in the pathogenesis of the multiple-organ damage and mortality, the possible beneficial effect of recombinant bactericidal/permeability-increasing protein (rBPl21), and whether neutralizing endotoxemia by rBPl21 treatment influences tumor necrosis factor (TNF) formation in rats after hemorrhagic shock and resuscitation. SUMMARY BACKGROUND DATA: Hypovolemic shock might be associated with bacterial or endotoxin translocation as well as systemic sepsis. Similar to bactericidal/permeability-increasing (BPl) protein, rBPl21 has been found to bind endotoxin and inhibit TNF production. METHODS: A rat model of prolonged hemorrhagic shock (30 to 35 mm Hg for 180 min) followed by adequate resuscitation was employed. Recombinant bactericidal/permeability-increasing protein was administered at 5 mg/kg intravenously. The control group was treated similarly to the BPl group, but received thaumatin as a protein-control preparation in the same dose as rBPl21. RESULTS: Immediately after resuscitation (230 min), plasma endotoxin levels in the control group (61.0 +/- 16.3 pg/mL) were almost neutralized by rBPl21 treatment (13.8 +/- 4.8 pg/mL, p < 0.05). Plasma TNF levels were not significantly influenced by rBPl21 treatment. The 48-hour survival rate was 68.8% in the treatment group versus 37.5% in the control group (p = 0.08). Microscopic histopathologic examination revealed relatively minor damage to various organs in the treatment group. CONCLUSIONS: These data suggest that hemorrhagic shock may lead to bacterial/endotoxin translocation with concomitant TNF formation, endogenous endotoxemia may play an important role in the pathogenesis of multiple-organ failure after shock and trauma, TNF formation at an early stage might be related mainly to mechanisms other than Kupffer's cells activation via lipopolysaccharide, and rBPl21 might be a useful therapeutic agent against endogenous bacteria

  10. 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

  11. AMP-activated protein kinase is required for exercise-induced peroxisome proliferator-activated receptor co-activator 1 translocation to subsarcolemmal mitochondria in skeletal muscle.

    PubMed

    Smith, Brennan K; Mukai, Kazutaka; Lally, James S; Maher, Amy C; Gurd, Brendon J; Heigenhauser, George J F; Spriet, Lawrence L; Holloway, Graham P

    2013-03-15

    In skeletal muscle, mitochondria exist as two subcellular populations known as subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS mitochondria preferentially respond to exercise training, suggesting divergent transcriptional control of the mitochondrial genomes. The transcriptional co-activator peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and mitochondrial transcription factor A (Tfam) have been implicated in the direct regulation of the mitochondrial genome in mice, although SS and IMF differences may exist, and the potential signalling events regulating the mitochondrial content of these proteins have not been elucidated. Therefore, we examined the potential for PGC-1α and Tfam to translocate to SS and IMF mitochondria in human subjects, and performed experiments in rodents to identify signalling mechanisms regulating these translocation events. Acute exercise in humans and rats increased PGC-1α content in SS but not IMF mitochondria. Acute exposure to 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside in rats recapitulated the exercise effect of increased PGC-1α protein within SS mitochondria only, suggesting that AMP-activated protein kinase (AMPK) signalling is involved. In addition, rendering AMPK inactive (AMPK kinase dead mice) prevented exercise-induced PGC-1α translocation to SS mitochondria, further suggesting that AMPK plays an integral role in these translocation events. In contrast to the conserved PGC-1α translocation to SS mitochondria across species (humans, rats and mice), acute exercise only increased mitochondrial Tfam in rats. Nevertheless, in rat resting muscle PGC-1α and Tfam co-immunoprecipate with α-tubulin, suggesting a common cytosolic localization. These data suggest that exercise causes translocation of PGC-1α preferentially to SS mitochondria in an AMPK-dependent manner.

  12. Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the Hepatitis C virus helicase†

    PubMed Central

    Matlock, Dennis L.; Yeruva, Laxmi; Byrd, Alicia K.; Mackintosh, Samuel G.; Langston, Clint; Brown, Carrie; Cameron, Craig E.; Fischer, Christopher J.; Raney, Kevin D.

    2010-01-01

    Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called non-structural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains respectively. The helicase domain of NS3 protein is referred as NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3’ to -5’ direction. The directionality for unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5’ end. NS3h can bind to the ssDNA and in the presence of ATP, move towards the 5’-end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h, 200 nM oligonucleotide) we find that NS3h translocates on ssDNA at a rate of 46 ± 5 nt s−1 and that it can move for 230 ± 60 nt before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5’-end. Upon addition of streptavidin, a ‘protein-block’ was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single cycle conditions, supporting the conclusion that NS3h is a relatively

  13. Small heat shock proteins translocate to the cytoskeleton in human skeletal muscle following eccentric exercise independently of phosphorylation.

    PubMed

    Frankenberg, Noni T; Lamb, Graham D; Overgaard, Kristian; Murphy, Robyn M; Vissing, Kristian

    2014-06-01

    Small heat shock proteins (sHSPs) are a subgroup of the highly conserved family of HSPs that are stress inducible and confer resistance to cellular stress and injury. This study aimed to quantitatively examine whether type of contraction (concentric or eccentric) affects sHSPs, HSP27 and αB-crystallin, localization, and phosphorylation in human muscle. Vastus lateralis muscle biopsies from 11 healthy male volunteers were obtained pre- and 3 h, 24 h, and 7 days following concentric (CONC), eccentric (ECC1), and repeated bout eccentric (ECC2) exercise. No changes were apparent in a control group (n = 5) who performed no exercise. Eccentric exercise induced muscle damage, as evidenced by increased muscle force loss, perceived muscle soreness, and elevated plasma creatine kinase and myoglobin levels. Total HSP27 and αB-crystallin amounts did not change following any type of exercise. Following eccentric exercise (ECC1 and ECC2) phosphorylation of HSP27 at serine 15 (pHSP27-Ser15) was increased approximately 3- to 6-fold at 3 h, and pαB-crystallin-Ser59 increased ~10-fold at 3 h. Prior to exercise most of the sHSP and psHSP pools were present in the cytosolic compartment. Eccentric exercise resulted in partial redistribution of HSP27 (~23%) from the cytosol to the cytoskeletal fraction (~28% for pHSP27-Ser15 and ~7% for pHSP27-Ser82), with subsequent full reversal within 24 h. αB-crystallin also showed partial redistribution from the cytosolic to cytoskeletal fraction (~18% of total) 3 h post-ECC1, but not after ECC2. There was no redistribution or phosphorylation of sHSPs with CONC. Eccentric exercise results in increased sHSP phosphorylation and translocation to the cytoskeletal fraction, but the sHSP translocation is not dependent on their phosphorylation.

  14. 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.

  15. Attachment of cell-binding ligands to arginine-rich cell penetrating peptides enables cytosolic translocation of complexed siRNA

    PubMed Central

    Zeller, Skye; Choi, Changseon; Uchil, Pradeep D.; Ban, Hongseok; Siefert, Alyssa; Fahmy, Tarek M.; Mothes, Walther; Lee, Sang Kyung; Kumar, Priti

    2014-01-01

    SUMMARY Cell penetrating peptides (CPPs) like nona-arginine (9R) poorly translocate siRNA into cells. Our studies demonstrate that attaching 9R to ligands that bind cell-surface receptors quantitatively increases siRNA uptake and importantly, allows functional delivery of complexed siRNA. The mechanism involved accumulation of ligand-9R:siRNA microparticles on the cell membrane, which induced transient membrane inversion at the site of ligand-9R binding and rapid siRNA translocation into the cytoplasm. siRNA release also occurred late after endocytosis when the ligand was attached to the L isoform of 9R, but not the protease-resistant 9DR, prolonging mRNA knockdown. This critically depended on endosomal proteolytic activity implying partial CPP degradation is required for endosome to cytosol translocation. The data demonstrate that ligand attachment renders simple polycationic CPPs effective for siRNA delivery by restoring their intrinsic property of translocation. PMID:25544044

  16. Proteomics: bases for protein complexity understanding.

    PubMed

    Rotilio, Domenico; Della Corte, Anna; D'Imperio, Marco; Coletta, Walter; Marcone, Simone; Silvestri, Cristian; Giordano, Lucia; Di Michele, Michela; Donati, Maria Benedetta

    2012-03-01

    In the post genomic era we became aware that the genomic sequence and protein functions cannot be correlated. One gene can encode multiple protein functions mainly because of mRNA splice variants, post translational modifications (PTM) and moonlighting functions. To study the whole population of proteins present in a cell to a specific time point and under defined conditions it is necessary to investigate the proteome. Comprehensive analysis of the proteome requires the use of emerging high technologies because of the complexity and wide dynamic range of protein concentrations. Proteomics provides the tools to study protein identification and quantitation, protein-protein interactions, protein modifications and localization. The most widespread strategy for studying global protein expression employs two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) allowing thousands of proteins to be resolved and their expression quantified. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has emerged as a high throughput technique for protein identification and characterization because of its high sensitivity, precision and accuracy. LC-MS/MS is well suited for accurate quantitation of protein expression levels, post-translational modifications and comparative and absolute quantitative analysis of peptides. Bioinformatic tools are required to elaborate the growing number of proteomic data. Here, we give an overview of the current status of the wide range of technologies that define and characterize the modern proteomics.

  17. Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the hepatitis C virus helicase.

    PubMed

    Matlock, Dennis L; Yeruva, Laxmi; Byrd, Alicia K; Mackintosh, Samuel G; Langston, Clint; Brown, Carrie; Cameron, Craig E; Fischer, Christopher J; Raney, Kevin D

    2010-03-16

    Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription, and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called nonstructural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains, respectively. The helicase domain of NS3 is termed NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3'-5' direction. The directionality of unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5' end. NS3h can bind to the ssDNA and in the presence of ATP move toward the 5' end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h and 200 nM oligonucleotide), we find that NS3h translocates on ssDNA at a rate of 46 +/- 5 nucleotides/s, and that it can move for 230 +/- 60 nucleotides before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5' end. Upon addition of streptavidin, a "protein block" was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single-cycle conditions, supporting the conclusion that NS3h is a relatively poor helicase

  18. Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation.

    PubMed

    Baytshtok, Vladimir; Chen, Jiejin; Glynn, Steven E; Nager, Andrew R; Grant, Robert A; Baker, Tania A; Sauer, Robert T

    2017-04-07

    The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.

  19. Whey protein hydrolysate increases translocation of GLUT-4 to the plasma membrane independent of insulin in wistar rats.

    PubMed

    Morato, Priscila Neder; Lollo, Pablo Christiano Barboza; Moura, Carolina Soares; Batista, Thiago Martins; Camargo, Rafael Ludemann; Carneiro, Everardo Magalhães; Amaya-Farfan, Jaime

    2013-01-01

    Whey protein (WP) and whey protein hydrolysate (WPH) have the recognized capacity to increase glycogen stores. The objective of this study was to verify if consuming WP and WPH could also increase the concentration of the glucose transporters GLUT-1 and GLUT-4 in the plasma membrane (PM) of the muscle cells of sedentary and exercised animals. Forty-eight Wistar rats were divided into 6 groups (n = 8 per group), were treated and fed with experimental diets for 9 days as follows: a) control casein (CAS); b) WP; c) WPH; d) CAS exercised; e) WP exercised; and f) WPH exercised. After the experimental period, the animals were sacrificed, muscle GLUT-1 and GLUT-4, p85, Akt and phosphorylated Akt were analyzed by western blotting, and the glycogen, blood amino acids, insulin levels and biochemical health indicators were analyzed using standard methods. Consumption of WPH significantly increased the concentrations of GLUT-4 in the PM and glycogen, whereas the GLUT-1 and insulin levels and the health indicators showed no alterations. The physical exercise associated with consumption of WPH had favorable effects on glucose transport into muscle. These results should encourage new studies dealing with the potential of both WP and WPH for the treatment or prevention of type II diabetes, a disease in which there is reduced translocation of GLUT-4 to the plasma membrane.

  20. 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.

  1. The 18-kDa translocator protein (TSPO) disrupts mammary epithelial morphogenesis and promotes breast cancer cell migration.

    PubMed

    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.

  2. 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

  3. Formation and cell translocation of carbon nanotube-fibrinogen protein corona

    NASA Astrophysics Data System (ADS)

    Chen, Ran; Radic, Slaven; Choudhary, Poonam; Ledwell, Kimberley G.; Huang, George; Brown, Jared M.; Chun Ke, Pu

    2012-09-01

    The binding of plasma fibrinogen with both single-walled and multi-walled carbon nanotubes (SWNTs and MWNTs) has been examined. Specifically, our absorbance study indicated that MWNTs were coated with multi-layers of fibrinogen to render a "hard protein corona," while SWNTs were adsorbed with thin layers of the protein to precipitate out of the aqueous phase. In addition, static quenching as a result of energy transfer from fluorescently labeled fibrinogen to their nanotube substrates was revealed by Stern-Volmer analysis. When exposed to HT-29 cells, the nanotubes and fibrinogen could readily dissociate, possibly stemming from their differential affinities for the amphiphilic membrane bilayer.

  4. 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.

  5. La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation.

    PubMed

    Pisanelli, Giuseppe; Laurent-Rolle, Maudry; Manicassamy, Balaji; Belicha-Villanueva, Alan; Morrison, Juliet; Lozano-Dubernard, Bernardo; Castro-Peralta, Felipa; Iovane, Giuseppe; García-Sastre, Adolfo

    2016-02-02

    La Piedad Michoacán Mexico Virus (LPMV) is a member of the Rubulavirus genus within the Paramyxoviridae family. LPMV is the etiologic agent of "blue eye disease", causing a significant disease burden in swine in Mexico with long-term implications for the agricultural industry. This virus mainly affects piglets and is characterized by meningoencephalitis and respiratory distress. It also affects adult pigs, causing reduced fertility and abortions in females, and orchitis and epididymitis in males. Viruses of the Paramyxoviridae family evade the innate immune response by targeting components of the interferon (IFN) signaling pathway. The V protein, expressed by most paramyxoviruses, is a well-characterized IFN signaling antagonist. Until now, there were no reports on the role of the LPMV-V protein in inhibiting the IFN response. In this study we demonstrate that LPMV-V protein antagonizes type I but not type II IFN signaling by binding STAT2, a component of the type I IFN cascade. Our results indicate that the last 18 amino acids of LPMV-V protein are required for binding to STAT2 in human and swine cells. While LPMV-V protein does not affect the protein levels of STAT1 or STAT2, it does prevent the IFN-induced phosphorylation and nuclear translocation of STAT1 and STAT2 thereby inhibiting cellular responses to IFN α/β.

  6. Pim-1 preserves mitochondrial morphology by inhibiting dynamin-related protein 1 translocation.

    PubMed

    Din, Shabana; Mason, Matthew; Völkers, Mirko; Johnson, Bevan; Cottage, Christopher T; Wang, Zeping; Joyo, Anya Y; Quijada, Pearl; Erhardt, Peter; Magnuson, Nancy S; Konstandin, Mathias H; Sussman, Mark A

    2013-04-09

    Mitochondrial morphological dynamics affect the outcome of ischemic heart damage and pathogenesis. Recently, mitochondrial fission protein dynamin-related protein 1 (Drp1) has been identified as a mediator of mitochondrial morphological changes and cell death during cardiac ischemic injury. In this study, we report a unique relationship between Pim-1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemic stress. Transgenic hearts overexpressing cardiac Pim-1 display reduction of total Drp1 protein levels, increased phosphorylation of Drp1-(S637), and inhibition of Drp1 localization to the mitochondria. Consistent with these findings, adenoviral-induced Pim-1 neonatal rat cardiomyocytes (NRCMs) retain a reticular mitochondrial phenotype after simulated ischemia (sI) and decreased Drp1 mitochondrial sequestration. Interestingly, adenovirus Pim-dominant negative NRCMs show increased expression of Bcl-2 homology 3 (BH3)-only protein p53 up-regulated modulator of apoptosis (PUMA), which has been previously shown to induce Drp1 accumulation at mitochondria and increase sensitivity to apoptotic stimuli. Overexpression of the p53 up-regulated modulator of apoptosis-dominant negative adenovirus attenuates localization of Drp1 to mitochondria in adenovirus Pim-dominant negative NRCMs promotes reticular mitochondrial morphology and inhibits cell death during sI. Therefore, Pim-1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to sI.

  7. 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...

  8. Imaging the dynamics of intracellular protein translocation by photoconversion of phamret-cybr/ROM.

    PubMed

    Yang, L; Matsuda, T; Raviraj, V; Ching, Y W; Braet, F; Nagai, T; Soon, L L

    2011-06-01

    Cybr/Reduced On-random Motile (ROM) is a scaffold protein, containing a postsynaptic density protein-95/discs-large/ZO-1 (PDZ) domain, a LEU region and a PDZ domain binding region at the C-terminus. In the immune system, Cybr/ROM was found to localize in vesicles and at the plasma membrane, through interactions with cytohesin-1. In this investigation, we reported Cybr/ROM as occurring in vesicles, the cytoplasm and at membrane ruffles of H1299 lung cancer cells. Its localization at the ruffles was dependent on intact actin structures as indicated by latrunculin A treatment, which abrogated ruffle formation and staining of Cybr/ROM at the cells' periphery. Transfection of truncation mutants consisting of either the PDZ or LEU domain showed that the LEU domain of ROM was localized to membrane ruffles, vesicles and the cytoplasm, whereas, the PDZ domain localized to the membrane ruffles and cytoplasm only. There was therefore, domain/molecular segregation of Cybr/ROM in different cellular compartments. Cybr/ROM was subcloned into a plasmid carrying the photoactivation-mediated resonance energy transfer (Phamret) protein. The photoconversion experiments demonstrated the diffusion of ROM from the cytoplasm to the membrane ruffling sites and conversely from membrane ruffles to the cytoplasm. Large variances in the transport velocity of Cybr/ROM in the cytoplasm suggested that its movements were facilitated by other mechanisms in addition to diffusion.

  9. Human translocation liposarcoma-CCAAT/enhancer binding protein (C/EBP) homologous protein (TLS-CHOP) oncoprotein prevents adipocyte differentiation by directly interfering with C/EBPbeta function.

    PubMed

    Adelmant, G; Gilbert, J D; Freytag, S O

    1998-06-19

    Human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is a fusion oncoprotein found specifically in a malignant tumor of adipose tissue and results from a t(12;16) translocation that fuses the amino-terminal part of TLS to the entire coding region of CHOP. Being that CHOP is a member of the C/EBP transcription factor family, proteins that comprise part of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked adipocyte differentiation by directly interfering with C/EBP function. Using a single-step retroviral infection protocol, either wild-type or mutant TLS-CHOP were co-expressed along with C/EBPbeta in naïve NIH3T3 cells, and their ability to inhibit C/EBPbeta-driven adipogenesis was determined. TLS-CHOP was extremely effective at blocking adipocyte differentiation when expressed at a level comparable to that observed in human myxoid liposarcoma. This effect of TLS-CHOP required a functional leucine zipper domain and correlated with its ability to heterodimerize with C/EBPbeta and inhibit C/EBPbeta DNA binding and transactivation activity in situ. In contrast, the TLS-CHOP basic region was dispensable, making it unlikely that the inhibitory effect of TLS-CHOP is attributable to unscheduled gene expression resulting from TLS-CHOP's putative transactivation activity. Another adipogenic transcription factor, PPARgamma2, was able to rescue TLS-CHOP-inhibited cells, indicating that TLS-CHOP interferes primarily with C/EBPbeta-driven adipogenesis and not with other requisite events of the adipocyte differentiation program. Together, the results demonstrate that TLS-CHOP blocks adipocyte differentiation by directly preventing C/EBPbeta from binding to and transactivating its target genes. Moreover, they provide strong support for the thesis that a blockade to normal differentiation is an important aspect of the cancer process.

  10. Quantification of Detergents Complexed with Membrane Proteins

    PubMed Central

    Chaptal, Vincent; Delolme, Frédéric; Kilburg, Arnaud; Magnard, Sandrine; Montigny, Cédric; Picard, Martin; Prier, Charlène; Monticelli, Luca; Bornert, Olivier; Agez, Morgane; Ravaud, Stéphanie; Orelle, Cédric; Wagner, Renaud; Jawhari, Anass; Broutin, Isabelle; Pebay-Peyroula, Eva; Jault, Jean-Michel; Kaback, H. Ronald; le Maire, Marc; Falson, Pierre

    2017-01-01

    Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies. PMID:28176812

  11. 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.

  12. 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

  13. 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

  14. New Anthocyanin-Human Salivary Protein Complexes.

    PubMed

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

    2015-08-04

    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.

  15. Heat Shock Protein translocation induced by membrane fluidization increases tumor-cell sensitivity to chemotherapeutic drugs.

    PubMed

    Dempsey, Nina C; Ireland, H Elyse; Smith, Carly M; Hoyle, Christine F; Williams, John H H

    2010-10-28

    Treatment of chronic lymphocytic leukemia (CLL) remains a challenge due to the frequency of drug resistance amongst patients. Improving the delivery of chemotherapeutic agents while reducing the expression of anti-apoptotic Heat Shock Proteins (HSPs) within the cancer cells may facilitate in overcoming this drug resistance. We demonstrate for the first time that sub-lethal doses of chemotherapeutic agents can be combined with membrane fluidizing treatments to produce a significant increase in drug efficacy and apoptosis in vitro. We show that fluidizers result in a transient decrease in intracellular HSPs, resulting in increased tumor-cell sensitivity and a membrane-associated induction of HSP gene expression.

  16. Assembly reflects evolution of protein complexes.

    PubMed

    Levy, Emmanuel D; Boeri Erba, Elisabetta; Robinson, Carol V; Teichmann, Sarah A

    2008-06-26

    A homomer is formed by self-interacting copies of a protein unit. This is functionally important, as in allostery, and structurally crucial because mis-assembly of homomers is implicated in disease. Homomers are widespread, with 50-70% of proteins with a known quaternary state assembling into such structures. Despite their prevalence, their role in the evolution of cellular machinery and the potential for their use in the design of new molecular machines, little is known about the mechanisms that drive formation of homomers at the level of evolution and assembly in the cell. Here we present an analysis of over 5,000 unique atomic structures and show that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity. Where quaternary structure is not conserved among the members of a protein family, a detailed investigation revealed well-defined evolutionary pathways by which proteins transit between different quaternary structure types. Furthermore, we show by perturbing subunit interfaces within complexes and by mass spectrometry analysis, that the (dis)assembly pathway mimics the evolutionary pathway. These data represent a molecular analogy to Haeckel's evolutionary paradigm of embryonic development, where an intermediate in the assembly of a complex represents a form that appeared in its own evolutionary history. Our model of self-assembly allows reliable prediction of evolution and assembly of a complex solely from its crystal structure.

  17. Hsp70 Protein Complexes as Drug Targets

    PubMed Central

    Assimon, Victoria A.; Gillies, Anne T.; Rauch, Jennifer N.; Gestwicki, Jason E.

    2013-01-01

    Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70’s interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, such as pro-folding, pro-degradation and pro-trafficking. Thus, a promising strategy may be to block protein-protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to those goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology. PMID:22920901

  18. Hsp70 protein complexes as drug targets.

    PubMed

    Assimon, Victoria A; Gillies, Anne T; Rauch, Jennifer N; Gestwicki, Jason E

    2013-01-01

    Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70's interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, including roles in pro-folding, pro-degradation and pro-trafficking pathways. Thus, a promising strategy may be to block protein- protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to these goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology.

  19. Anti-stress effects of ONO-2952, a novel translocator protein 18 kDa antagonist, in rats.

    PubMed

    Mitsui, Katsukuni; Niwa, Tomohiro; Kawahara, Yuji; Morimoto, Noriko; Ohmoto, Kazuyuki; Kato, Masashi; Yamaura, Yoshiyuki; Yoshimoto, Naoki; Suna, Hideaki; Katsumata, Seishi

    2015-12-01

    Accumulating evidence has shown the pathophysiological significance of the translocator protein 18 kDa (TSPO) in the central nervous system. In this study, we evaluated the beneficial effects of ONO-2952, a novel TSPO antagonist in rat stress models. ONO-2952 potently bound both rat and human TSPO (Ki=0.330-9.30 nmol/L) with high selectivity over other receptors, transporters, ion channels and enzymes. ONO-2952 inhibited both neurosteroid accumulation and noradrenaline release in the brain of rats exposed to acute stress. The inhibitory effect of ONO-2952 on stress-induced noradrenaline release was attenuated by co-treatment with the TSPO agonist CB34 in a dose-dependent manner. ONO-2952, at 0.3 mg/kg or higher, dose-dependently suppressed restraint stress-induced defecation in rats with brain TSPO occupancy of more than 50%. In addition, ONO-2952, at 1 mg/kg or higher, suppressed conditioned fear stress-induced freezing behavior in rats with an efficacy equivalent to that of diazepam, given orally at 3 mg/kg. Results of the passive avoidance learning test revealed that ONO-2952, unlike diazepam, did not affect learning and memory even at doses 10 times higher than its effective doses in the stress models. The present findings indicate that ONO-2952 is a promising candidate for the treatment of stress-related disorders.

  20. 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

  1. Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor).

    PubMed

    Sileikyte, Justina; Petronilli, Valeria; Zulian, Alessandra; Dabbeni-Sala, Federica; Tognon, Giuseppe; Nikolov, Peter; Bernardi, Paolo; Ricchelli, Fernanda

    2011-01-14

    We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

  2. Characterization of protein complexes using targeted proteomics.

    PubMed

    Gomez, Yassel Ramos; Gallien, Sebastien; Huerta, Vivian; van Oostrum, Jan; Domon, Bruno; Gonzalez, Luis Javier

    2014-01-01

    Biological systems are not only controlled by the abundance of individual proteins, but also by the formation of complexes and the dynamics of protein-protein interactions. The identification of the components of protein complexes can be obtained by shotgun proteomics using affinity purification coupled to mass spectrometry. Such studies include the analyses of several samples and experimental controls in order to discriminate true specific interactions from unspecific interactions and contaminants. However, shotgun proteomics have limited quantification capabilities for low abundant proteins on large sample sets due to the undersampling and the stochastic precursor ion selection. In this context, targeted proteomics constitutes a powerful analytical tool to systematically detect and quantify peptides in multiple samples, for instance those obtained from affinity purification experiments. Hypothesis-driven strategies have mainly relied on the selected reaction monitoring (SRM) technique performed on triple quadrupole instruments, which enables highly selective and sensitive measurements of peptides, acting as surrogates of the pre-selected proteins, over a wide range of concentrations. More recently, novel quantitative methods based on high resolution instruments, such as the parallel reaction monitoring (PRM) technique implemented on the quadrupole-orbitrap instrument, have arisen and provided alternatives to perform quantitative analyses with enhanced selectivity.The application of targeted proteomics to protein-protein interaction experiments from plasma and other physiological fluid samples and the inclusion of parallel reaction monitoring (PRM), combined with other recent technology developments opens a vast area for clinical application of proteomics. It is anticipated that it will reveal valuable information about specific, individual, responses against drugs, exogenous proteins or pathogens.

  3. Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation.

    PubMed Central

    Konrad, Zvia; Eichler, Jerry

    2002-01-01

    Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg(2+)-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [(3)H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane. PMID:12069685

  4. A quantitative method to track protein translocation between intracellular compartments in real-time in live cells using weighted local variance image analysis.

    PubMed

    Calmettes, Guillaume; Weiss, James N

    2013-01-01

    The genetic expression of cloned fluorescent proteins coupled to time-lapse fluorescence microscopy has opened the door to the direct visualization of a wide range of molecular interactions in living cells. In particular, the dynamic translocation of proteins can now be explored in real time at the single-cell level. Here we propose a reliable, easy-to-implement, quantitative image processing method to assess protein translocation in living cells based on the computation of spatial variance maps of time-lapse images. The method is first illustrated and validated on simulated images of a fluorescently-labeled protein translocating from mitochondria to cytoplasm, and then applied to experimental data obtained with fluorescently-labeled hexokinase 2 in different cell types imaged by regular or confocal microscopy. The method was found to be robust with respect to cell morphology changes and mitochondrial dynamics (fusion, fission, movement) during the time-lapse imaging. Its ease of implementation should facilitate its application to a broad spectrum of time-lapse imaging studies.

  5. Drug ligand-induced activation of translocator protein (TSPO) stimulates steroid production by aged brown Norway rat Leydig cells.

    PubMed

    Chung, J Y; Chen, H; Midzak, A; Burnett, A L; Papadopoulos, V; Zirkin, B R

    2013-06-01

    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.

  6. Interaction graph mining for protein complexes using local clique merging.

    PubMed

    Li, Xiao-Li; Tan, Soon-Heng; Foo, Chuan-Sheng; Ng, See-Kiong

    2005-01-01

    While recent technological advances have made available large datasets of experimentally-detected pairwise protein-protein interactions, there is still a lack of experimentally-determined protein complex data. To make up for this lack of protein complex data, we explore the mining of existing protein interaction graphs for protein complexes. This paper proposes a novel graph mining algorithm to detect the dense neighborhoods (highly connected regions) in an interaction graph which may correspond to protein complexes. Our algorithm first locates local cliques for each graph vertex (protein) and then merge the detected local cliques according to their affinity to form maximal dense regions. We present experimental results with yeast protein interaction data to demonstrate the effectiveness of our proposed method. Compared with other existing techniques, our predicted complexes can match or overlap significantly better with the known protein complexes in the MIPS benchmark database. Novel protein complexes were also predicted to help biologists in their search for new protein complexes.

  7. Aminophospholipid translocation in erythrocytes: Evidence for the involvement of a specific transporter and an endofacial protein

    SciTech Connect

    Connor, J.; Schroit, A.J. )

    1990-01-09

    The transport of exogenously supplied fluorescent analogues of aminophospholipids from the outer to inner leaflet in red blood cells (RBC) is dependent upon the oxidative status of membrane sulfhydryls. Oxidation of a sulfhydryl on a 32-kDa membrane protein by pyridyldithioethylamine (PDA) has been previously shown to inhibit the transport of NBD-labeled phosphatidylserine (NBD-PS). In the present study, other sulfhydryl oxidants were examined to determine whether additional sites are involved in the transport process. The results show that diamide inhibits the transport of NBD-PS via a mechanism that is independent of the 32-kDa site. This is shown by the inability of diamide to block labeling of the 32-kDa sulfhydryl with {sup 125}I-labeled PDA and to protect against PDA-mediated inhibition of NBD-PS transport. Diamide-mediated inhibition, but not PDA-mediated inhibition, could be reversed by reduction with cysteamine or endogenous glutathione. Once established, the asymmetric distribution of NBD-PS could not be altered by oxidation of either site. These data indicate that a second site critical to the transport of aminophospholipids residues on the endofacial surface and suggest that the transport of aminophospholipids across the bilayer membrane of RBC depends on a coordinated and complementary process between a cytoskeletal component and the 32-kDa membrane polypeptide; both must be operative for transport to proceed.

  8. 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.

  9. 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

  10. Evidence that transporters associated with antigen processing translocate a major histocompatibility complex class I-binding peptide into the endoplasmic reticulum in an ATP-dependent manner.

    PubMed Central

    Androlewicz, M J; Anderson, K S; Cresswell, P

    1993-01-01

    We have investigated the role of the putative peptide transporters associated with antigen processing (TAP) by using a permeabilized-cell system. The main objective was to determine whether these molecules, which bear homology to the ATP-binding cassette family of transporters, translocate antigenic peptides across the endoplasmic reticulum membrane for assembly with major histocompatibility complex (MHC) class I molecules and beta 2-microglobulin light chain. The pore-forming toxin streptolysin O was used to generate permeabilized cells, and peptide translocation was determined by measuring the amount of added radiolabeled peptide bound to endogenous class I molecules. No radiolabeled peptide was associated with MHC class I glycoproteins from unpermeabilized cells. We found that efficient peptide binding to MHC class I molecules in permeabilized cells is both transporter dependent and ATP dependent. In antigen-processing mutant cells lacking a functional transporter, uptake occurs only through a less-efficient transporter and ATP-independent pathway. In addition, short peptides (8-10 amino acids) known to bind MHC class I molecules compete efficiently with a radiolabeled peptide for TAP-dependent translocation, whereas longer peptides and a peptide derived from an endoplasmic reticulum signal sequence do not compete efficiently. This result indicates that the optimal substrates for TAP possess the characteristics of MHC-binding peptides. Images Fig. 2 Fig. 3 Fig. 4 PMID:8415666

  11. BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells

    PubMed Central

    Kim, Nam-Hee; Yoshimaru, Tetsuro; Chen, Yi-An; Matsuo, Taisuke; Komatsu, Masato; Miyoshi, Yasuo; Tanaka, Eiji; Sasa, Mitsunori; Mizuguchi, Kenji; Katagiri, Toyomasa

    2015-01-01

    We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) binds Prohibitin 2 (PHB2) in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2)-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA) family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα). These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6) binding region(s) of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells. PMID:26052702

  12. Flutriciclamide (18F-GE180) PET: First-in-Human PET Study of Novel Third-Generation In Vivo Marker of Human Translocator Protein.

    PubMed

    Fan, Zhen; Calsolaro, Valeria; Atkinson, Rebecca A; Femminella, Grazia D; Waldman, Adam; Buckley, Christopher; Trigg, William; Brooks, David J; Hinz, Rainer; Edison, Paul

    2016-11-01

    Neuroinflammation is associated with neurodegenerative disease. PET radioligands targeting the 18-kDa translocator protein (TSPO) have been used as in vivo markers of neuroinflammation, but there is an urgent need for novel probes with improved signal-to-noise ratio. Flutriciclamide ((18)F-GE180) is a recently developed third-generation TSPO ligand. In this first study, we evaluated the optimum scan duration and kinetic modeling strategies for (18)F-GE180 PET in (older) healthy controls.

  13. 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.

  14. Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate-dependent manner.

    PubMed

    Wawra, Stephan; Bain, Judith; Durward, Elaine; de Bruijn, Irene; Minor, Kirsty L; Matena, Anja; Löbach, Lars; Whisson, Stephen C; Bayer, Peter; Porter, Andrew J; Birch, Paul R J; Secombes, Chris J; van West, Pieter

    2012-02-07

    The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate-modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host-microbe interactions.

  15. Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate–dependent manner

    PubMed Central

    Wawra, Stephan; Bain, Judith; Durward, Elaine; de Bruijn, Irene; Minor, Kirsty L.; Matena, Anja; Löbach, Lars; Whisson, Stephen C.; Bayer, Peter; Porter, Andrew J.; Birch, Paul R. J.; Secombes, Chris J.; van West, Pieter

    2012-01-01

    The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate–modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host–microbe interactions. PMID:22308362

  16. Thermodynamics of interfacial changes in a protein-protein complex.

    PubMed

    Das, Amit; Chakrabarti, Jaydeb; Ghosh, Mahua

    2014-03-04

    Recent experiments with biomacromolecular complexes suggest that structural modifications at the interfaces are vital for stability of the complexes and the functions of the biomacromolecules. Although several qualitative aspects about such interfaces are known from structural data, quantification of the interfacial changes is lacking. In this work, we study the thermodynamic changes at the interface in the complex between an enzyme, Nuclease A (NucA), and a specific inhibitor protein, NuiA. We calculate the conformational free energy and conformational entropy costs from histograms of the dihedral angles generated from all-atom molecular dynamics simulations on the complex and the free proteins. We extract the conformational thermodynamic parameters for changes in the tertiary structure of NuiA. We show that the binding is dominated by the interfacial changes, where the basic residues of NucA and acidic residues of NuiA are highly ordered and stabilized via strong electrostatic interactions. Our results correlate well with known information from structural studies. The tight interfacial structure is reflected in the significant changes in the structure and dynamics of the water molecules at the enzyme-inhibitor interface. The interfacial water molecules contribute significantly to the entropy loss for the overall complexation.

  17. Increased 5-hydroxymethylcytosine and Ten-eleven Translocation Protein Expression in Ultraviolet B-irradiated HaCaT Cells

    PubMed Central

    Wang, Dan; Huang, Jin-Hua; Zeng, Qing-Hai; Gu, Can; Ding, Shu; Lu, Jian-Yun; Chen, Jing; Yang, Sheng-Bo

    2017-01-01

    Background: DNA hydroxymethylation refers to a chemical modification process in which 5-methylcytosine (5mC) is catalyzed to 5- hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family proteins. Recent studies have revealed that aberrant TETs expression or 5hmC level may play important roles in the occurrence and development of various pathological and physiological processes including cancer and aging. This study aimed to explore the relation between aberrant DNA hydroxymethylation with skin photoaging and to investigate the levels of TETs, 5mC, and 5hmC expression 24 h after 40 mJ/cm2 and 80 mJ/cm2 doses of ultraviolet B (UVB) irradiation to HaCaT cells. Methods: To explore whether aberrant DNA hydroxymethylation is also related to skin photoaging, 40 mJ/cm2 and 80 mJ/cm2 doses of UVB were chosen to treat keratinocytes (HaCaT cells). After 24 h of UVB irradiation, 5mC and 5hmC levels were determined by immunohistochemistry (IHC) and immunofluorescence (IF), and at the same time, the expression levels of matrix metalloproteinase 1 (MMP-1) and TETs were assessed by reverse transcription-polymerase chain reaction or Western blot analysis. Results: After 40 mJ/cm2 and 80 mJ/cm2 doses of UVB exposure, both IHC and IF results showed that 5hmC levels increased significantly, while the 5mC levels did not exhibit significant changes in HaCaT cells, compared with HaCat cells without UVB exposure. Moreover, compared with HaCat cells without UVB exposure, the levels of TET1, TET2, and TET3 mRNA and protein expression were significantly upregulated (mRNA: P = 0.0022 and 0.0043 for TET1; all P < 0.0001 for TET2; all P = 0.0006 for TET3; protein: P = 0.0012 and 0.0006 for TET1; all P = 0.0022 for TET2; and all P = 0.0002 for TET3), and the levels of MMP-1 mRNA expression increased dose dependently in 40 mJ/cm2 and 80 mJ/cm2 UVB-irradiated groups. Conclusion: UVB radiation could cause increased 5hmC and TET expression, which might become a novel biomarker in UVB

  18. 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.

  19. Epigenetic Silencing of the Human 18 kDa Translocator Protein in a T Cell Leukemia Cell Line.

    PubMed

    Middleton, Ryan J; Kam, Winnie Wai-Ying; Liu, Guo-Jun; Banati, Richard B

    2017-02-01

    The mitochondrial membrane 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is constitutively expressed in most organs, most abundantly in hormonal tissue and cells of mononuclear phagocyte lineage, while in the brain, TSPO expression is induced in the wake of injury, inflammation, and neurodegeneration. Increased TSPO expression is also prominent in several cancerous tissues where it appears to correlate with the degree of malignancy. Currently, TSPO is thus actively investigated as a generic biomarker for disease activity and a therapeutic target for a wide range of diseases. In this study, we report a Jurkat human T cell leukemia cell line that has only trace expression of TSPO mRNA. Through the use of bisulphite genomic sequencing, we show that the Jurkat TSPO promoter is highly methylated except for CpG sites that are adjacent to the transcription start site. Control measurements in HEK-293, HeLa, and U87-MG cells with high TSPO mRNA expression showed low levels of TSPO promoter methylation. Demethylation with 5-aza-2'-deoxycytidine (5-aza-dC) caused a dose-dependent increase in TSPO mRNA with a corresponding demethylation of the TSPO promoter in Jurkat cells. Treating HeLa and U87-MG cells with 5-aza-dC caused no change in the level of TSPO mRNA. These observations confirm the epigenetic regulation of TSPO and suggest it to be a more common mechanism by which the differential expression of TSPO in various cell types and in health and disease may be explained.

  20. Apoptosis induction by erucylphosphohomocholine via the 18 kDa mitochondrial translocator protein: implications for cancer treatment.

    PubMed

    Veenman, Leo; Gavish, Moshe; Kugler, Wilfried

    2014-05-01

    Many types of cancer, for example glioblastoma, show resistance against current anti-cancer treatments. One reason is that they are not capable to effectively activate their intracellular cell death pathways. Novel treatments designed to overcome these deficiencies in cancer cells present promising concepts to eradicate chemotherapy-resistant cancer cells. One of these approaches includes the membrane seeking compound erucylphosphohomocholine (ErPC3) which is part of the latest generation of alkylphospholipid analogs developed over the last two-and-a-half decades. ErPC3 exerts potent antineoplastic effects in animal models and against established cancer cell lines including, for example, glioblastoma and different types of leukemia, while sparing their normal counterparts. Starting with a historical survey, we report here on the anticancer activity of ErPC3 and on ErPC3's established mechanisms of action. We cover the current knowledge on the induction of mitochondrial apoptosis by ErPC3, including its interaction with the 18 kDa translocator protein (TSPO). In addition we discuss other signaling pathways modulated by ErPC3. Interaction with the TSPO leads to activation of the mitochondrial apoptosis cascade. This includes cardiolipin oxidation at mitochondrial levels, collapse of the mitochondrial membrane potential, and release of cytochrome c, the initiating steps of the mitochondrial apoptosis cascade. Other pathways modulated by ErPC3 include different kinases for the PI3K/Akt/mTOR and the MAP kinase pathways. Furthermore, ErPC3's cytotoxic actions may include its effects on phosphatidylcholine synthesis to inhibit the endoplasmic reticulum enzyme CTP:phosphocholine cytidyltransferase. These basic research data hopefully will lead to effective approaches toward exploitation of ErPC3 for the treatment of cancer.

  1. Accumulation and transformation of inorganic and organic arsenic in rice and role of thiol-complexation to restrict their translocation to shoot

    PubMed Central

    Mishra, Seema; Mattusch, Jürgen; Wennrich, Rainer

    2017-01-01

    Environmental contamination of arsenic (As) and its accumulation in rice (Oryza sativa L.) is of serious human health concern. In planta speciation of As is an important tool to understand As metabolism in plants. In the present study, we investigated root to shoot As translocation and speciation in rice exposed to inorganic and methylated As. Arsenate (AsV) and methylarsonate (MAV) were efficiently reduced to arsenite (AsIII) and MAIII, respectively in rice root and shoot but no trivalent form of dimethylarsinate (DMAV) was detected. Further, up to 48 and 83% of root As in AsV and MAV exposed plants, respectively were complexed with various thiols showing up to 20 and 16 As species, respectively. Several mixed As- and MA-complexes with hydroxymethyl-phytochelatin, DesGly-phytochelatin, hydroxymethyl-GSH and cysteine were identified in rice. Despite high complexation in roots, more As was translocated to shoots in MAV exposed plants than AsV, with shoot/root As transfer factor being in order DMAV > MAV > AsV. Moreover, in shoots 78% MAIII and 71% AsIII were present as weakly bound species which is alarming, as MAIII has been found to be more cytotoxic than AsIII for human and it could also be an important factor inducing straighthead (spikelet sterility disorder) in rice. PMID:28094280

  2. 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.

  3. 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.

  4. Identification of novel protein-protein interactions at the cytosolic surface of the Sec63 complex in the yeast ER membrane.

    PubMed

    Willer, Martin; Jermy, Andrew J; Young, Barry P; Stirling, Colin J

    2003-01-30

    Precursors of secretory proteins are targeted to the membrane of the endoplasmic reticulum by specific protein complexes that recognize their signal sequence. All eukaryotic cells investigated so far have been found to possess the signal recognition particle (SRP) that targets the majority of precursors to the translocation machinery. In Saccharomyces cerevisiae a number of proteins are translocated independently of SRP. These precursors rely on a different signal sequence-binding complex, which includes Sec62p, Sec63p, Sec71p and Sec72p. Identifying interactions between individual components of this tetrameric protein complex is important in the understanding of its function. We demonstrate a specific interaction between the only two essential proteins in this complex, Sec62p and Sec63p. Second, we show evidence of homodimerization of Sec72p molecules and further identify the YLR301w gene product as a novel in vivo interacting partner of Sec72p. Finally, we determine the authentic N-terminus of Sec62p and describe interacting subdomains of both Sec62p and Sec63p.

  5. CmRBP50 Protein Phosphorylation Is Essential for Assembly of a Stable Phloem-mobile High-affinity Ribonucleoprotein Complex*

    PubMed Central

    Li, Pingfang; Ham, Byung-Kook; Lucas, William J.

    2011-01-01

    RNA-binding proteins (RBPs) form ribonucleoprotein (RNP) complexes that play crucial roles in RNA processing for gene regulation. The angiosperm sieve tube system contains a unique population of transcripts, some of which function as long-distance signaling agents involved in regulating organ development. These phloem-mobile mRNAs are translocated as RNP complexes. One such complex is based on a phloem RBP named Cucurbita maxima RNA-binding protein 50 (CmRBP50), a member of the polypyrimidine track binding protein family. The core of this RNP complex contains six additional phloem proteins. Here, requirements for assembly of this CmRBP50 RNP complex are reported. Phosphorylation sites on CmRBP50 were mapped, and then coimmunoprecipitation and protein overlay studies established that the phosphoserine residues, located at the C terminus of CmRBP50, are critical for RNP complex assembly. In vitro pull-down experiments revealed that three phloem proteins, C. maxima phloem protein 16, C. maxima GTP-binding protein, and C. maxima phosphoinositide-specific phospholipase-like protein, bind directly with CmRBP50. This interaction required CmRBP50 phosphorylation. Gel mobility-shift assays demonstrated that assembly of the CmRBP50-based protein complex results in a system having enhanced binding affinity for phloem-mobile mRNAs carrying polypyrimidine track binding motifs. This property would be essential for effective long-distance translocation of bound mRNA to the target tissues. PMID:21572046

  6. 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.

  7. 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 ...

  8. 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

  9. 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

  10. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    PubMed Central

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-01-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes. PMID:8611143

  11. Cephaloridine induces translocation of protein kinase C delta into mitochondria and enhances mitochondrial generation of free radicals in the kidney cortex of rats causing renal dysfunction.

    PubMed

    Kohda, Yuka; Gemba, Munekazu

    2005-05-01

    We have previously reported that the enhancement of free radical generation in mitochondria isolated from the kidney cortex of rats exposed to cephaloridine (CER) is probably mediated by the activation of protein kinase C (PKC). We examined which isoenzymes of PKC might be involved in the development of nephrotoxicity induced by CER in rats. The CER-induced renal dysfunction observed 24 h after its injection was prevented by a potent antioxidant DPPD and well-known PKC inhibitors like H-7 and rottlerin. At 1.5 and 3.5 h after the CER injection, the free radical generation was increased markedly and this was associated with translocation of PKCdelta into the mitochondria of renal cortex tissue. Pretreatment of rats with H-7, a PKC inhibitor, significantly inhibited the CER-derived increase in mitochondrial generation of free radicals, suggesting that H-7 probably gets into the mitochondria and inhibits the activity of translocated PKC within the mitochondria. It was also shown that pretreatment of rats with rottlerin, a specific inhibitor of PKCdelta, suppressed the early translocation of PKCdelta into mitochondria and inhibited the CER-derived development of renal dysfunction. These results suggest that the CER-derived early translocation of PKCdelta into mitochondria probably leads to the enhanced production of free radicals through the mitochondrial respiratory chain during the development of the nephrotoxicity caused by CER. Understanding the role of PKCdelta in mitochondria may provide an important clue to the molecular mechanisms of mitochondrial production of reactive oxygen species and the free radical-induced renal failure in rats treated with CER.

  12. 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

  13. Analysis of DNA-protein complexes induced by chemical carcinogens

    SciTech Connect

    Costa, M. )

    1990-11-01

    DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.

  14. Increase of calcium levels in epithelial cells induces translocation of calcium-binding proteins migration inhibitory factor-related protein 8 (MRP8) and MRP14 to keratin intermediate filaments.

    PubMed

    Goebeler, M; Roth, J; van den Bos, C; Ader, G; Sorg, C

    1995-07-15

    Migration inhibitory factor-related protein 8 (MRP8) and MRP14, two S-100-like Ca(2+)-binding proteins, have been described in cells of the epithelial lineage where they are either expressed constitutively (e.g. by mucosal squamous epithelium) or induced during disease (e.g. in keratinocytes during the course of psoriasis). Their biological function, however, is not yet clear. Recent studies have provided evidence that S-100-like proteins may interact with cytoskeletal components; we have therefore studied the biochemical properties and subcellular distribution of MRP8 and MRP14 in epithelial cells. TR146 human squamous carcinoma cells, which were found to express MRP8 and MRP14 in Northern and Western blot studies, were chosen for analysis. Cross-linking experiments using bis(sulphosuccinimidyl)suberate followed by SDS/PAGE and Western blot analysis revealed formation of heteromeric MRP8-MRP14 complexes. On subjecting TR146 cell lysates to two-dimensional gel electrophoresis and Western blotting, four distinct MRP14 isoforms could be identified resembling those described earlier in macrophages. A differential centrifugation technique revealed a Ca(2+)-dependent translocation of MRP8-MRP14 from the cytoplasm to the membrane and the Nonidet P40-insoluble cytoskeletal fraction. Double-label immunofluorescence microscopy of Ca2+ ionophore A23187-stimulated TR146 cells and cytochalasin B and demecolcine cytoskeleton disruption studies identified these structures as keratin intermediate filaments. Ca(2+)-dependent binding of MRP8-MRP14 to keratin filaments was additionally confirmed by an in vitro binding assay. In conclusion, our data suggest that MRP8 and MRP14 may be involved in Ca(2+)-dependent reorganization of cytoskeletal filaments in epithelial cells, which could be of importance for events associated with differentiation and inflammatory activation.

  15. 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.

  16. An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism

    PubMed Central

    Lee, Hyeon-Cheol; Portnoff, Alyse D.; Rocco, Mark A.; DeLisa, Matthew P.

    2014-01-01

    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. PMID:25531212

  17. Elongation factor G initiates translocation through a power stroke.

    PubMed

    Chen, Chunlai; Cui, Xiaonan; Beausang, John F; Zhang, Haibo; Farrell, Ian; Cooperman, Barry S; Goldman, Yale E

    2016-07-05

    During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G's GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

  18. 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

  19. Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage.

    PubMed

    Rolletschek, Hardy; Nguyen, Thuy H; Häusler, Rainer E; Rutten, Twan; Göbel, Cornelia; Feussner, Ivo; Radchuk, Ruslana; Tewes, Annegret; Claus, Bernhard; Klukas, Christian; Linemann, Ute; Weber, Hans; Wobus, Ulrich; Borisjuk, Ljudmilla

    2007-08-01

    The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.

  20. Rust Secreted Protein Ps87 Is Conserved in Diverse Fungal Pathogens and Contains a RXLR-like Motif Sufficient for Translocation into Plant Cells

    PubMed Central

    Gu, Biao; Kale, Shiv D.; Wang, Qinhu; Wang, Dinghe; Pan, Qiaona; Cao, Hua; Meng, Yuling; Kang, Zhensheng; Tyler, Brett M.; Shan, Weixing

    2011-01-01

    Background Effector proteins of biotrophic plant pathogenic fungi and oomycetes are delivered into host cells and play important roles in both disease development and disease resistance response. How obligate fungal pathogen effectors enter host cells is poorly understood. The Ps87 gene of Puccinia striiformis encodes a protein that is conserved in diverse fungal pathogens. Ps87 homologs from a clade containing rust fungi are predicted to be secreted. The aim of this study is to test whether Ps87 may act as an effector during Puccinia striiformis infection. Methodology/Principal Findings Yeast signal sequence trap assay showed that the rust protein Ps87 could be secreted from yeast cells, but a homolog from Magnaporthe oryzae that was not predicted to be secreted, could not. Cell re-entry and protein uptake assays showed that a region of Ps87 containing a conserved RXLR-like motif [K/R]RLTG was confirmed to be capable of delivering oomycete effector Avr1b into soybean leaf cells and carrying GFP into soybean root cells. Mutations in the Ps87 motif (KRLTG) abolished the protein translocation ability. Conclusions/Significance The results suggest that Ps87 and its secreted homologs could utilize similar protein translocation machinery as those of oomycete and other fungal pathogens. Ps87 did not show direct suppression activity on plant defense responses. These results suggest Ps87 may represent an “emerging effector” that has recently acquired the ability to enter plant cells but has not yet acquired the ability to alter host physiology. PMID:22076138

  1. In vitro translocation experiments with RxLR-reporter fusion proteins of Avr1b from Phytophthora sojae and AVR3a from Phytophthora infestans fail to demonstrate specific autonomous uptake in plant and animal cells.

    PubMed

    Wawra, Stephan; Djamei, Armin; Albert, Isabell; Nürnberger, Thorsten; Kahmann, Regine; van West, Pieter

    2013-05-01

    Plant-pathogenic oomycetes have a large set of secreted effectors that can be translocated into their host cells during infection. One group of these effectors are the RxLR effectors for which it has been shown, in a few cases, that the RxLR motif is important for their translocation. It has been suggested that the RxLR-leader sequences alone are enough to translocate the respective effectors into eukaryotic cells through binding to surface-exposed phosphoinositol-3-phosphate. These conclusions were primary based on translocation experiments conducted with recombinant fusion proteins whereby the RxLR leader of RxLR effectors (i.e., Avr1b from Phytophthora sojae) were fused to the green fluorescent protein reporter-protein. However, we failed to observe specific cellular uptake for a comparable fusion protein where the RxLR leader of the P. infestans AVR3a was fused to monomeric red fluorescent protein. Therefore, we reexamined the ability of the reported P. sojae AVR1b RxLR leader to enter eukaryotic cells. Different relevant experiments were performed in three independent laboratories, using fluorescent reporter fusion constructs of AVR3a and Avr1b proteins in a side-by-side comparative study on plant tissue and human and animal cells. We report that we were unable to obtain conclusive evidence for specific RxLR-mediated translocation.

  2. 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.

  3. 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.

  4. Caffeine prevents kidney stone formation by translocation of apical surface annexin A1 crystal-binding protein into cytoplasm: In vitro evidence

    PubMed Central

    Peerapen, Paleerath; Thongboonkerd, Visith

    2016-01-01

    Recent large 3 cohorts have shown that caffeinated beverage consumption was associated with lower risk of kidney stone disease. However, its protective mechanisms remained unknown and had not been previously investigated. We thus evaluated protective effects of caffeine (1 μM–10 mM) on calcium oxalate monohydrate (COM) kidney stone formation, using crystallization, crystal growth, cell-crystal adhesion, Western blotting, and immunofluorescence assays. The results showed that caffeine reduced crystal number but, on the other hand, increased crystal size, resulting in unchanged crystal mass, consistent with crystal growth that was not affected by caffeine. However, caffeine significantly decreased crystal-binding capacity of MDCK renal tubular cells in a dose-dependent manner. Western blotting and immunofluorescence study of COM crystal-binding proteins revealed significantly decreased level of annexin A1 on apical surface and its translocation into cytoplasm of the caffeine-treated cells, but no significant changes in other COM crystal-binding proteins (annexin A2, α-enolase, HSP70, and HSP90) were observed. Moreover, caffeine decreased intracellular [Ca2+] but increased [Ca2+] secretory index. Taken together, our findings showed an in vitro evidence of the protective mechanism of caffeine against kidney stone formation via translocation of annexin A1 from apical surface into cytoplasm to reduce the crystal-binding capacity of renal tubular epithelial cells. PMID:27924845

  5. Construction of ontology augmented networks for protein complex prediction.

    PubMed

    Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian

    2013-01-01

    Protein complexes are of great importance in understanding the principles of cellular organization and function. The increase in available protein-protein interaction data, gene ontology and other resources make it possible to develop computational methods for protein complex prediction. Most existing methods focus mainly on the topological structure of protein-protein interaction networks, and largely ignore the gene ontology annotation information. In this article, we constructed ontology augmented networks with protein-protein interaction data and gene ontology, which effectively unified the topological structure of protein-protein interaction networks and the similarity of gene ontology annotations into unified distance measures. After constructing ontology augmented networks, a novel method (clustering based on ontology augmented networks) was proposed to predict protein complexes, which was capable of taking into account the topological structure of the protein-protein interaction network, as well as the similarity of gene ontology annotations. Our method was applied to two different yeast protein-protein interaction datasets and predicted many well-known complexes. The experimental results showed that (i) ontology augmented networks and the unified distance measure can effectively combine the structure closeness and gene ontology annotation similarity; (ii) our method is valuable in predicting protein complexes and has higher F1 and accuracy compared to other competing methods.

  6. Separation of membrane protein complexes by native LDS-PAGE.

    PubMed

    Arnold, Janine; Shapiguzov, Alexey; Fucile, Geoffrey; Rochaix, Jean-David; Goldschmidt-Clermont, Michel; Eichacker, Lutz Andreas

    2014-01-01

    Gel electrophoresis has become one of the most important methods for the analysis of proteins and protein complexes in a molecular weight range of 1-10(7) kDa. The separation of membrane protein complexes remained challenging to standardize until the demonstration of Blue Native PAGE in 1991 [1] and Clear Native PAGE in 1994 [2]. We present a robust protocol for high-resolution separation of photosynthetic complexes from Arabidopsis thaliana using lithium dodecyl sulfate as anion in a modified Blue Native PAGE (LDS-PAGE). Here, non-covalently bound chlorophyll is used as a sensitive probe to characterize the assembly/biogenesis of the pigment-protein complexes essential for photosynthesis. The high fluorescence yield recorded from chlorophyll-binding protein complexes can also be used to establish the separation of native protein complexes as an electrophoretic standard.

  7. Structural insights into ribosome translocation

    PubMed Central

    Ling, Clarence

    2016-01-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF‐G) in bacteria and elongation factor 2 (EF‐2) in eukaryotes. Recent structural and single‐molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the ‘head’ domain of small ribosomal subunit undergoes forward‐ and back‐swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF‐G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF‐G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620–636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  8. 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

  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. Structure, dynamics, assembly, and evolution of protein complexes.

    PubMed

    Marsh, Joseph A; Teichmann, Sarah A

    2015-01-01

    The assembly of individual proteins into functional complexes is fundamental to nearly all biological processes. In recent decades, many thousands of homomeric and heteromeric protein complex structures have been determined, greatly improving our understanding of the fundamental principles that control symmetric and asymmetric quaternary structure organization. Furthermore, our conception of protein complexes has moved beyond static representations to include dynamic aspects of quaternary structure, including conformational changes upon binding, multistep ordered assembly pathways, and structural fluctuations occurring within fully assembled complexes. Finally, major advances have been made in our understanding of protein complex evolution, both in reconstructing evolutionary histories of specific complexes and in elucidating general mechanisms that explain how quaternary structure tends to evolve. The evolution of quaternary structure occurs via changes in self-assembly state or through the gain or loss of protein subunits, and these processes can be driven by both adaptive and nonadaptive influences.

  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. Alphavirus M1 induces apoptosis of malignant glioma cells via downregulation and nucleolar translocation of p21WAF1/CIP1 protein.

    PubMed

    Hu, Jun; Cai, Xiao-Feng; Yan, Guangmei

    2009-10-15

    Alphavirus, a genus of arthropod-borne togavirus, is well-known for its pro-apoptotic capability. However, the underlying mechanism remains to be further clarified. Here, we have identified that M1, an alphavirus isolated in 1960s, targeted C6 malignant glioma cells for apoptosis. Flow cytometry analysis showed that more cells enter S-phase post M1 infection, and subsequently undergo a classic apoptosis. To elucidate the mechanism of S-phase arrest and its relationship to apoptosis, we tested the expression of several critical cell cycle regulatory proteins and found elevated phosphorylation of cyclin-dependent kinase 2 (CDK2), decreased expression of cyclin A and proliferating cell nuclear antigen (PCNA). Notably, the protein level of p21(WAF1/CIP1) was downregulated earliest and most effectively among all tested changes of cell cycle regulators, though its mRNA level was strongly upregulated. To evaluate the role of p21(WAF1/CIP1) in S-phase accumulation and subsequent apoptosis, we confirmed that exogenous p21(WAF1/CIP1) overexpression or treatment with roscovitine (a selective chemical inhibitor of CDK2) efficiently protected against apoptosis with a reduced S-phase accumulation. Thus, it is indicated that the downregulation of p21(WAF1/CIP1) mediated C6 apoptosis via overactivation of CDK2. In addition, confocal microscopy showed that p21(WAF1/CIP1) totally translocated to nucleolus during M1-induced C6 apoptosis. Altogether, downregulation and nucleolar translocation of the p21(WAF1/CIP1) protein played an active role in M1-induced C6 apoptosis.

  14. Hysteresis as a Marker for Complex, Overlapping Landscapes in Proteins

    PubMed Central

    Andrews, Benjamin T.; Capraro, Dominique T.; Sulkowska, Joanna I.; Onuchic, José N.; Jennings, Patricia A.

    2013-01-01

    Topologically complex proteins fold by multiple routes as a result of hard-to-fold regions of the proteins. Oftentimes these regions are introduced into the protein scaffold for function and increase frustration in the otherwise smooth-funneled landscape. Interestingly, while functional regions add complexity to folding landscapes, they may also contribute to a unique behavior referred to as hysteresis. While hysteresis is predicted to be rare, it is observed in various proteins, including proteins containing a unique peptide cyclization to form a fluorescent chromophore as well as proteins containing a knotted topology in their native fold. Here, hysteresis is demonstrated to be a consequence of the decoupling of unfolding events from the isomerization or hula-twist of a chromophore in one protein and the untying of the knot in a second protein system. The question now is- can hysteresis be a marker for the interplay of landscapes where complex folding and functional regions overlap? PMID:23525263

  15. A proteomic strategy for global analysis of plant protein complexes.

    PubMed

    Aryal, Uma K; Xiong, Yi; McBride, Zachary; Kihara, Daisuke; Xie, Jun; Hall, Mark C; Szymanski, Daniel B

    2014-10-01

    Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

  16. 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.

  17. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

    PubMed

    Krogan, Nevan J; Cagney, Gerard; Yu, Haiyuan; Zhong, Gouqing; Guo, Xinghua; Ignatchenko, Alexandr; Li, Joyce; Pu, Shuye; Datta, Nira; Tikuisis, Aaron P; Punna, Thanuja; Peregrín-Alvarez, José M; Shales, Michael; Zhang, Xin; Davey, Michael; Robinson, Mark D; Paccanaro, Alberto; Bray, James E; Sheung, Anthony; Beattie, Bryan; Richards, Dawn P; Canadien, Veronica; Lalev, Atanas; Mena, Frank; Wong, Peter; Starostine, Andrei; Canete, Myra M; Vlasblom, James; Wu, Samuel; Orsi, Chris; Collins, Sean R; Chandran, Shamanta; Haw, Robin; Rilstone, Jennifer J; Gandi, Kiran; Thompson, Natalie J; Musso, Gabe; St Onge, Peter; Ghanny, Shaun; Lam, Mandy H Y; Butland, Gareth; Altaf-Ul, Amin M; Kanaya, Shigehiko; Shilatifard, Ali; O'Shea, Erin; Weissman, Jonathan S; Ingles, C James; Hughes, Timothy R; Parkinson, John; Gerstein, Mark; Wodak, Shoshana J; Emili, Andrew; Greenblatt, Jack F

    2006-03-30

    Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

  18. 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.

  19. 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.

  20. 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

  1. RNA binding protein Musashi-1 directly targets Msi2 and Erh during early testis germ cell development and interacts with IPO5 upon translocation to the nucleus.

    PubMed

    Sutherland, Jessie M; Sobinoff, Alexander P; Fraser, Barbara A; Redgrove, Kate A; Davidson, Tara-Lynne; Siddall, Nicole A; Koopman, Peter; Hime, Gary R; McLaughlin, Eileen A

    2015-07-01

    Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the process of gamete development, male germ cells experience extended periods of inactive transcription despite requirements for continued growth and differentiation. Spermatogenesis therefore provides an ideal model to study the effects of posttranscriptional control on gene regulation. During spermatogenesis posttranscriptional regulation is orchestrated by abundantly expressed RNA-binding proteins. One such group of RNA-binding proteins is the Musashi family, previously identified as a critical regulator of testis germ cell development and meiosis in Drosophila and also shown to be vital to sperm development and reproductive potential in the mouse. We focus in depth on the role and function of the vertebrate Musashi ortholog Musashi-1 (MSI1). Through detailed expression studies and utilizing our novel transgenic Msi1 testis-specific overexpression model, we have identified 2 unique RNA-binding targets of MSI1 in spermatogonia, Msi2 and Erh, and have demonstrated a role for MSI1 in translational regulation. We have also provided evidence to suggest that nuclear import protein, IPO5, facilitates the nuclear translocation of MSI1 to the transcriptionally silenced XY chromatin domain in meiotic pachytene spermatocytes, resulting in the release of MSI1 RNA-binding targets. This firmly establishes MSI1 as a master regulator of posttranscriptional control during early spermatogenesis and highlights the significance of the subcellular localization of RNA binding proteins in relation to their function.

  2. Native Mass Spectrometry of Photosynthetic Pigment-Protein Complexes

    PubMed Central

    Zhang, Hao; Cui, Weidong; Gross, Michael L.; Blankenship, Robert E.

    2013-01-01

    Native mass spectrometry, or as is sometimes called “native electrospray (ESI)” allows proteins in their native or near-native protein in solution to be introduced into gas phase and interrogated by MS. This approach is now a powerful tool to investigate protein complexes. This article reviews the background of native MS of protein complexes and describes its strengths, taking photosynthetic pigment-protein complexes as examples. Native MS can be utilized in combination with other MS-based approaches to obtain complementary information to that provided by tools such as X-ray crystallography and NMR spectroscopy to understand the structure-function relationships of protein complexes. When additional information beyond that provided by native MS is required, other MS-based strategies can be successfully applied to augment the results of native MS. PMID:23337874

  3. Advances in protein complex analysis using mass spectrometry

    PubMed Central

    Gingras, Anne-Claude; Aebersold, Ruedi; Raught, Brian

    2005-01-01

    Proteins often function as components of larger complexes to perform a specific function, and formation of these complexes may be regulated. For example, intracellular signalling events often require transient and/or regulated protein–protein interactions for propagation, and protein binding to a specific DNA sequence, RNA molecule or metabolite is often regulated to modulate a particular cellular function. Thus, characterizing protein complexes can offer important insights into protein function. This review describes recent important advances in mass spectrometry (MS)-based techniques for the analysis of protein complexes. Following brief descriptions of how proteins are identified using MS, and general protein complex purification approaches, we address two of the most important issues in these types of studies: specificity and background protein contaminants. Two basic strategies for increasing specificity and decreasing background are presented: whereas (1) tandem affinity purification (TAP) of tagged proteins of interest can dramatically improve the signal-to-noise ratio via the generation of cleaner samples, (2) stable isotopic labelling of proteins may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques. Examples, as well as advantages and disadvantages of each approach, are presented. PMID:15611014

  4. Luminogenic "clickable" lanthanide complexes for protein labeling.

    PubMed

    Candelon, Nicolas; Hădade, Niculina D; Matache, Mihaela; Canet, Jean-Louis; Cisnetti, Federico; Funeriu, Daniel P; Nauton, Lionel; Gautier, Arnaud

    2013-10-14

    Development of lanthanide-based luminescent "switch-on" systems via azide-alkyne [3+2] cycloaddition is described. We used these for non-specific protein labeling and as tags for specific and selective activity-based protein labeling.

  5. 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

  6. Multiscale Model for the Assembly Kinetics of Protein Complexes.

    PubMed

    Xie, Zhong-Ru; Chen, Jiawen; Wu, Yinghao

    2016-02-04

    The assembly of proteins into high-order complexes is a general mechanism for these biomolecules to implement their versatile functions in cells. Natural evolution has developed various assembling pathways for specific protein complexes to maintain their stability and proper activities. Previous studies have provided numerous examples of the misassembly of protein complexes leading to severe biological consequences. Although the research focusing on protein complexes has started to move beyond the static representation of quaternary structures to the dynamic aspect of their assembly, the current understanding of the assembly mechanism of protein complexes is still largely limited. To tackle this problem, we developed a new multiscale modeling framework. This framework combines a lower-resolution rigid-body-based simulation with a higher-resolution Cα-based simulation method so that protein complexes can be assembled with both structural details and computational efficiency. We applied this model to a homotrimer and a heterotetramer as simple test systems. Consistent with experimental observations, our simulations indicated very different kinetics between protein oligomerization and dimerization. The formation of protein oligomers is a multistep process that is much slower than dimerization but thermodynamically more stable. Moreover, we showed that even the same protein quaternary structure can have very diverse assembly pathways under different binding constants between subunits, which is important for regulating the functions of protein complexes. Finally, we revealed that the binding between subunits in a complex can be synergistically strengthened during assembly without considering allosteric regulation or conformational changes. Therefore, our model provides a useful tool to understand the general principles of protein complex assembly.

  7. Carbachol- and elevated Ca(2+)-induced translocation of functionally active protein kinase C to the brush border of rabbit ileal Na+ absorbing cells.

    PubMed Central

    Cohen, M E; Wesolek, J; McCullen, J; Rys-Sikora, K; Pandol, S; Rood, R P; Sharp, G W; Donowitz, M

    1991-01-01

    Protein kinase C is involved in mediating the effects of elevated Ca2+ in ileal villus Na+ absorbing cells to inhibit NaCl absorption. The present studies were undertaken to understand the mechanism by which this occurs. The effects of carbachol and the calcium ionophore A23187, agents which elevate intracellular Ca2+ and inhibit NaCl absorption in ileal villus cells, were studied. Carbachol treatment of villus cells caused a rapid decrease in protein kinase C activity in cytosol, with an accompanying increase in microvillus membrane C kinase. Exposure of the villus cells to calcium ionophore also caused a quantitatively similar decrease in cytosol C kinase and increase in C kinase activity in the microvillus membrane. This increase caused by carbachol and Ca2+ ionophore was specific for the microvillus membrane. In fact, 30 s and 10 min after exposure of the cells to carbachol, basolateral membrane protein kinase C decreased, in a time-dependent manner; whereas 10 min of Ca2+ ionophore exposure did not alter basolateral C kinase. Exposure of villus cells to Ca2+ ionophore or carbachol caused similar increases in microvillus membrane diacylglycerol content. As judged by the ability to inhibit Na+/H+ exchange measured in ileal villus cell brush border membrane vesicles, the protein kinase C which translocated to the microvillus membrane was functionally significant. Inhibition of Na+/H+ exchange required ATP and was reversed by the protein kinase C antagonist H-7. In conclusion, the effect of carbachol and Ca2+ ionophore in regulation of ileal NaCl absorption is associated with an increase in microvillus membrane diacylglycerol content and functionally active protein kinase C. The effects of both carbachol and Ca2+ ionophore are different on brush border and basolateral membrane distribution of protein kinase C. Images PMID:1885773

  8. Protein-protein interactions in complex cosolvent solutions.

    PubMed

    Javid, Nadeem; Vogtt, Karsten; Krywka, Chris; Tolan, Metin; Winter, Roland

    2007-04-02

    The effects of various kosmotropic and chaotropic cosolvents and salts on the intermolecular interaction potential of positively charged lysozyme is evaluated at varying protein concentrations by using synchrotron small-angle X-ray scattering in combination with liquid-state theoretical approaches. The experimentally derived static structure factors S(Q) obtained without and with added cosolvents and salts are analysed with a statistical mechanical model based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) potential, which accounts for repulsive and attractive interactions between the protein molecules. Different cosolvents and salts influence the interactions between protein molecules differently as a result of changes in the hydration level or solvation, in charge screening, specific adsorption of the additives at the protein surface, or increased hydrophobic interactions. Intermolecular interaction effects are significant above protein concentrations of 1 wt %, and with increasing protein concentration, the repulsive nature of the intermolecular pair potential V(r) increases markedly. Kosmotropic cosolvents like glycerol and sucrose exhibit strong concentration-dependent effects on the interaction potential, leading to an increase of repulsive forces between the protein molecules at low to medium high osmolyte concentrations. Addition of trifluoroethanol exhibits a multiphasic effect on V(r) when changing its concentration. Salts like sodium chloride and potassium sulfate exhibit strong concentration-dependent changes of the interaction potential due to charge screening of the positively charged protein molecules. Guanidinium chloride (GdmCl) at low concentrations exhibits a similar charge-screening effect, resulting in increased attractive interactions between the protein molecules. At higher GdmCl concentrations, V(r) becomes more repulsive in nature due to the presence of high concentrations of Gdm(+) ions binding to the protein molecules. Our findings also

  9. The cancer-associated K351N mutation affects the ubiquitination and the translocation to mitochondria of p53 protein.

    PubMed

    Muscolini, Michela; Montagni, Elisa; Palermo, Vanessa; Di Agostino, Silvia; Gu, Wei; Abdelmoula-Souissi, Salma; Mazzoni, Cristina; Blandino, Giovanni; Tuosto, Loretta

    2011-11-18

    Stress-induced monoubiquitination of p53 is a crucial event for the nuclear-cytoplasm-mitochondria trafficking and transcription-independent pro-apoptotic functions of p53. Although an intact ubiquitination pathway and a functional nuclear export sequence are required for p53 nuclear export, the role of specific residues within this region in regulating both processes remains largely unknown. Here we characterize the mechanisms accounting for the nuclear accumulation of a new point mutation (Lys-351 to Asn) in the nuclear export sequence of p53 identified in a cisplatin-resistant ovarian carcinoma cell line (A2780 CIS). We found that K351N substitution abrogates the monoubiquitination of p53 induced by both Mdm2 and MSL2 E3-ligases. As a consequence, cells expressing p53 K351N mutant showed defects in cisplatin-induced translocation of p53 to mitochondria, Bax oligomerization, and mitochondrial membrane depolarization. These data identify K351N as a critical mutation of p53 that contributes to the development and maintenance of resistance to cisplatin.

  10. Identifying protein complexes in protein-protein interaction networks by using clique seeds and graph entropy.

    PubMed

    Chen, Bolin; Shi, Jinhong; Zhang, Shenggui; Wu, Fang-Xiang

    2013-01-01

    The identification of protein complexes plays a key role in understanding major cellular processes and biological functions. Various computational algorithms have been proposed to identify protein complexes from protein-protein interaction (PPI) networks. In this paper, we first introduce a new seed-selection strategy for seed-growth style algorithms. Cliques rather than individual vertices are employed as initial seeds. After that, a result-modification approach is proposed based on this seed-selection strategy. Predictions generated by higher order clique seeds are employed to modify results that are generated by lower order ones. The performance of this seed-selection strategy and the result-modification approach are tested by using the entropy-based algorithm, which is currently the best seed-growth style algorithm to detect protein complexes from PPI networks. In addition, we investigate four pairs of strategies for this algorithm in order to improve its accuracy. The numerical experiments are conducted on a Saccharomyces cerevisiae PPI network. The group of best predictions consists of 1711 clusters, with the average f-score at 0.68 after removing all similar and redundant clusters. We conclude that higher order clique seeds can generate predictions with higher accuracy and that our improved entropy-based algorithm outputs more reasonable predictions than the original one.

  11. Measurement of protein-ligand complex formation.

    PubMed

    Lowe, Peter N; Vaughan, Cara K; Daviter, Tina

    2013-01-01

    Experimental approaches to detect, measure, and quantify protein-ligand binding, along with their theoretical bases, are described. A range of methods for detection of protein-ligand interactions is summarized. Specific protocols are provided for a nonequilibrium procedure pull-down assay, for an equilibrium direct binding method and its modification into a competition-based measurement and for steady-state measurements based on the effects of ligands on enzyme catalysis.

  12. The Chaperone-Mediated Autophagy Receptor Organizes in Dynamic Protein Complexes at the Lysosomal Membrane ▿ †

    PubMed Central

    Bandyopadhyay, Urmi; Kaushik, Susmita; Varticovski, Lyuba; Cuervo, Ana Maria

    2008-01-01

    Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which specific cytosolic proteins are sent to lysosomes for degradation. Substrate proteins bind to the lysosomal membrane through the lysosome-associated membrane protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene, and this binding is limiting for their degradation via CMA. However, the mechanisms of substrate binding and uptake remain unknown. We report here that LAMP-2A organizes at the lysosomal membrane into protein complexes of different sizes. The assembly and disassembly of these complexes are a very dynamic process directly related to CMA activity. Substrate proteins only bind to monomeric LAMP-2A, while the efficient translocation of substrates requires the formation of a particular high-molecular-weight LAMP-2A complex. The two major chaperones related to CMA, hsc70 and hsp90, play critical roles in the functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we have identified a novel function for hsc70 in the disassembly of LAMP-2A from these complexes, whereas the presence of lysosome-associated hsp90 is essential to preserve the stability of LAMP-2A at the lysosomal membrane. PMID:18644871

  13. The Metabotropic Glutamate Receptor mGlu7 Activates Phospholipase C, Translocates Munc-13-1 Protein, and Potentiates Glutamate Release at Cerebrocortical Nerve Terminals*

    PubMed Central

    Martín, Ricardo; Durroux, Thierry; Ciruela, Francisco; Torres, Magdalena; Pin, Jean-Philippe; Sánchez-Prieto, José

    2010-01-01

    At synaptic boutons, metabotropic glutamate receptor 7 (mGlu7 receptor) serves as an autoreceptor, inhibiting glutamate release. In this response, mGlu7 receptor triggers pertussis toxin-sensitive G protein activation, reducing presynaptic Ca2+ influx and the subsequent depolarization evoked release. Here we report that receptor coupling to signaling pathways that potentiate release can be seen following prolonged exposure of nerve terminals to the agonist l-(+)-phosphonobutyrate, l-AP4. This novel mGlu7 receptor response involves an increase in the release induced by the Ca2+ ionophore ionomycin, suggesting a mechanism that is independent of Ca2+ channel activity, but dependent on the downstream exocytotic release machinery. The mGlu7 receptor-mediated potentiation resists exposure to pertussis toxin, but is dependent on phospholipase C, and increased phosphatidylinositol (4,5)-bisphosphate hydrolysis. Furthermore, the potentiation of release does not depend on protein kinase C, although it is blocked by the diacylglycerol-binding site antagonist calphostin C. We also found that activation of mGlu7 receptors translocate the active zone protein essential for synaptic vesicle priming, munc13-1, from soluble to particulate fractions. We propose that the mGlu7 receptor can facilitate or inhibit glutamate release through multiple pathways, thereby exerting homeostatic control of presynaptic function. PMID:20375012

  14. The 18-kDa Translocator Protein Inhibits Vascular Cell Adhesion Molecule-1 Expression via Inhibition of Mitochondrial Reactive Oxygen Species

    PubMed Central

    Joo, Hee Kyoung; Lee, Yu Ran; Kang, Gun; Choi, Sunga; Kim, Cuk-Seong; Ryoo, Sungwoo; Park, Jin Bong; Jeon, Byeong Hwa

    2015-01-01

    Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10–100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO (0.1–0.5 μM), a specific mitochondrial antioxidants, and cyclosporin A (1–5 μM), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam (1–50 μM), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells. PMID:26608360

  15. 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

  16. Conservation of Telomere protein complexes: Shuffling through Evolution

    PubMed Central

    Linger, Benjamin R.; Price, Carolyn M.

    2009-01-01

    The rapid evolution of telomere proteins has hindered identification of orthologs from diverse species and created the impression that certain groups of eukaryotes have largely non-overlapping sets of telomere proteins. However, the recent identification of additional telomere proteins from various model organisms has dispelled this notion by expanding our understanding of the composition, architecture and range of telomere protein complexes present in individual species. It is now apparent that versions of the budding yeast CST complex and mammalian shelterin are present in multiple phyla. While the precise subunit composition and architecture of these complexes vary between species, the general function is often conserved. Despite the overall conservation of telomere protein complexes, there is still considerable species specific variation, with some organisms having lost a particular subunit or even an entire complex. In some cases, complex components appear to have migrated between the telomere and the telomerase RNP. Finally, gene duplication has created telomere protein paralogs with novel functions. While one paralog may be part of a conserved telomere protein complex and have the expected function, the other paralog may serve in a completely different aspect of telomere biology. PMID:19839711

  17. Characterization of Novel Molecular Mechanisms Favoring Rac1 Membrane Translocation.

    PubMed

    Castro-Castro, Antonio; Muriel, Olivia; Del Pozo, Miguel A; Bustelo, Xosé R

    2016-01-01

    The Rac1 GTPase plays key roles in cytoskeletal organization, cell motility and a variety of physiological and disease-linked responses. Wild type Rac1 signaling entails dissociation of the GTPase from cytosolic Rac1-Rho GDP dissociation inhibitor (GDI) complexes, translocation to membranes, activation by exchange factors, effector binding, and activation of downstream signaling cascades. Out of those steps, membrane translocation is the less understood. Using transfections of a expression cDNA library in cells expressing a Rac1 bioreporter, we previously identified a cytoskeletal feedback loop nucleated by the F-actin binding protein coronin 1A (Coro1A) that promotes Rac1 translocation to the plasma membrane by facilitating the Pak-dependent dissociation of Rac1-Rho GDI complexes. This screening identified other potential regulators of this process, including WDR26, basigin, and TMEM8A. Here, we show that WDR26 promotes Rac1 translocation following a Coro1A-like and Coro1A-dependent mechanism. By contrast, basigin and TMEM8A stabilize Rac1 at the plasma membrane by inhibiting the internalization of caveolin-rich membrane subdomains. This latter pathway is F-actin-dependent but Coro1A-, Pak- and Rho GDI-independent.

  18. Characterization of Novel Molecular Mechanisms Favoring Rac1 Membrane Translocation

    PubMed Central

    Castro-Castro, Antonio; Muriel, Olivia; del Pozo, Miguel A.

    2016-01-01

    The Rac1 GTPase plays key roles in cytoskeletal organization, cell motility and a variety of physiological and disease-linked responses. Wild type Rac1 signaling entails dissociation of the GTPase from cytosolic Rac1-Rho GDP dissociation inhibitor (GDI) complexes, translocation to membranes, activation by exchange factors, effector binding, and activation of downstream signaling cascades. Out of those steps, membrane translocation is the less understood. Using transfections of a expression cDNA library in cells expressing a Rac1 bioreporter, we previously identified a cytoskeletal feedback loop nucleated by the F-actin binding protein coronin 1A (Coro1A) that promotes Rac1 translocation to the plasma membrane by facilitating the Pak-dependent dissociation of Rac1-Rho GDI complexes. This screening identified other potential regulators of this process, including WDR26, basigin, and TMEM8A. Here, we show that WDR26 promotes Rac1 translocation following a Coro1A-like and Coro1A-dependent mechanism. By contrast, basigin and TMEM8A stabilize Rac1 at the plasma membrane by inhibiting the internalization of caveolin-rich membrane subdomains. This latter pathway is F-actin-dependent but Coro1A-, Pak- and Rho GDI-independent. PMID:27835684

  19. Alkyl Cinnamates Induce Protein Kinase C Translocation and Anticancer Activity against Breast Cancer Cells through Induction of the Mitochondrial Pathway of Apoptosis

    PubMed Central

    Deka, Suman Jyoti; Mamdi, Narsimha; Manna, Debasis

    2016-01-01

    Purpose The protein kinase C (PKC) family of serine-threonine kinases plays an important role in cancer cell progression. Thus, molecules that target PKC have potential as anticancer agents. The current study aims to understand the treatment of breast cancer cells with alkyl cinnamates. We have also explored the mechanistic details of their anticancer action and the underlying molecular signaling. Methods 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure the viability of MDAMB-231 breast cancer cells to assess the anticancer activity of these compounds. In addition, flow cytometry was performed to study the effect of alkyl cinnamates on the cell cycle and apoptosis. Immunoblotting and immunofluorescence techniques were performed to study PKC translocation, cytochrome c release, and modulation of the mitochondrial membrane potential in breast cancer cells targeted with alkyl cinnamates. Results The PKC agonist DM-2-8 translocated 16.6%±1.7% PKCα from cytosol to the plasma membrane and showed excellent anticancer activity with an half maximal inhibitory concentration (IC50) of 4.13±0.27 µg/mL against cancer cells. The treated cells had an abnormal morphology and exhibited cell cycle defects with G2/M arrest and reduced S phase. Cancer cells treated with DM-2-3, DM-2-4, or DM-2-8 underwent apoptosis as the major pathway of cell death, further confirmed by genomic DNA fragmentation. Furthermore, the mitochondrial membrane potential was perturbed, indicating involvement of the mitochondrial pathway of apoptosis. Immunolocalization studies revealed cytochrome c release from mitochondria to cytosol. Cancer cells treated with DM-2-8 and curcumin showed activation of caspase-9 and caspase-3 as downstream molecular components of the apoptotic pathway. Alkyl cinnamates also caused oxidative stress, which regulates the apoptotic machinery (DNA fragmentation), cell death, and morphological abnormalities in cancer cells

  20. The retromer complex - endosomal protein recycling and beyond.

    PubMed

    Seaman, Matthew N J

    2012-10-15

    The retromer complex is a vital element of the endosomal protein sorting machinery that is conserved across all eukaryotes. Retromer is most closely associated with the endosome-to-Golgi retrieval pathway and is necessary to maintain an active pool of hydrolase receptors in the trans-Golgi network. Recent progress in studies of retromer have identified new retromer-interacting proteins, including the WASH complex and cargo such as the Wntless/MIG-14 protein, which now extends the role of retromer beyond the endosome-to-Golgi pathway and has revealed that retromer is required for aspects of endosome-to-plasma membrane sorting and regulation of signalling events. The interactions between the retromer complex and other macromolecular protein complexes now show how endosomal protein sorting is coordinated with actin assembly and movement along microtubules, and place retromer squarely at the centre of a complex set of protein machinery that governs endosomal protein sorting. Dysregulation of retromer-mediated endosomal protein sorting leads to various pathologies, including neurodegenerative diseases such as Alzheimer disease and spastic paraplegia and the mechanisms underlying these pathologies are starting to be understood. In this Commentary, I will highlight recent advances in the understanding of retromer-mediated endosomal protein sorting and discuss how retromer contributes to a diverse set of physiological processes.

  1. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

    PubMed

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

    2016-02-02

    The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.

  2. 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.

  3. Herpes simplex virus 1 VP22 regulates translocation of multiple viral and cellular proteins and promotes neurovirulence.

    PubMed

    Tanaka, Michiko; Kato, Akihisa; Satoh, Yuko; Ide, Takahiro; Sagou, Ken; Kimura, Kayo; Hasegawa, Hideki; Kawaguchi, Yasushi

    2012-05-01

    Herpes simplex virus 1 (HSV-1) protein VP22, encoded by the UL49 gene, is a major virion tegument protein. In the present study, we showed that VP22 was required for efficient redistribution of viral proteins VP16, VP26, ICP0, ICP4, and ICP27 and of cellular protein Hsc-70 to the cytoplasm of infected cells. We found that two dileucine motifs in VP22, at amino acids 235 and 236 and amino acids 251 and 252, were necessary for VP22 regulation of the proper cytoplasmic localization of these viral and cellular proteins. The dileucine motifs were also required for proper cytoplasmic localization of VP22 itself and for optimal expression of viral proteins VP16, VP22, ICP0, UL41, and glycoprotein B. Interestingly, a recombinant mutant virus with alanines substituted for the dileucines at amino acids 251 and 252 had a 50% lethal dose (LD(50)) for neurovirulence in mice following intracerebral inoculation about 10(3)-fold lower than the LD(50) of the repaired virus. Furthermore, the replication and spread of this mutant virus in the brains of mice following intracerebral inoculation were significantly impaired relative to those of the repaired virus. The ability of VP22 to regulate the localization and expression of various viral and cellular proteins, as shown in this study, was correlated with an increase in viral replication and neurovirulence in the experimental murine model. Thus, HSV-1 VP22 is a significant neurovirulence factor in vivo.

  4. Computational approaches for detecting protein complexes from protein interaction networks: a survey

    PubMed Central

    2010-01-01

    Background Most proteins form macromolecular complexes to perform their biological functions. However, experimentally determined protein complex data, especially of those involving more than two protein partners, are relatively limited in the current state-of-the-art high-throughput experimental techniques. Nevertheless, many techniques (such as yeast-two-hybrid) have enabled systematic screening of pairwise protein-protein interactions en masse. Thus computational approaches for detecting protein complexes from protein interaction data are useful complements to the limited experimental methods. They can be used together with the experimental methods for mapping the interactions of proteins to understand how different proteins are organized into higher-level substructures to perform various cellular functions. Results Given the abundance of pairwise protein interaction data from high-throughput genome-wide experimental screenings, a protein interaction network can be constructed from protein interaction data by considering individual proteins as the nodes, and the existence of a physical interaction between a pair of proteins as a link. This binary protein interaction graph can then be used for detecting protein complexes using graph clustering techniques. In this paper, we review and evaluate the state-of-the-art techniques for computational detection of protein complexes, and discuss some promising research directions in this field. Conclusions Experimental results with yeast protein interaction data show that the interaction subgraphs discovered by various computational methods matched well with actual protein complexes. In addition, the computational approaches have also improved in performance over the years. Further improvements could be achieved if the quality of the underlying protein interaction data can be considered adequately to minimize the undesirable effects from the irrelevant and noisy sources, and the various biological evidences can be better

  5. Strategies for crystallization of large membrane protein complexes

    NASA Astrophysics Data System (ADS)

    Yoshikawa, Shinya; Shinzawa-Itoh, Kyoko; Ueda, Hidefumi; Tsukihara, Tomitake; Fukumoto, Yoshihisa; Kubota, Tomomi; Kawamoto, Masahide; Fukuyama, Keiichi; Matsubara, Hiroshi

    1992-08-01

    Crystalline cytochrome c oxidase and ubiquinol: cytochrome c oxidoreductase which diffracted X-rays at 7-8A˚resolution were obtained from bovine heart mitochondria. The methods for the purification and crystallization of these enzymes indicate that large membrane protein complexes are easier to purify and crystallize than smaller homologous membrane protein complexes, because the former have more hydrophilic surface than the latter. Bulky and polydispersed detergents such as Brij-35 and Tween 20 attached to the isolated complex are not always obstructive to crystallization if they are effective for stabilizing the complexes.

  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. Sizing Large Proteins and Protein Complexes by Electrospray Ionization Mass Spectrometry and Ion Mobility

    PubMed Central

    Kaddis, Catherine S.; Lomeli, Shirley H.; Yin, Sheng; Berhane, Beniam; Apostol, Marcin I.; Kickhoefer, Valerie A.; Rome, Leonard H.; Loo, Joseph A.

    2009-01-01

    Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method termed GEMMA (Gas-Phase Electrophoretic Mobility Molecular Analysis), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared to other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and x-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm3. Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water. PMID:17434746

  8. Nanoparticle-protein complexes mimicking corona formation in ocular environment.

    PubMed

    Jo, Dong Hyun; Kim, Jin Hyoung; Son, Jin Gyeong; Dan, Ki Soon; Song, Sang Hoon; Lee, Tae Geol; Kim, Jeong Hun

    2016-12-01

    Nanoparticles adsorb biomolecules to form corona upon entering the biological environment. In this study, tissue-specific corona formation is provided as a way of controlling protein interaction with nanoparticles in vivo. In the vitreous, the composition of the corona was determined by the electrostatic and hydrophobic properties of the associated proteins, regardless of the material (gold and silica) or size (20- and 100-nm diameter) of the nanoparticles. To control protein adsorption, we pre-incubate 20-nm gold nanoparticles with 5 selectively enriched proteins from the corona, formed in the vitreous, to produce nanoparticle-protein complexes. Compared to bare nanoparticles, nanoparticle-protein complexes demonstrate improved binding to vascular endothelial growth factor (VEGF) in the vitreous. Furthermore, nanoparticle-protein complexes retain in vitro anti-angiogenic properties of bare nanoparticles. In particular, priming the nanoparticles (gold and silica) with tissue-specific corona proteins allows nanoparticle-protein complexes to exert better in vivo therapeutic effects by higher binding to VEGF than bare nanoparticles. These results suggest that controlled corona formation that mimics in vivo processes may be useful in the therapeutic use of nanomaterials in local environment.

  9. Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle.

    PubMed Central

    Nagley, P; Farrell, L B; Gearing, D P; Nero, D; Meltzer, S; Devenish, R J

    1988-01-01

    A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the F0/F1-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a multicopy expression vector (pLF1) that replicates in the nucleus of yeast cells and that carries an inserted DNA segment, specifying a precursor protein (N9/Y8) consisting of subunit 8 fused to an N-terminal cleavable transit peptide (the leader sequence from Neurospora crassa ATPase subunit 9). The successful incorporation of the imported subunit 8 into functional ATPase complexes after transformation with pLF1 expressing N9/Y8 was indicated by the efficient genetic complementation of respiratory growth defects of aap1 mit- mutants, which lack endogenous subunit 8. The reconstitution of ATPase function was confirmed by biochemical assays of ATPase performance in mitochondria and by immunochemical analyses that demonstrated the assembly of the cytoplasmically synthesized subunit 8 into the ATPase complex. Reconstitution of ATPase function required the cytoplasmically synthesized subunit to have a transit peptide. The strategy for importation and reconstitution developed for subunit 8 leads to a systematic approach to the directed manipulation of mitochondrially encoded membrane-associated proteins that has general implications for exploring membrane biogenesis mechanistically and evolutionarily. Images PMID:2895470

  10. Unwinding protein complexes in ALTernative telomere maintenance.

    PubMed

    Bhattacharyya, Saumitri; Sandy, April; Groden, Joanna

    2010-01-01

    Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA-binding proteins and a number of three dimensional nucleic acid structures including G-quartets and D-loops. A number of studies suggest that the BLM and WRN recQ-like helicases play important roles in recombination-mediated mechanisms of telomere elongation or Alternative Lengthening of Telomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT-associated nuclear bodies in telomerase-negative immortalized cell lines and interact with the telomere-specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double-strand break repair processes such as non-homologous end joining, homologous recombination-mediated repair, resolution of stalled replication forks and synthesis-dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ-like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec-Q-like helicases and their role in telomere elongation during ALT processes.

  11. Mitogen-activated protein kinase 6 mediates nuclear translocation of ORE3 to promote ORE9 gene expression in methyl jasmonate-induced leaf senescence.

    PubMed

    Zhang, Yushan; Liu, Jian; Chai, Jinyu; Xing, Da

    2016-01-01

    Methyl jasmonate (MeJA) is a potent promoter of plant senescence. ORESARA3 (ORE3)/ETHYLENE INSENSITIVE2 (EIN2), a protein similar to the members of the disease-related Nramp metal transporter family, is involved in cross-talk among several senescence processes related to abscisic acid, ethylene, MeJA, age and darkness. Nevertheless, the mechanism involved in the regulation of ORE3/EIN2 in exogenous MeJA-induced leaf senescence remains unclear. The C-terminal end of ORE3/EIN2 (CEND) was cleaved from ORE3/EIN2 located in the endoplasmic reticulum and then transferred to the nucleus during MeJA-induced senescence. Further analyses showed that mitogen-activated protein kinase 6 (MPK6) promoted CEND cleavage and nuclear translocation. Nuclear CEND accumulated ETHYLENE INSENSITIVE3 (EIN3), a transcription factor that accelerates MeJA-induced leaf senescence wherein ORESARA9 (ORE9) expression was suppressed in ein3, ore3, and mpk6 mutant plants. ChIP experiments revealed that EIN3 bound directly to the ORE9 promoter and this binding was enhanced in MeJA-induced leaf senescence. This study revealed the effect of the signalling pathway involving MPK6-ORE3-EIN3-ORE9 on regulating leaf senescence and provided insights into the mechanism of MeJA in promoting leaf senescence in Arabidopsis thaliana.

  12. 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.

  13. 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.

  14. Intramolecular Disulfide Bond of Tim22 Protein Maintains Integrity of the TIM22 Complex in the Mitochondrial Inner Membrane*

    PubMed Central

    Okamoto, Hiroaki; Miyagawa, Akiko; Shiota, Takuya; Tamura, Yasushi; Endo, Toshiya

    2014-01-01

    Mitochondrial proteins require protein machineries called translocators in the outer and inner membranes for import into and sorting to their destination submitochondrial compartments. Among them, the TIM22 complex mediates insertion of polytopic membrane proteins into the inner membrane, and Tim22 constitutes its central insertion channel. Here we report that the conserved Cys residues of Tim22 form an intramolecular disulfide bond. By comparison of Tim22 Cys → Ser mutants with wild-type Tim22, we show that the disulfide bond of Tim22 stabilizes Tim22 especially at elevated temperature through interactions with Tim18, which are also important for the stability of the TIM22 complex. We also show that lack of the disulfide bond in Tim22 impairs the assembly of TIM22 pathway substrate proteins into the inner membrane especially when the TIM22 complex handles excess amounts of substrate proteins. Our findings provide a new insight into the mechanism of the maintenance of the structural and functional integrity of the TIM22 complex. PMID:24385427

  15. From quantitative protein complex analysis to disease mechanism.

    PubMed

    Texier, Y; Kinkl, N; Boldt, K; Ueffing, M

    2012-12-15

    Interest in the field of cilia biology and cilia-associated diseases - ciliopathies - has strongly increased over the last few years. Proteomic technologies, especially protein complex analysis by affinity purification-based methods, have been used to decipher various basic but also disease-associated mechanisms. This review focusses on some selected recent studies using affinity purification-based protein complex analysis, thereby exemplifying the great possibilities this technology offers.

  16. Burkholderia pseudomallei type III secreted protein BipC: role in actin modulation and translocation activities required for the bacterial intracellular lifecycle

    PubMed Central

    Kang, Wen Tyng; Vellasamy, Kumutha Malar; Rajamani, Lakshminarayanan; Beuerman, Roger W.

    2016-01-01

    Melioidosis, an infection caused by the facultative intracellular pathogen Burkholderia pseudomallei, has been classified as an emerging disease with the number of patients steadily increasing at an alarming rate. B. pseudomalleipossess various virulence determinants that allow them to invade the host and evade the host immune response, such as the type III secretion systems (TTSS). The products of this specialized secretion system are particularly important for the B. pseudomallei infection. Lacking in one or more components of the TTSS demonstrated different degrees of defects in the intracellular lifecycle of B. pseudomallei. Further understanding the functional roles of proteins involved in B. pseudomallei TTSS will enable us to dissect the enigma of B. pseudomallei-host cell interaction. In this study, BipC (a translocator), which was previously reported to be involved in the pathogenesis of B. pseudomallei, was further characterized using the bioinformatics and molecular approaches. The bipCgene, coding for a putative invasive protein, was first PCR amplified from B. pseudomallei K96243 genomic DNA and cloned into an expression vector for overexpression in Escherichia coli. The soluble protein was subsequently purified and assayed for actin polymerization and depolymerization. BipC was verified to subvert the host actin dynamics as demonstrated by the capability to polymerize actin in vitro. Homology modeling was also attempted to predict the structure of BipC. Overall, our findings identified that the protein encoded by the bipC gene plays a role as an effector involved in the actin binding activity to facilitate internalization of B. pseudomalleiinto the host cells. PMID:28028452

  17. A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators.

    PubMed Central

    Ueda, K; Miyake, K; Horinouchi, S; Beppu, T

    1993-01-01

    Mutants of Streptomyces griseus deficient in A-factor production are sporulation negative, since A-factor is an essential hormonal regulator for the induction of morphological and physiological differentiation in this bacterium. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this mutant strain. Subcloning experiments and nucleotide sequencing showed that two open reading frames, ORF1 with 656 amino acids and ORF2 with 201 amino acids, were required in order to induce sporulation. The amino acid sequence of ORF1 significantly resembled that of the Escherichia coli HlyB protein, a member of a family of bacterial membrane proteins engaged in ATP-dependent secretion mechanisms. Conserved features of this surface translocator family, such as the transmembrane structure predicted by their hydropathy profiles and the amino acid sequence forming an ATP-binding fold, were also conserved in ORF1. The ORF1 gene appeared to constitute a transcriptional unit with an additional upstream gene encoding ORF3, which was greatly similar to ORF1 in size and amino acid sequence. The other protein, ORF2, showed significant end-to-end homology with the E. coli uhpA product, a regulatory protein for the uptake of sugar phosphates. Like UhpA as a response regulator of a bacterial two-component regulatory system, ORF2 contained a helix-turn-helix DNA-binding domain at its COOH-terminal portion and an Asp residue (Asp-54) probably to be phosphorylated at its NH2-terminal portion. An amino acid replacement from Asp-54 to Asn resulted in the loss of the ability of ORF2 to induce sporulation in strain HH1. Images PMID:8458843

  18. Dystrophin complex functions as a scaffold for signalling proteins.

    PubMed

    Constantin, Bruno

    2014-02-01

    Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part

  19. Mass Spectrometry of Protein-Ligand Complexes: Enhanced Gas Phase Stability of Ribonuclease-Nucleotide Complexes

    PubMed Central

    Yin, Sheng; Xie, Yongming; Loo, Joseph A.

    2008-01-01

    Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas phase stabilities of noncovalent complexes that can result. PMID:18565758

  20. Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

    SciTech Connect

    Bordner, Andrew J.; Gorin, Andrey A.

    2008-05-12

    Here, protein-protein interactions are ubiquitous and essential for cellular processes. High-resolution X-ray crystallographic structures of protein complexes can elucidate the details of their function and provide a basis for many computational and experimental approaches. Here we demonstrate that existing annotations of protein complexes, including those provided by the Protein Data Bank (PDB) itself, contain a significant fraction of incorrect annotations. Results: We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster is relevant based on a diverse set of properties; and (4) finally combining these scores for each entry in order to predict the complex structure. Unlike previous annotation methods, consistent prediction of complexes with identical or almost identical protein content is insured. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions.

  1. Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

    DOE PAGES

    Bordner, Andrew J.; Gorin, Andrey A.

    2008-05-12

    Here, protein-protein interactions are ubiquitous and essential for cellular processes. High-resolution X-ray crystallographic structures of protein complexes can elucidate the details of their function and provide a basis for many computational and experimental approaches. Here we demonstrate that existing annotations of protein complexes, including those provided by the Protein Data Bank (PDB) itself, contain a significant fraction of incorrect annotations. Results: We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster ismore » relevant based on a diverse set of properties; and (4) finally combining these scores for each entry in order to predict the complex structure. Unlike previous annotation methods, consistent prediction of complexes with identical or almost identical protein content is insured. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions.« less

  2. Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen

    PubMed Central

    Rabideau, Amy E.; Liao, Xiaoli; Akçay, Gizem; Pentelute, Bradley L.

    2015-01-01

    A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LFN) into the cytosol via PA pore. Here, we move beyond natural amino acids and systematically investigate the translocation of polypeptide cargo containing non-canonical amino acids and functionalities through PA pore. Our results indicate translocation is not perturbed with alterations to the peptide backbone or side-chain. Moreover, despite their structural complexity, we found that the small molecule drugs, doxorubicin and monomethyl auristatin F (MMAF) translocated efficiently through PA pore. However, we found cyclic peptides and the small molecule drug docetaxel abrogated translocation due to their large size and structural rigidity. For cargos that reached the cytosol, we demonstrated that each remained intact after translocation. These studies show PA is capable of translocating non-canonical cargo provided it is in a conformational state conducive for passage through the narrow pore. PMID:26178180

  3. Adenosine deaminase complexing protein in cancer studies.

    PubMed

    Ten Kate, J; Dinjens, W N; Meera Khan, P; Bosman, F T

    1986-01-01

    ADCP is a dimeric glycoprotein of about 200KD, for which the physiological role is still obscure. This protein occurs mainly in a membrane bound form in various human tissues. In this paper we review the current literature on ADCP in cancer studies. Soluble ADCP was described to be consistently decreased or absent in cancers of lung, liver, kidney and colon. These findings could not be confirmed by immunohistochemical and quantitative biochemical studies in a series of colorectal-, prostatic-, and renal carcinomas. Only in a third of these tumors a decrease could be demonstrated, whereas in the other cases unaltered or even increased amounts were observed. However, in virally transformed human fibroblasts a consistent decrease or complete absence of ADCP was seen, while primary fibroblasts were found to contain high amounts of this protein. Recently, the use of ADCP as a differentiation marker in colonic cancer has been advocated. Furthermore the presence of ADCP in the serum of renal adenocarcinoma patients was found to be indicative of a better chance of five year survival. These studies suggest that ADCP may be a differentiation marker useful for immunohistochemical characterization of colonic and renal carcinomas as well as a serum marker useful for follow-up studies of these types of cancer, analogous to CEA. Finally, ADCP has been found to be selectively expressed by certain T-cell subsets and henceforth may be useful in the studies on leukemias.

  4. Stimulation of non-amyloidogenic processing of amyloid-β protein precursor by cryptotanshinone involves activation and translocation of ADAM10 and PKC-α.

    PubMed

    Durairajan, Siva Sundara Kumar; Liu, Liang-Feng; Lu, Jia-Hong; Koo, Irene; Maruyama, Kei; Chung, Sookja K; Huang, Jian-Dong; Li, Min

    2011-01-01

    Cerebral deposition of amyloid-β peptide (Aβ) plaques is now considered the central feature of Alzheimer's disease. Recent studies suggest that cryptotanshinone (CTS) extracted from the root of Salvia miltiorrhiza Bunge could be used for the prevention and treatment of Alzheimer's disease. In this study, we investigated the role of CTS on non-amyloidogenic processing of amyloid-β protein precursor (AβPP) as well as its regulation by protein kinase C (PKC). Treatment with CTS dose-dependently and significantly reduced both intracellular and secreted levels of Aβ40 and Aβ42 in N2a mouse neuroblastoma cells stably expressing human SwedishAβPP (N2a-SwedAβPP). Using N2a-SwedAβPP and human neuroblastoma SHSY5Y cells, it was demonstrated that CTS significantly and dose-dependently increased the production of sAβPPα and C-terminal fragment-α (CTF-α) from AβPP. At the same time, CTS specifically increased the maturation of "a disintegrin and metalloproteinase-10" (ADAM10), an α-secretase candidate. The increase of sAβPPα secretion by CTS was blocked by the hydroxamate-based inhibitors GI254023X and GW280264X, and by the PKC-α inhibitor GÖ6976, suggesting involvement of the ADAM10 and PKC-α in CTS-induced α-secretase cleavage. In other experiments, CTS induced the phosphorylation of PKC-α indicating that PKC-α is involved in CTS-induced sAβPPα secretion. Furthermore, treatment of neuroblastoma cells with CTS induced the co-translocation of ADAM10 and PKC-α to the cell membrane, the site at which AβPP was cleaved, and this translocation was significantly reduced by GÖ6976. These results suggest that CTS-induced sAβPPα secretion is regulated by a PKC-α and ADAM10 cascade in neuroblastoma cells and may be involved in the lowering of Aβ production.

  5. 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

  6. Semiquinone and Cluster N6 Signals in His-tagged Proton-translocating NADH:Ubiquinone Oxidoreductase (Complex I) from Escherichia coli*

    PubMed Central

    Narayanan, Madhavan; Gabrieli, David J.; Leung, Steven A.; Elguindy, Mahmoud M.; Glaser, Carl A.; Saju, Nitha; Sinha, Subhash C.; Nakamaru-Ogiso, Eiko

    2013-01-01

    NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ∼15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mm−1 cm−1 at 274 nm and 50.3 mm−1 cm−1 at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 μmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ∼12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed. PMID:23543743

  7. A Novel Role for Protein Kinase Kin2 in Regulating HAC1 mRNA Translocation, Splicing, and Translation

    PubMed Central

    Anshu, Ashish; Mannan, M. Amin-ul; Chakraborty, Abhijit; Chakrabarti, Saikat

    2014-01-01

    A signaling network called the unfolded protein response (UPR) resolves the protein-folding defects in the endoplasmic reticulum (ER) from yeasts to humans. In the yeast Saccharomyces cerevisiae, the UPR activation involves (i) aggregation of the ER-resident kinase/RNase Ire1 to form an Ire1 focus, (ii) targeting HAC1 pre-mRNA toward the Ire1 focus that cleaves out an inhibitory intron from the mRNA, and (iii) translation of Hac1 protein from the spliced mRNA. Targeting HAC1 mRNA to the Ire1 focus requires a cis-acting bipartite element (3′BE) located at the 3′ untranslated leader. Here, we report that the 3′BE plays an additional role in promoting translation from the spliced mRNA. We also report that a high dose of either of two paralogue kinases, Kin1 and Kin2, overcomes the defective UPR caused by a mutation in the 3′BE. These results define a novel role for Kin kinases in the UPR beyond their role in cell polarity and exocytosis. Consistently, targeting, splicing, and translation of HAC1 mRNA are substantially reduced in the kin1Δ kin2Δ strain. Furthermore, we show that Kin2 kinase domain itself is sufficient to activate the UPR, suggesting that Kin2 initiates a signaling cascade to ensure an optimum UPR. PMID:25348718

  8. Luminescent iridium(III) complexes as novel protein staining agents.

    PubMed

    Jia, Junli; Fei, Hao; Zhou, Ming

    2012-05-01

    This article reports a new class of luminescent metal complexes, biscyclometalated iridium(III) complexes with an ancillary bathophenanthroline disulfonate ligand, for staining protein bands that are separated by electrophoresis. The performances of these novel staining agents have been studied in comparison with tris(bathophenanthroline disulfonate) ruthenium(II) tetrasodium salt (i.e. RuBPS) using a commercially available imaging system. The staining agents showed different limits of detection, linear dynamic ranges, and protein-to-protein variations. The overall performances of all three stains were found to be better than or equivalent to RuBPS under the experimental conditions.

  9. 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.

  10. Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

    PubMed Central

    Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.

    2013-01-01

    Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423

  11. 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

  12. Activation of the c-abl oncogene by viral transduction or chromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties.

    PubMed Central

    Davis, R L; Konopka, J B; Witte, O N

    1985-01-01

    The v-abl protein of Abelson murine leukemia virus is a tyrosine-specific kinase. Its normal cellular homolog, murine c-abl, does not possess detectable tyrosine kinase activity in vitro. Previously, we have detected tyrosine kinase activity in vitro for an altered c-abl gene product (c-abl P210) in the K562 human chronic myelogenous leukemia cell line. The expression of this variant c-abl gene product correlates with chromosomal translocation and amplification of the c-abl gene in K562 cells. Like v-abl, c-abl P210 is a fusion protein containing non-abl sequences near the amino terminus of c-abl. We compared the in vitro tyrosine kinase activity of c-abl P210 with that of wild-type murine v-abl. The remarkable similarities of these two proteins with respect to cis-acting autophosphorylation, trans-acting phosphorylation of exogenous substrates, and kinase inhibition, using site-directed abl-specific antisera, suggested that c-abl P210 could function similarly to v-abl in vivo. In addition, c-abl P210 possessed an associated serine kinase activity in immunoprecipitates. The serine kinase activity was not inhibited by site-directed, abl-specific antisera that inhibit the tyrosine kinase activity, suggesting that the serine kinase activity is not an intrinsic property of c-abl P210. Thus, the activation of the c-abl gene in a human leukemia cell line may have functional consequences analogous to activation of the c-abl gene in Abelson murine leukemia virus. Images PMID:4039028

  13. Conjugative type IV secretion in Gram-positive pathogens: TraG, a lytic transglycosylase and endopeptidase, interacts with translocation channel protein TraM.

    PubMed

    Kohler, Verena; Probst, Ines; Aufschnaiter, Andreas; Büttner, Sabrina; Schaden, Lisa; Rechberger, Gerald N; Koraimann, Günther; Grohmann, Elisabeth; Keller, Walter

    2017-02-17

    Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Gram-positive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501ΔtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial-2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system.

  14. 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

    SciTech Connect

    Nucifora, G.; Begy, C.R.; Rowley, J.D. ); Erickson, P.; Drabkin, H.A. )

    1993-08-15

    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. The authors 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 have now been localized to ban 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[prime] 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. 23 refs., 6 figs.

  15. 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

  16. Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes

    SciTech Connect

    Martinovic, Suzana; Pasa-Tolic, Liljiana; Smith, Richard D.

    2004-10-01

    Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of non-covalent complexes on the intact complex level.

  17. 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.

  18. Global landscape of HIV-human protein complexes.

    PubMed

    Jäger, Stefanie; Cimermancic, Peter; Gulbahce, Natali; Johnson, Jeffrey R; McGovern, Kathryn E; Clarke, Starlynn C; Shales, Michael; Mercenne, Gaelle; Pache, Lars; Li, Kathy; Hernandez, Hilda; Jang, Gwendolyn M; Roth, Shoshannah L; Akiva, Eyal; Marlett, John; Stephens, Melanie; D'Orso, Iván; Fernandes, Jason; Fahey, Marie; Mahon, Cathal; O'Donoghue, Anthony J; Todorovic, Aleksandar; Morris, John H; Maltby, David A; Alber, Tom; Cagney, Gerard; Bushman, Frederic D; Young, John A; Chanda, Sumit K; Sundquist, Wesley I; Kortemme, Tanja; Hernandez, Ryan D; Craik, Charles S; Burlingame, Alma; Sali, Andrej; Frankel, Alan D; Krogan, Nevan J

    2011-12-21

    Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

  19. 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.

  20. 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

  1. 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

  2. TWEAK affects keratinocyte G2/M growth arrest and induces apoptosis through the translocation of the AIF protein to the nucleus.

    PubMed

    Sabour Alaoui, Sanaa; Dessirier, Valérie; de Araujo, Elisabeth; Alexaki, Vassilia-Ismini; Pelekanou, Vassiliki; Lkhider, Mustapha; Stathopoulos, Efstathios N; Castanas, Elias; Bagot, Martine; Bensussan, Armand; Tsapis, Andreas

    2012-01-01

    The soluble TNF-like weak inducer of apoptosis (TWEAK, TNFSF12) binds to the fibroblast growth factor-inducible 14 receptor (FN14, TNFRSF12A) on the cell membrane and induces multiple biological responses, such as proliferation, migration, differentiation, angiogenesis and apoptosis. Previous reports show that TWEAK, which does not contain a death domain in its cytoplasmic tail, induces the apoptosis of tumor cell lines through the induction of TNFα secretion. TWEAK induces apoptosis in human keratinocytes. Our experiments clearly demonstrate that TWEAK does not induce the secretion of TNFα or TRAIL proteins. The use of specific inhibitors and the absence of procaspase-3 cleavage suggest that the apoptosis of keratinocytes follows a caspase- and cathepsin B-independent pathway. Further investigation showed that TWEAK induces a decrease in the mitochondrial membrane potential of keratinocytes. Confocal microscopy showed that TWEAK induces the cleavage and the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus, thus initiating caspase-independent apoptosis. Moreover, TWEAK induces FOXO3 and GADD45 expression, cdc2 phosphorylation and cdc2 and cyclinB1 degradation, resulting in the arrest of cell growth at the G2/M phase. Finally, we report that TWEAK and FN14 are normally expressed in the basal layer of the physiological epidermis and are greatly enhanced in benign (psoriasis) and malignant (squamous cell carcinoma) skin pathologies that are characterized by an inflammatory component. TWEAK might play an essential role in skin homeostasis and pathology.

  3. 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.

  4. The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

    PubMed Central

    Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

    1995-01-01

    The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626033

  5. The influence of clozapine treatment and other antipsychotics on the 18 kDa translocator protein, formerly named the peripheral-type benzodiazepine receptor, and steroid production.

    PubMed

    Danovich, Lena; Veenman, Leo; Leschiner, Svetlana; Lahav, Michal; Shuster, Vered; Weizman, Abraham; Gavish, Moshe

    2008-01-01

    It has been shown that the atypical antipsychotic drug clozapine increases the levels of the neurosteroid allopregnanolone in the rat brain. The 18 kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, has been demonstrated to be involved in the process of steroid biosynthesis, in peripheral steroidogenic tissues as well as in glia cells in the brain. In the current study, we investigated the influence of chronic treatment with clozapine and other antipsychotics (thioridazine,sulpiride and risperidone) on TSPO binding in cell cultures and rat tissues. Clozapine significantly increased TSPO binding density in C6 rat glioma cells and in MA-10 mouse Leydig tumor cells, while the antipsychotic sulpiride had no effect on TSPO binding density in both cell lines. In addition, clozapine, but not sulpiride, significantly increased progesterone synthesis by MA-10 Leydig tumor cells. In an animal experiment, male Sprague-Dawley rats were treated with clozapine (20 mg/kg), risperidone (0.5 mg/kg), thioridazine (20 mg/kg), or sulpiride (20 mg/kg) for 21 days, followed by 7 days of withdrawal. Clozapine induced significant increases in TSPO binding in brain and peripheral steroidogenic tissues, whereas the other antipsychotics did not show such pronounced effects on TSPO binding. Our results suggest that TSPO may be involved in the modulation of steroidogenesis by clozapine.

  6. The antidepressant-like activity of AC-5216, a ligand for 18KDa translocator protein (TSPO), in an animal model of diabetes mellitus

    PubMed Central

    Qiu, Zhi-Kun; He, Jia-Li; Liu, Xu; Zhang, Guan-Hua; Zeng, Jia; Nie, Hong; Shen, Yong-Gang; Chen, Ji-Sheng

    2016-01-01

    Diabetes mellitus is a chronic disease that is associated with depression. Also, depression is common in adults with type 2 diabetes mellitus (T2DM). Translocator protein (18kDa) (TSPO) and allopregnanolone play an important role in the depression treatment. However, few studies have evaluated TSPO and allopregnanolone in the treatment of depression in T2DM. AC-5216, a ligand for TSPO, produces anxiolytic- and antidepressant-like effects in animal models. The present study aimed to explore antidepressant-like effects of AC-5216 on diabetic rats. Following the development of diabetic model induced by high fat diet (HFD) feeding and streptozotocin (STZ), AC-5216 (0.3 and 1 mg/kg, i.g.) elicited the antidepressant-like effects in behavioral tests while these activities were blocked by TSPO antagonist PK11195 (3 mg/kg, i.p.). The levels of allopregnanolone in the prefrontal cortex and hippocampus were increased by AC-5216 (0.3 and 1 mg/kg, i.g.), which was antagonized by PK11195 (3 mg/kg, i.p.). The increased plasma glucose (PG) and decreased insulin (INS) in HFD-STZ rats were reversed by AC-5216 (0.3 and 1 mg/kg, i.g.). This study indicates that the antidepressant-like effects of AC-5216 on HFD-STZ rats, suggesting that TSPO may represent a novel therapeutic target for depression in T2DM. PMID:27886206

  7. Membrane translocation of protein kinase Ctheta during T lymphocyte activation requires phospholipase C-gamma-generated diacylglycerol.

    PubMed

    Díaz-Flores, Ernesto; Siliceo, María; Martínez-A, Carlos; Mérida, Isabel

    2003-08-01

    Protein kinase C (PKC) is the only PKC isoform recruited to the immunological synapse after T cell receptor stimulation, suggesting that its activation mechanism differs from that of the other isoforms. Previous studies have suggested that this selective PKC recruitment may operate via a Vav-regulated, cytoskeletal-dependent mechanism, independent of the classical phospholipase C/diacylglycerol pathway. Here, we demonstrate that, together with tyrosine phosphorylation of PKC in the regulatory domain, binding of phospholipase C-dependent diacylglycerol is required for PKC recruitment to the T cell synapse. In addition, we demonstrate that diacylglycerol kinase alpha-dependent diacylglycerol phosphorylation provides the negative signal required for PKC inactivation, ensuring fine control of the T cell activation response.

  8. 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

  9. 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

  10. Machine Learning Approaches for Predicting Protein Complex Similarity.

    PubMed

    Farhoodi, Roshanak; Akbal-Delibas, Bahar; Haspel, Nurit

    2017-01-01

    Discriminating native-like structures from false positives with high accuracy is one of the biggest challenges in protein-protein docking. While there is an agreement on the existence of a relationship between various favorable intermolecular interactions (e.g., Van der Waals, electrostatic, and desolvation forces) and the similarity of a conformation to its native structure, the precise nature of this relationship is not known. Existing protein-protein docking methods typically formulate this relationship as a weighted sum of selected terms and calibrate their weights by using a training set to evaluate and rank candidate complexes. Despite improvements in the predictive power of recent docking methods, producing a large number of false positives by even state-of-the-art methods often leads to failure in predicting the correct binding of many complexes. With the aid of machine learning methods, we tested several approaches that not only rank candidate structures relative to each other but also predict how similar each candidate is to the native conformation. We trained a two-layer neural network, a multilayer neural network, and a network of Restricted Boltzmann Machines against extensive data sets of unbound complexes generated by RosettaDock and PyDock. We validated these methods with a set of refinement candidate structures. We were able to predict the root mean squared deviations (RMSDs) of protein complexes with a very small, often less than 1.5 Å, error margin when trained with structures that have RMSD values of up to 7 Å. In our most recent experiments with the protein samples having RMSD values up to 27 Å, the average prediction error was still relatively small, attesting to the potential of our approach in predicting the correct binding of protein-protein complexes.

  11. Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150.

    PubMed

    Elsworth, Brendan; Sanders, Paul R; Nebl, Thomas; Batinovic, Steven; Kalanon, Ming; Nie, Catherine Q; Charnaud, Sarah C; Bullen, Hayley E; de Koning Ward, Tania F; Tilley, Leann; Crabb, Brendan S; Gilson, Paul R

    2016-11-01

    The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.

  12. 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

  13. Expression of the translocator protein of 18 kDa by microglia, macrophages and astrocytes based on immunohistochemical localization in abnormal human brain

    PubMed Central

    Cosenza-Nashat, Melissa; Zhao, Meng-Liang; Suh, Hyeon-Sook; Morgan, Janet; Natividad, Ryan; Morgello, Susan; Lee, Sunhee C.

    2009-01-01

    Aims Microglia are involved in neurodegeneration, are prime targets for anti-inflammatory therapy and are potential biomarkers of disease progression. For example, positron emission tomography (PET) imaging employing radioligands for the mitochondrial translocator protein of 18 kDa (TSPO, formerly known as the peripheral benzodiazepine receptor) is being scrutinized to detect neuroinflammation in various diseases. TSPO is presumably present in activated microglia, but may be present in other neural cells. Methods We sought to elucidate the protein expression in normal human CNS, several neurological diseases (HIV encephalitis, Alzheimer’s disease, multiple sclerosis and stroke) and SIV encephalitis by performing immunohistochemistry with two anti-TSPO antibodies. Results Although the overall parenchymal staining was minimal in normal brain, endothelial and smooth muscle cells, subpial glia, intravascular monocytes and ependymal cells were TSPO positive. In disease states, elevated TSPO was present in parenchymal microglia, macrophages and some hypertrophic astrocytes, but the distribution of TSPO varied depending on the disease, disease stage and proximity to the lesion or relation to infection. Staining with the two antibodies correlated well in white matter, but one antibody also stained cortical neurons. Quantitative analysis demonstrated a significant increase in TSPO in the white matter of HIV encephalitis compared to brains without encephalitis. TSPO expression was also increased in SIV encephalitis. Conclusions This report provides the first comprehensive immunohistochemical analysis of the expression of TSPO. The results are useful for informing the usage of PET as an imaging modality and have an impact on the potential use of TSPO as an anti-inflammatory pharmacological target. PMID:19077109

  14. β-Barrel membrane protein assembly by the Bam complex.

    PubMed

    Hagan, Christine L; Silhavy, Thomas J; Kahne, Daniel

    2011-01-01

    β-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. β-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts β-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for β-barrel assembly. Ultimately, studies of the mechanism of β-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.

  15. Protein-complex structure completion using IPCAS (Iterative Protein Crystal structure Automatic Solution).

    PubMed

    Zhang, Weizhe; Zhang, Hongmin; Zhang, Tao; Fan, Haifu; Hao, Quan

    2015-07-01

    Protein complexes are essential components in many cellular processes. In this study, a procedure to determine the protein-complex structure from a partial molecular-replacement (MR) solution is demonstrated using a direct-method-aided dual-space iterative phasing and model-building program suite, IPCAS (Iterative Protein Crystal structure Automatic Solution). The IPCAS iteration procedure involves (i) real-space model building and refinement, (ii) direct-method-aided reciprocal-space phase refinement and (iii) phase improvement through density modification. The procedure has been tested with four protein complexes, including two previously unknown structures. It was possible to use IPCAS to build the whole complex structure from one or less than one subunit once the molecular-replacement method was able to give a partial solution. In the most challenging case, IPCAS was able to extend to the full length starting from less than 30% of the complex structure, while conventional model-building procedures were unsuccessful.

  16. Surface translocation and tri-iodothyronine uptake of mutant MCT8 proteins are cell type-dependent.

    PubMed

    Kinne, Anita; Roth, Stephan; Biebermann, Heike; Köhrle, Josef; Grüters, Annette; Schweizer, Ulrich

    2009-12-01

    Mutations in the gene encoding the thyroid hormone transporter, monocarboxylate transporter 8 (MCT8), underlie severe mental retardation. We wanted to understand the functional consequences of a series of missense mutations in MCT8 in order to identify therapeutic options for affected patients. We established cell lines stably expressing 12 MCT8 variants in JEG1 and MDCK1 cells. The cell lines were characterized according to MCT8 mRNA and protein expression, tri-iodothyronine (T(3)) transport activity, substrate K(M) characteristics, surface expression, and responsiveness to T(3) preincubation and chemical chaperones. Functional activities of ins235V and L568P MCT8 mutants depend on the cell type in which they are expressed. These mutants and R271H exhibited considerable transport activity when present at the cell surface as verified by surface biotinylation and kinetic analysis. Most mutants, however, were inactive in T(3) transport even when present at the cell surface (e.g. S194F, A224V, DeltaF230, L512P). Preincubation of G558D with T(3) increased T(3) uptake in MDCK1 cells to a small, but significant, extent. Chemical chaperones were ineffective. The finding that the cell type determines surface expression and T(3) transport activities of missense mutants in MCT8 may be important to understand phenotypic variability among carriers of different mutations. In particular, the clinical observation that the severity of derangements of thyroid hormone levels does not correlate with mental impairments of the patients may be based on different residual activity of mutant MCT8 in different cell types.

  17. 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

  18. Subcellular localization of RNA degrading proteins and protein complexes in prokaryotes.

    PubMed

    Evguenieva-Hackenberg, Elena; Roppelt, Verena; Lassek, Christian; Klug, Gabriele

    2011-01-01

    The archaeal exosome is a prokaryotic protein complex with RNA processing and degrading activities. Recently it was shown that the exosome is localized at the periphery of the cell in the thermoacidophilic archaeon Sulfolobus solfataricus. This localization is most likely mediated by the archaeal DnaG protein and depends on (direct or indirect) hydrophobic interactions with the membrane. A localization of RNA degrading proteins and protein complexes was also demonstrated in several bacteria. In bacteria a subcellular localization was also shown for substrates of these proteins and protein complexes, i.e. chromosomally encoded mRNAs and a small RNA. Thus, despite the missing compartmentalization, a spatial organization of RNA processing and degradation exists in prokaryotic cells. Recent data suggest that the spatial organization contributes to the temporal regulation of these processes.

  19. 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.

  20. 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.

  1. EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways.

    PubMed

    Liu, Wei; Chen, Chunlai; Kavaliauskas, Darius; Knudsen, Charlotte R; Goldman, Yale E; Cooperman, Barry S

    2015-10-30

    The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and Pi release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.

  2. Molecular dynamics simulations of a membrane protein/amphipol complex.

    PubMed

    Perlmutter, Jason D; Popot, Jean-Luc; Sachs, Jonathan N

    2014-10-01

    Amphipathic polymers known as "amphipols" provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.

  3. Platelet-activating factor (PAF)-dependent biochemical, morphologic, and physiologic responses of human platelets: Demonstration of translocation of protein kinase C associated with protein phosphorylation

    SciTech Connect

    Block, L.H.; Abraham, W.M.; Groscurth, P.; Qiao, B.Y.; Perruchoud, A.P. )

    1989-10-01

    Platelet-activating factor (PAF) is a potent stimulus for platelet aggregation and secretion. PAF has been shown to stimulate the phosphatidylinositol (PI) pathway in platelets, which implies that PAF should activate protein kinase C. In this study, measurements of PI metabolites, the elevation of intracellular free calcium concentration, (Ca2+)i, the activation of protein kinase C, and the phosphorylation of platelet proteins (using a two-dimensional gel electrophoretic technique) were performed before and after the addition of 10(-8) M PAF to human platelets. These findings were correlated with morphologic changes in the platelets as determined by immunoelectron microscopic studies on the cytoskeleton and by X-ray analysis of dense bodies. The results show that PAF stimulates the production of PI metabolites and causes an increase in the membrane-associated activity of protein kinase C. These changes are accompanied by a rise in the (Ca2+)i and protein phosphorylation. The increase in protein kinase C activity reaches a maximum at approximately 60 s, a time frame that is consistent with the protein phosphorylation and the subsequent morphologic and secretory events. X-ray analysis revealed two types of dense bodies containing various amounts of calcium which appeared to be released sequentially after PAF activation. These results suggest that the protein phosphorylation that controls the physiologic events resulting from PAF activation of human platelets is catalyzed by protein kinase C.

  4. Platelet-activating factor (PAF)-dependent biochemical, morphologic, and physiologic responses of human platelets: demonstration of translocation of protein kinase C associated with protein phosphorylation.

    PubMed

    Block, L H; Abraham, W M; Groscurth, P; Qiao, B Y; Perruchoud, A P

    1989-10-01

    Platelet-activating factor (PAF) is a potent stimulus for platelet aggregation and secretion. PAF has been shown to stimulate the phosphatidylinositol (PI) pathway in platelets, which implies that PAF should activate protein kinase C. In this study, measurements of PI metabolites, the elevation of intracellular free calcium concentration, (Ca2+)i, the activation of protein kinase C, and the phosphorylation of platelet proteins (using a two-dimensional gel electrophoretic technique) were performed before and after the addition of 10(-8) M PAF to human platelets. These findings were correlated with morphologic changes in the platelets as determined by immunoelectron microscopic studies on the cytoskeleton and by X-ray analysis of dense bodies. The results show that PAF stimulates the production of PI metabolites and causes an increase in the membrane-associated activity of protein kinase C. These changes are accompanied by a rise in the (Ca2+)i and protein phosphorylation. The increase in protein kinase C activity reaches a maximum at approximately 60 s, a time frame that is consistent with the protein phosphorylation and the subsequent morphologic and secretory events. X-ray analysis revealed two types of dense bodies containing various amounts of calcium which appeared to be released sequentially after PAF activation. These results suggest that the protein phosphorylation that controls the physiologic events resulting from PAF activation of human platelets is catalyzed by protein kinase C.

  5. 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.

  6. 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.

  7. Nuclear translocation of the 1,25D{sub 3}-MARRS (membrane associated rapid response to steroids) receptor protein and NF{kappa}B in differentiating NB4 leukemia cells

    SciTech Connect

    Wu, Wenqing; Beilhartz, Greg; Roy, Yvette; Richard, Cynthia L.; Curtin, Maureen; Brown, Lauren; Cadieux, Danielle; Coppolino, Marc; Farach-Carson, Mary C.; Nemere, Ilka; Meckling, Kelly A.

    2010-04-15

    1,25 Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D{sub 3}, which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D{sub 3}-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NF{kappa}B). In unstimulated cells, 1,25D{sub 3}-MARRS can be co-immunoprecipitated with antibodies directed at NF{kappa}B, and NF{kappa}B is co-precipitated when antibodies against 1,25D{sub 3}-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D{sub 3}-MARRS and NF{kappa}B begin translocating to the nucleus within minutes of co-stimulation with 1,25D{sub 3} and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D{sub 3}-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NF{kappa}B and other factors with which it may be interacting.

  8. Oxidative stress-induced apoptotic insults to rat osteoblasts are attenuated by nitric oxide pretreatment via GATA-5-involved regulation of Bcl-X L gene expression and protein translocation.

    PubMed

    Wu, Gong-Jhe; Wang, Weu; Lin, Yi-Ling; Liu, Shing Hwa; Chen, Ruei-Ming

    2016-04-01

    Nitric oxide (NO) has biphasic effects on regulating osteoblast survival and death. This study was aimed to evaluate the effects of NO pretreatment on hydrogen peroxide (HP)-induced insults of rat osteoblasts and the possible mechanisms. Exposure of osteoblasts prepared from rat calvarias to HP significantly increased intracellular reactive oxygen species levels, decreased alkaline phosphatase activity and cell survival, and ultimately induced cell apoptosis. However, NO pretreatment lowered HP-induced oxidative stress and apoptotic insults. In parallel, HP increased Bax levels and its translocation from the cytoplasm to mitochondria. NO pretreatment caused significant attenuations in HP-induced modulations in Bax synthesis and translocation. In contrast, pretreatment with NO enhanced levels and translocation of antiapoptotic Bcl-XL protein in rat osteoblasts. RNA analyses further revealed that HP inhibited Bcl-XL mRNA expression without affecting Bax mRNA levels. In comparison, NO induced Bcl-XL mRNA production and alleviated HP-caused inhibition of this mRNA expression. As to the mechanism, HP suppressed RNA and protein levels of transcription factor GATA-5 in rat osteoblasts. Pretreatment with NO induced GATA-5 mRNA and protein expressions and simultaneously attenuated HP-induced inhibition of this gene's expression. Consequently, GATA-5 knockdown using RNA interference inhibited Bcl-XL mRNA expression and concurrently lowered NO's protection against HP-induced apoptotic insults. Therefore, this study showed that NO can protect rat osteoblasts from HP-induced apoptotic insults. The protective mechanisms are mediated by GATA-5-mediated transcriptional induction of Bcl-X L gene, and translocational modulation of Bcl-XL and Bax proteins.

  9. The bcl-2 candidate proto-oncogene product is a 24-kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18) translocation.

    PubMed Central

    Chen-Levy, Z; Nourse, J; Cleary, M L

    1989-01-01

    We have identified a 24-kilodalton protein that is the product of the human bcl-2 gene, implicated as an oncogene because of its presence at the site of t(14;18) translocation breakpoints. The Bcl-2 protein was detected by specific, highly sensitive rabbit antibodies and was shown to be present in a number of human lymphoid cell lines and tissues, as well as in mouse B cells transfected with a bcl-2 cDNA construct. Characterization of the Bcl-2 protein demonstrated that it has a lipophilic nature and is associated with membrane structures, probably by means of its hydrophobic carboxy-terminal membrane-spanning domain. In t(14;18)-carrying cell lines, the protein is predominantly localized to the perinuclear endoplasmic reticulum, with a minor fraction in the plasma membrane. These properties, together with the observations that Bcl-2 does not have a characteristic signal peptide and is not glycosylated, suggest that it is an integral-membrane protein that spans the bilayer at its C-terminal hydrophobic region but is exposed only at the cytoplasmic surface. The relative abundance of the Bcl-2 protein in various human lymphoid cell lines correlated with transcription of the bcl-2 gene. The protein was abundant in all t(14;18)-carrying cell lines and lymphomas and was also found at lower levels in pre-B-cell lines and nonmalignant lymphoid tissues that do not carry t(14;18) translocations. These results suggest that the Bcl-2 protein is functional in normal B lymphocytes and that a quantitative difference in its expression may play a role in the pathogenesis of lymphomas carrying the t(14;18) translocation. Images PMID:2651903

  10. Single-protein study of photoresistance of pigment-protein complex in lipid bilayer

    NASA Astrophysics Data System (ADS)

    Uchiyama, Daisuke; Hoshino, Hajime; Otomo, Kohei; Kato, Taro; Onda, Ken-ichi; Watanabe, Akira; Oikawa, Hiroyuki; Fujiyoshi, Satoru; Matsushita, Michio; Nango, Mamoru; Watanabe, Natsuko; Sumino, Ayumi; Dewa, Takehisa

    2011-07-01

    Photoresistance of a pigment-binding membrane protein, light-harvesting 2 (LH2) complex from the photosynthetic bacterium, Rhodopseudomonas acidophila, was investigated by fluorescence of single LH2 complexes at a temperature of 296 K. Before irreversibly stopping fluorescence, a single LH2 complex in phospholipid bilayer of dimyristoylphosphatidylcholine (DMPC) emitted on average 4 times more fluorescence photons than a complex in detergent micelle of octylglucopyranoside (OG). Fluorescence-excitation spectrum of single LH2 complexes taken at 5 K showed that the LH2 complex is structurally less perturbed in DMPC bilayer than in OG micelle, suggesting that structural instability reduces photoresistance of LH2.

  11. 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.

  12. 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

  13. Heat capacity changes in carbohydrates and protein-carbohydrate complexes.

    PubMed

    Chavelas, Eneas A; García-Hernández, Enrique

    2009-05-13

    Carbohydrates are crucial for living cells, playing myriads of functional roles that range from being structural or energy-storage devices to molecular labels that, through non-covalent interaction with proteins, impart exquisite selectivity in processes such as molecular trafficking and cellular recognition. The molecular bases that govern the recognition between carbohydrates and proteins have not been fully understood yet. In the present study, we have obtained a surface-area-based model for the formation heat capacity of protein-carbohydrate complexes, which includes separate terms for the contributions of the two molecular types. The carbohydrate model, which was calibrated using carbohydrate dissolution data, indicates that the heat capacity contribution of a given group surface depends on its position in the saccharide molecule, a picture that is consistent with previous experimental and theoretical studies showing that the high abundance of hydroxy groups in carbohydrates yields particular solvation properties. This model was used to estimate the carbohydrate's contribution in the formation of a protein-carbohydrate complex, which in turn was used to obtain the heat capacity change associated with the protein's binding site. The model is able to account for protein-carbohydrate complexes that cannot be explained using a previous model that only considered the overall contribution of polar and apolar groups, while allowing a more detailed dissection of the elementary contributions that give rise to the formation heat capacity effects of these adducts.

  14. A Novel Four-Way Complex Variant Translocation Involving Chromosome 46,XY,t(4;9;19;22)(q25:q34;p13.3;q11.2) in a Chronic Myeloid Leukemia Patient

    PubMed Central

    Asif, Muhammad; Jamal, Mohammad Sarwar; Khan, Abdul Rehman; Naseer, Muhammad Imran; Hussain, Abrar; Choudhry, Hani; Malik, Arif; Khan, Shahida Aziz; Mahmoud, Maged Mostafa; Ali, Ashraf; Iram, Saima; Kamran, Kashif; Iqbal, Asim; Abduljaleel, Zainularifeen; Pushparaj, Peter Natesan; Rasool, Mahmood

    2016-01-01

    Philadelphia (Ph) chromosome (9;22)(q34;q11) is well established in more than 90% of chronic myeloid leukemia (CML) patients, and the remaining 5–8% of CML patients show variant and complex translocations, with the involvement of third, fourth, or fifth chromosome other than 9;22. However, in very rare cases, the fourth chromosome is involved. Here, we found a novel case of four-way Ph+ chromosome translocation involving 46,XY,t(4;9;19;22)(q25:q34;p13.3;q11.2) with CML in the chronic phase. Complete blood cell count of the CML patient was carried out to obtain total leukocytes count, hemoglobin, and platelets. Fluorescence in situ hybridization technique was used for the identification of BCR–ABL fusion gene, and cytogenetic test for the confirmation of Ph (9;22)(q34;q11) and the mechanism of variant translocation in the bone marrow. The patient is successfully treated with a dose of 400 mg/day imatinib mesylate (Gleevec). We observed a significant decrease in white blood cell count of 11.7 × 109/L after 48-month follow-up. Patient started feeling better generally. There was a reduction in the swelling of the body, fatigue, and anxiety. PMID:27303656

  15. Physiology in conservation translocations

    PubMed Central

    Tarszisz, Esther; Dickman, Christopher R.; Munn, Adam J.

    2014-01-01

    Conservation translocations aim to restore species to their indigenous ranges, protect populations from threats and/or reinstate ecosystem functions. They are particularly important for the conservation and management of rare and threatened species. Despite tremendous efforts and advancement in recent years, animal conservation translocations generally have variable success, and the reasons for this are often uncertain. We suggest that when little is known about the physiology and wellbeing of individuals either before or after release, it will be difficult to determine their likelihood of survival, and this could limit advancements in the science of translocations for conservation. In this regard, we argue that physiology offers novel approaches that could substantially improve translocations and associated practices. As a discipline, it is apparent that physiology may be undervalued, perhaps because of the invasive nature of some physiological measurement techniques (e.g. sampling body fluids, surgical implantation). We examined 232 publications that dealt with translocations of terrestrial vertebrates and aquatic mammals and, defining ‘success’ as high or low, determined how many of these studies explicitly incorporated physiological aspects into their protocols and monitoring. From this review, it is apparent that physiological evaluation before and after animal releases could progress and improve translocation/reintroduction successes. We propose a suite of physiological measures, in addition to animal health indices, for assisting conservation translocations over the short term and also for longer term post-release monitoring. Perhaps most importantly, we argue that the incorporation of physiological assessments of animals at all stages of translocation can have important welfare implications by helping to reduce the total number of animals used. Physiological indicators can also help to refine conservation translocation methods. These approaches fall

  16. Electrophoretic separation of proteins via complexation with a polyelectrolyte

    NASA Astrophysics Data System (ADS)

    Baskin, E. M.; Shklovskii, B. I.; Zilberstein, G. V.

    2003-01-01

    We suggest to augment standard isoelectric focusing for separation of proteins in a gradient of pH by a similar focusing in the presence of a strongly charged polyelectrolyte (PE). Proteins which have the same isoelectric point but different “hidden” charges of both signs in this point make complexes with PE, which focus in different pH. This is a result of charge inversion of such proteins by adsorbed PE molecules, which is sensitive to the hidden charge. Hence, the hidden charge is a new separation parameter.

  17. Characterization of Type Three Secretion System Translocator Interactions with Phospholipid Membranes.

    PubMed

    Adam, Philip R; Barta, Michael L; Dickenson, Nicholas E

    2017-01-01

    In vitro characterization of type III secretion system (T3SS) translocator proteins has proven challenging due to complex purification schemes and their hydrophobic nature that often requires detergents to provide protein solubility and stability. Here, we provide experimental details for several techniques that overcome these hurdles, allowing for the direct characterization of the Shigella translocator protein IpaB with respect to phospholipid membrane interaction. The techniques specifically discussed in this chapter include membrane interaction/liposome flotation, liposome sensitive fluorescence quenching, and protein-mediated liposome disruption assays. These assays have provided valuable insight into the role of IpaB in T3SS-mediated phospholipid membrane interactions by Shigella and should readily extend to other members of this important class of proteins.

  18. Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion.

    PubMed

    Ekiert, Damian C; Cox, Jeffery S

    2014-10-14

    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.

  19. Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    DOE PAGES

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-10-01

    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 in this paper, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), whichmore » 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. Furthermore, 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.« less

  20. Unwrapping of DNA-protein complexes under external stretching.

    PubMed

    Sakaue, Takahiro; Löwen, Hartmut

    2004-08-01

    A DNA-protein complex modeled by a semiflexible chain and an attractive spherical core is studied in the situation when an external stretching force is acting on one end monomer of the chain while the other end monomer is kept fixed in space. Without a stretching force, the chain is wrapped around the core. By applying an external stretching force, unwrapping of the complex is induced. We study the statics and dynamics of the unwrapping process by computer simulations and simple phenomenological theory. We find two different scenarios depending on the chain stiffness: For a flexible chain, the extension of the complex scales linearly with the external force applied. The sphere-chain complex is disordered; i.e., there is no clear winding of the chain around the sphere. For a stiff chain, on the other hand, the complex structure is ordered, which is reminiscent of nucleosome. There is a clear winding number, and the unwrapping process under external stretching is discontinuous with jumps of the distance-force curve. This is associated with discrete unwinding processes of the complex. Our predictions are of relevance for experiments, which measure force-extension curves of DNA-protein complexes, such as nucleosome, using optical tweezers.

  1. Neuroinflammation in healthy aging: a PET study using a novel Translocator Protein 18kDa (TSPO) radioligand, [(18)F]-FEPPA.

    PubMed

    Suridjan, I; Rusjan, P M; Voineskos, A N; Selvanathan, T; Setiawan, E; Strafella, A P; Wilson, A A; Meyer, J H; Houle, S; Mizrahi, R

    2014-01-01

    One of the cellular markers of neuroinflammation is increased microglia activation, characterized by overexpression of mitochondrial 18kDa Translocator Protein (TSPO). TSPO expression can be quantified in-vivo using the positron emission tomography (PET) radioligand [(18)F]-FEPPA. This study examined microglial activation as measured with [(18)F]-FEPPA PET across the adult lifespan in a group of healthy volunteers. We performed genotyping for the rs6971 TS.PO gene polymorphism to control for the known variability in binding affinity. Thirty-three healthy volunteers (age range: 19-82years; 22 high affinity binders (HAB), 11 mixed affinity binders (MAB)) underwent [(18)F]-FEPPA PET scans, acquired on the High Resolution Research Tomograph (HRRT) and analyzed using a 2-tissue compartment model. Regression analyses were performed to examine the effect of age adjusting for genetic status on [(18)F]-FEPPA total distribution volumes (VT) in the hippocampus, temporal, and prefrontal cortex. We found no significant effect of age on [(18)F]-FEPPA VT (F (1,30)=0.918; p=0.346), and a significant effect of genetic polymorphism (F (1,30)=8.767; p=0.006). This is the first in-vivo study to evaluate age-related changes in TSPO binding, using the new generation TSPO radioligands. Increased neuroinflammation, as measured with [(18)F]-FEPPA PET was not associated with normal aging, suggesting that healthy elderly individuals may serve as useful benchmark against patients with neurodegenerative disorders where neuroinflammation may be present.

  2. The 18 kDa translocator protein (peripheral benzodiazepine receptor) expression in the bone of normal, osteoprotegerin or low calcium diet treated mice.

    PubMed

    Kam, Winnie Wai-Ying; Meikle, Steven R; Zhou, Hong; Zheng, Yu; Blair, Julie M; Seibel, Marcus; Dunstan, Colin R; Banati, Richard B

    2012-01-01

    The presence of the translocator protein (TSPO), previously named as the mitochondrial or peripheral benzodiazepine receptor, in bone cells was studied in vitro and in situ using RT-qPCR, and receptor autoradiography using the selective TSPO ligand PK11195.In vitro, the TSPO is highly expressed in osteoblastic and osteoclastic cells.In situ, constitutive expression of TSPO is found in bone marrow and trabecular bone, e.g., spongiosa. Mice with a reduction of bone turnover induced by a 4-day treatment of osteoprotegerin reduces [(3)H]PK11195 binding in the spongiosa (320±128 Bq x mg(-1), 499±106 Bq x mg(-1) in saline-treated controls). In contrast, mice with an increase in bone turnover caused by a 4-day low calcium diet increases [(3)H]PK11195 binding in the spongiosa (615±90 Bq x mg(-1)). Further, our study includes technical feasibility data on [(18)F]fluoride microPET imaging of rodent bone with altered turnover. Despite [(18)F]fluoride having high uptake, the in vivo signal differences were small. Using a phantom model, we describe the spillover effect and partial volume loss that affect the quantitative microPET imaging of the small bone structures in experimental mouse models. In summary, we demonstrate the expression of TSPO in small rodent bone tissues, including osteoblasts and osteoclasts. A trend increase in TSPO expression was observed in the spongiosa from low to high bone turnover conditions. However, despite the potential utility of TSPO expression as an in vivo biomarker of bone turnover in experimental rodent models, our small animal PET imaging data using [(18)F]fluoride show that even under the condition of a good biological signal-to-noise ratio and high tracer uptake, the currently achievable instrument sensitivity and spatial resolution is unlikely to be sufficient to detect subtle differences in small structures, such as mouse bone.

  3. 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.

  4. Protein corona – from molecular adsorption to physiological complexity

    PubMed Central

    Docter, Dominic; Maskos, Michael

    2015-01-01

    Summary 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. PMID:25977856

  5. Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain

    SciTech Connect

    Radzimanowski, Jens; Beyreuther, Konrad; Sinning, Irmgard; Wild, Klemens

    2008-05-01

    Alzheimer’s disease is characterized by proteolytic processing of the amyloid precursor protein (APP), which releases the aggregation-prone amyloid-β (Aβ) peptide and liberates the intracellular domain (AICD) that interacts with various adaptor proteins. The crystallized AICD–Fe65-PTB2 complex is of central importance for APP translocation, nuclear signalling, processing and Aβ generation. Alzheimer’s disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid β peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 Å resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

  6. 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…

  7. Role of the import motor in insertion of transmembrane segments by the mitochondrial TIM23 complex.

    PubMed

    Popov-Čeleketić, Dušan; Waegemann, Karin; Mapa, Koyeli; Neupert, Walter; Mokranjac, Dejana

    2011-06-01

    The TIM23 complex mediates translocation of proteins across, and their lateral insertion into, the mitochondrial inner membrane. Translocation of proteins requires both the membrane-embedded core of the complex and its ATP-dependent import motor. Insertion of some proteins, however, occurs in the absence of ATP, questioning the need for the import motor during lateral insertion. We show here that the import motor associates with laterally inserted proteins even when its ATPase activity is not required. Furthermore, our results suggest a role for the import motor in lateral insertion. Thus, the import motor is involved in ATP-dependent translocation and ATP-independent lateral insertion.

  8. A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

    PubMed Central

    Lockard, Meghan A.; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2011-01-01

    We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit. PMID:21642284

  9. A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.

    PubMed

    Lockard, Meghan A; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B; Terwilliger, Thomas C; Waldo, Geoffrey S

    2011-07-01

    We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1-10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon(®) resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length 'breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit.

  10. Comparative Study of Elastic Network Model and Protein Contact Network for Protein Complexes: The Hemoglobin Case

    PubMed Central

    Di Paola, Luisa; Liang, Zhongjie; Giuliani, Alessandro

    2017-01-01

    The overall topology and interfacial interactions play key roles in understanding structural and functional principles of protein complexes. Elastic Network Model (ENM) and Protein Contact Network (PCN) are two widely used methods for high throughput investigation of structures and interactions within protein complexes. In this work, the comparative analysis of ENM and PCN relative to hemoglobin (Hb) was taken as case study. We examine four types of structural and dynamical paradigms, namely, conformational change between different states of Hbs, modular analysis, allosteric mechanisms studies, and interface characterization of an Hb. The comparative study shows that ENM has an advantage in studying dynamical properties and protein-protein interfaces, while PCN is better for describing protein structures quantitatively both from local and from global levels. We suggest that the integration of ENM and PCN would give a potential but powerful tool in structural systems biology. PMID:28243596

  11. Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry.

    PubMed

    Li, Huilin; Sheng, Yuewei; McGee, William; Cammarata, Michael; Holden, Dustin; Loo, Joseph A

    2017-03-07

    Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

  12. Transmembrane protein 147 (TMEM147) is a novel component of the Nicalin-NOMO protein complex.

    PubMed

    Dettmer, Ulf; Kuhn, Peer-Hendrik; Abou-Ajram, Claudia; Lichtenthaler, Stefan F; Krüger, Marcus; Kremmer, Elisabeth; Haass, Christian; Haffner, Christof

    2010-08-20

    Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely gamma-secretase and the Nicalin-NOMO (Nodal modulator) complex. The gamma-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the beta-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A approximately 22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to gamma-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with gamma-secretase.

  13. Transmembrane Protein 147 (TMEM147) Is a Novel Component of the Nicalin-NOMO Protein Complex*

    PubMed Central

    Dettmer, Ulf; Kuhn, Peer-Hendrik; Abou-Ajram, Claudia; Lichtenthaler, Stefan F.; Krüger, Marcus; Kremmer, Elisabeth; Haass, Christian; Haffner, Christof

    2010-01-01

    Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely γ-secretase and the Nicalin-NOMO (Nodal modulator) complex. The γ-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the β-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200–220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A ∼22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to γ-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with γ-secretase. PMID:20538592

  14. Guanine-nucleotide exchange on ribosome-bound elongation factor G initiates the translocation of tRNAs

    PubMed Central

    Zavialov, Andrey V; Hauryliuk, Vasili V; Ehrenberg, Måns

    2005-01-01

    Background During the translation of mRNA into polypeptide, elongation factor G (EF-G) catalyzes the translocation of peptidyl-tRNA from the A site to the P site of the ribosome. According to the 'classical' model, EF-G in the GTP-bound form promotes translocation, while hydrolysis of the bound GTP promotes dissociation of the factor from the post-translocation ribosome. According to a more recent model, EF-G operates like a 'motor protein' and drives translocation of the peptidyl-tRNA after GTP hydrolysis. In both the classical and motor protein models, GDP-to-GTP exchange is assumed to occur spontaneously on 'free' EF-G even in the absence of a guanine-nucleotide exchange factor (GEF). Results We have made a number of findings that challenge both models. First, free EF-G in the cell is likely to be in the GDP-bound form. Second, the ribosome acts as the GEF for EF-G. Third, after guanine-nucleotide exchange, EF-G in the GTP-bound form moves the tRNA2-mRNA complex to an intermediate translocation state in which the mRNA is partially translocated. Fourth, subsequent accommodation of the tRNA2-mRNA complex in the post-translocation state requires GTP hydrolysis. Conclusion These results, in conjunction with previously published cryo-electron microscopy reconstructions of the ribosome in various functional states, suggest a novel mechanism for translocation of tRNAs on the ribosome by EF-G. Our observations suggest that the ribosome is a universal guanosine-nucleotide exchange factor for EF-G as previously shown for the class-II peptide-release factor 3. PMID:15985150

  15. Modeling of Protein Binary Complexes Using Structural Mass Spectrometry Data

    SciTech Connect

    Amisha Kamal,J.; Chance, M.

    2008-01-01

    In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints--positive and/or negative--in the docking step and are also used to decide the type of energy filter--electrostatics or desolvation--in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure.

  16. Exploration of the Dynamic Properties of Protein Complexes Predicted from Spatially Constrained Protein-Protein Interaction Networks

    PubMed Central

    Yen, Eric A.; Tsay, Aaron; Waldispuhl, Jerome; Vogel, Jackie

    2014-01-01

    Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and visualization of the hidden

  17. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*

    PubMed Central

    Turk, Rolf; Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Pospisil, Tyler C.; Jones, Kayla S.; Campbell, Kevin P.; Wright, Michael E.

    2016-01-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  18. Heterodimeric Drosophila gap gene protein complexes acting as transcriptional repressors.

    PubMed Central

    Sauer, F; Jäckle, H

    1995-01-01

    The Drosophila gap gene Krüppel (Kr) encodes a transcriptional regulator. It acts both as an integral part of the Drosophila segmentation gene in the early blastoderm and in a variety of tissues and organs at later stages of embryogenesis. In transfected tissue culture cells, the Kr protein (Kr) was shown to both activate and repress gene expression in a concentration-dependent manner when acting from a single binding site close to the promoter. Here we show that KR can associate with the transcription factors encoded by the gap genes knirps (kni) and hunchback (hb) which affect KR-dependent gene expression in Drosophila tissue culture cells. The association of DNA-bound hb protein or free kni protein with distinct but different regions of KR results in the formation of DNA-bound transcriptional repressor complexes. Our results suggest that individual transcription factors can associate to form protein complexes which act as direct repressors of transcription. The interactions shown here add an unexpected level of complexity to the control of gene expression. Images PMID:7588607

  19. Characterization of the human GARP (Golgi associated retrograde protein) complex

    SciTech Connect

    Liewen, Heike; Meinhold-Heerlein, Ivo; Oliveira, Vasco; Schwarzenbacher, Robert; Luo Guorong; Wadle, Andreas; Jung, Martin; Pfreundschuh, Michael; Stenner-Liewen, Frank . E-mail: stenlie@t-online.de

    2005-05-15

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.

  20. Changes in protein structure at the interface accompanying complex formation.

    PubMed

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H; Chakrabarti, Pinak

    2015-11-01

    Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein-Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial.

  1. The RCP-Rab11 complex regulates endocytic protein sorting.

    PubMed

    Peden, Andrew A; Schonteich, Eric; Chun, John; Junutula, Jagath R; Scheller, Richard H; Prekeris, Rytis

    2004-08-01

    Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP-Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes.

  2. 2D depiction of nonbonding interactions for protein complexes.

    PubMed

    Zhou, Peng; Tian, Feifei; Shang, Zhicai

    2009-04-30

    A program called the 2D-GraLab is described for automatically generating schematic representation of nonbonding interactions across the protein binding interfaces. The input file of this program takes the standard PDB format, and the outputs are two-dimensional PostScript diagrams giving intuitive and informative description of the protein-protein interactions and their energetics properties, including hydrogen bond, salt bridge, van der Waals interaction, hydrophobic contact, pi-pi stacking, disulfide bond, desolvation effect, and loss of conformational entropy. To ensure these interaction information are determined accurately and reliably, methods and standalone programs employed in the 2D-GraLab are all widely used in the chemistry and biology community. The generated diagrams allow intuitive visualization of the interaction mode and binding specificity between two subunits in protein complexes, and by providing information on nonbonding energetics and geometric characteristics, the program offers the possibility of comparing different protein binding profiles in a detailed, objective, and quantitative manner. We expect that this 2D molecular graphics tool could be useful for the experimentalists and theoreticians interested in protein structure and protein engineering.

  3. The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes.

    PubMed

    Paleotti, Olivia; Macia, Eric; Luton, Frederic; Klein, Stephanie; Partisani, Mariagrazia; Chardin, Pierre; Kirchhausen, Tom; Franco, Michel

    2005-06-03

    The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.

  4. Regulation of nuclear translocation of nuclear factor-kappaB relA: evidence for complex dynamics at the single-cell level.

    PubMed Central

    Schooley, Kenneth; Zhu, Ping; Dower, Steven K; Qwarnström, Eva E

    2003-01-01

    We have analysed activation of nuclear factor-kappaB (NF-kappaB) in response to interleukin-1 (IL-1) in human fibroblasts by tracking intracellular distribution and levels of endogenous relA, NF-kappaB1 and inhibitor of kappaB (I-kappaB) alpha using semi-quantitative confocal microscopy. Nuclear translocation of endogenous relA correlated with I-kappaBalpha degradation during stimulation with IL-1, whereas no effects were seen on levels or localization of NF-kappaB1. During pathway activation, relA was transported up a concentration gradient, resulting in a 3-4-fold increase in nuclear levels, but without any significant decrease in cytoplasmic concentration. IL-1 stimulation caused translocation of only 20% of the relA, but resulted in degradation of up to 70% of the cytoplasmic I-kappaBalpha. RelA nuclear translocation in fibroblasts correlated with DNA-binding activity measured by electrophoretic mobility shift assay (EMSA), both with respect to kinetics and IL-1 concentration-dependence. Clonal populations of cells demonstrated a marked degree of heterogeneity in the response to IL-1. The single-cell assay revealed the presence of responder and non-responder subpopulations, with an enhanced proportion of responder cells, and prolonged responses at higher concentrations of IL-1. Comparing different cell types demonstrated that whereas HepG2 cells, as fibroblasts, showed good correlation between nuclear translocation of relA and activation of DNA binding by relA-containing dimers, EL4 thymoma cells showed no effect on relA localization, even during induction of significant levels NF-kappaB activity, as measured by EMSA. The analysis shows that stimulation by IL-1 results in transient perturbation of the NF-kappaB system, which cycles between the resting and active states with net redistribution of a minor proportion of its DNA-binding component. In addition, it demonstrates significant cell-to-cell variations, as well as cell-type-specific differences in net rel

  5. Homology modelling of protein-protein complexes: a simple method and its possibilities and limitations

    PubMed Central

    Launay, Guillaume; Simonson, Thomas

    2008-01-01

    Background Structure-based computational methods are needed to help identify and characterize protein-protein complexes and their function. For individual proteins, the most successful technique is homology modelling. We investigate a simple extension of this technique to protein-protein complexes. We consider a large set of complexes of known structures, involving pairs of single-domain proteins. The complexes are compared with each other to establish their sequence and structural similarities and the relation between the two. Compared to earlier studies, a simpler dataset, a simpler structural alignment procedure, and an additional energy criterion are used. Next, we compare the Xray structures to models obtained by threading the native sequence onto other, homologous complexes. An elementary requirement for a successful energy function is to rank the native structure above any threaded structure. We use the DFIREβ energy function, whose quality and complexity are typical of the models used today. Finally, we compare near-native models to distinctly non-native models. Results If weakly stable complexes are excluded (defined by a binding energy cutoff), as well as a few unusual complexes, a simple homology principle holds: complexes that share more than 35% sequence identity share similar structures and interaction modes; this principle was less clearcut in earlier studies. The energy function was then tested for its ability to identify experimental structures among sets of decoys, produced by a simple threading procedure. On average, the experimental structure is ranked above 92% of the alternate structures. Thus, discrimination of the native structure is good but not perfect. The discrimination of near-native structures is fair. Typically, a single, alternate, non-native binding mode exists that has a native-like energy. Some of the associated failures may correspond to genuine, alternate binding modes and/or native complexes that are artefacts of the crystal

  6. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  7. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    NASA Astrophysics Data System (ADS)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  8. A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

    PubMed Central

    Yeakley, J M; Morfin, J P; Rosenfeld, M G; Fu, X D

    1996-01-01

    A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8755518

  9. Changes in protein structure at the interface accompanying complex formation

    PubMed Central

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H.; Chakrabarti, Pinak

    2015-01-01

    Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein–Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial. PMID:26594372

  10. Protein Complex Affinity Capture from Cryomilled Mammalian Cells.

    PubMed

    LaCava, John; Jiang, Hua; Rout, Michael P

    2016-12-09

    Affinity capture is an effective technique for isolating endogenous protein