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Sample records for 18kda translocator protein

  1. The effects of PK11195 on meningioma was associated with allopregnanolone biosynthesis, which was mediated by translocator protein 18 KDa.

    PubMed

    Gao, Zhuo-Wei; Huang, Jing-Bin; Lin, Qing; Qin, Qiang; Liang, Yao-Jun; Zhou, Lu; Luo, Min

    2016-01-01

    Meningioma is one of the common brain tumors in adults. It had been shown that the allopregnanolone biosynthesis was associated with tumorigenesis and PK11195, the translocator protein 18 KDa (TSPO) antagonist, had the effects of the allopregnanolone biosynthesis. However, little is known about the association between the effects of PK11195 on meningioma and the allopregnanolone biosynthesis. To evaluate this, the meningioma cell line IOMM-LEE was applied. Cell viability and proliferation were determined by CCK-8 assay. The IC50 of PK11195 on the IOMM-LEE was 1.505 ± 0.08 nM. The cell viability and proliferation of AC-5216 (TSPO selective ligand, 2 and 4 nM) was blocked by PK11195 (1.5 nM). Further, we evaluated the role of allopregnanolone biosynthesis in the effects of TSPO on meningioma. Enzyme-Linked ImmunoSorbent Assay (ELISA) was used in the measurement of the allopregnanolone level. It showed that the allopregnanolone level was increased by AC-5216 (2 and 4 nM) and the increase was reversed by PK11195 (1.5 nM). Collectedly, it firstly indicated that the effects of PK11195 on meningioma were relevant to the decrease of allopregnanolone biosynthesis, which was mediated by TSPO.

  2. 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

  3. CLONING AND EXPRESSION OF THE TRANSLOCATOR PROTEIN (18 KDA), VOLTAGE-DEPENDENT ANION CHANNEL, AND DIAZEPAM BINDING INHIBITOR IN THE GONAD OF LARGEMOUTH BASS (MICROPTERUS SALMOIDES) ACROSS THE REPRODUCTIVE CYCLE

    PubMed Central

    Doperalski, Nicholas J.; Martyniuk, Christopher J.; Prucha, Melinda S.; Kroll, Kevin J.; Denslow, Nancy D.; Barber, David S.

    2011-01-01

    Cholesterol transport across the mitochondrial membrane is rate-limiting for steroidogenesis in vertebrates. Previous studies in fish have characterized expression of the steroidogenic acute regulatory protein, however the function and regulation of other genes and proteins involved in piscine cholesterol transport have not been evaluated. In the current study, mRNA sequences of the 18 kDa translocator protein (tspo; formerly peripheral benzodiazepine receptor), voltage-dependent anion channel (vdac), and diazepam binding inhibitor (dbi; also acyl-CoA binding protein) were cloned from largemouth bass. Gonadal expression was examined across reproductive stages to determine if expression is correlated with changes in steroid levels and with indicators of reproductive maturation. In testis, transcript abundance of tspo and dbi increased with reproductive maturation (6- and 23-fold maximal increase, respectively) and expression of tspo and dbi was positively correlated with reproductive stage, gonadosomatic index (GSI), and circulating levels of testosterone. Testis vdac expression was positively correlated with reproductive stage and GSI. In females, gonadal tspo and vdac expression was negatively correlated with GSI and levels of plasma testosterone and 17β-estradiol. Ovarian dbi expression was not correlated with indicators of reproductive maturation. These studies represent the first investigation of the steroidogenic role of tspo, vdac, and dbi in fish. Findings suggest that cholesterol transport in largemouth bass testis, but not ovary, may be transcriptionally-regulated, however further investigation will be necessary to fully elucidate the role of these genes in largemouth bass steroidogenesis. PMID:21600210

  4. Translocator protein: pharmacology and steroidogenesis.

    PubMed

    Midzak, Andrew; Zirkin, Barry; Papadopoulos, Vassilios

    2015-08-01

    The translocator protein (TSPO; 18k Da) is an evolutionarily conserved outer mitochondrial membrane (OMM) protein highly expressed in steroid-synthesizing cells and found to possess a number of physiological and drug-binding partners. Extensive pharmacological, biochemical and cell biological research over the years has led to a model of TSPO involvement in mitochondrial cholesterol transport and promotion of steroid synthesis, a model guiding the design of drugs useful in stimulating neurosteroid synthesis and alleviating psychopathological symptoms. The involvement of TSPO in these processes has been called into question; however, with the publication of TSPO-deletion mouse models which saw no changes in steroid production. Here, we review work characterizing TSPO in steroidogenesis and offer perspective to research into TSPO pharmacology and its involvement in steroid biosynthesis. © 2015 Authors; published by Portland Press Limited.

  5. Computer simulations and theory of protein translocation.

    PubMed

    Makarov, Dmitrii E

    2009-02-17

    The translocation of proteins through pores is central to many biological phenomena, such as mitochondrial protein import, protein degradation, and delivery of protein toxins to their cytosolic targets. Because proteins typically have to pass through constrictions that are too narrow to accommodate folded structures, translocation must be coupled to protein unfolding. The simplest model that accounts for such co-translocational unfolding assumes that both translocation and unfolding are accomplished by pulling on the end of the polypeptide chain mechanically. In this Account, we describe theoretical studies and computer simulations of this model and discuss how the time scales of translocation depend on the pulling force and on the protein structure. Computationally, this is a difficult problem because biologically or experimentally relevant time scales of translocation are typically orders of magnitude slower than those accessible by fully atomistic simulations. For this reason, we explore one-dimensional free energy landscapes along suitably defined translocation coordinates and discuss various approaches to their computation. We argue that the free energy landscape of translocation is often bumpy because confinement partitions the protein's configuration space into distinct basins of attraction separated by large entropic barriers. Favorable protein-pore interactions and nonnative interactions within the protein further contribute to the complexity. Computer simulations and simple scaling estimates show that forces of just 2-6 pN are often sufficient to ensure transport of unstructured polypeptides, whereas much higher forces are typically needed to translocate folded protein domains. The unfolding mechanisms found from simulations of translocation are different from those observed in the much better understood case of atomic force microscopy (AFM) pulling studies, in which proteins are unraveled by stretching them between their N- and C-termini. In contrast to

  6. 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.

  7. Ratcheting up protein translocation with anthrax toxin.

    PubMed

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

    2012-05-01

    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. Copyright © 2012 The Protein Society.

  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. 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

  10. 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.

  11. 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

  12. Translocation of knotted proteins through a pore

    NASA Astrophysics Data System (ADS)

    Szymczak, P.

    2014-09-01

    We report the results of molecular dynamics simulations of translocation of knotted proteins through pores. The protein is pulled into the pore with a constant force, which in many cases leads to the tightening of the knot. Since the radius of tightened knot is larger than that of the pore opening, the tight knot can block the pore thus preventing further translocation of the chain. Analyzing six different proteins, we show that the stuck probability increases with the applied force and that final positions of the tightened knot along the protein backbone are not random but are usually associated with sharp turns in the polypeptide chain. The combined effect of the confining geometry of the pore and the inhomogeneous character of the protein chain leads thus to the appearance of topological traps, which can immobilize the knot and lead to the jamming of the pore.

  13. 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.

  14. The Tat-dependent protein translocation pathway.

    PubMed

    Hou, Bo; Brüser, Thomas

    2011-12-01

    The twin-arginine translocation (Tat) pathway is found in bacteria, archaea, and plant chloroplasts, where it is dedicated to the transmembrane transport of fully folded proteins. These proteins contain N-terminal signal peptides with a specific Tat-system binding motif that is recognized by the transport machinery. In contrast to other protein transport systems, the Tat system consists of multiple copies of only two or three usually small (∼8-30 kDa) membrane proteins that oligomerize to two large complexes that transiently interact during translocation. Only one of these complexes includes a polytopic membrane protein, TatC. The other complex consists of TatA. Tat systems of plants, proteobacteria, and several other phyla contain a third component, TatB. TatB is evolutionarily and structurally related to TatA and usually forms tight complexes with TatC. Minimal two-component Tat systems lacking TatB are found in many bacterial and archaeal phyla. They consist of a 'bifunctional' TatA that also covers TatB functionalities, and a TatC. Recent insights into the structure and interactions of the Tat proteins have various important implications.

  15. Computational and theoretical insights into protein and peptide translocation.

    PubMed

    Makarov, Dmitrii E

    2014-03-01

    Biological processes such as protein degradation and mitochondrial protein import require protein passage, or translocation, across narrow pores. In addition to its biological significance, protein translocation through biological or engineered nanopores offers a powerful analytic tool for biophysics and nanotechnology. This mini-review discusses the physical mechanisms of protein translocation, as revealed by computational and theoretical studies. A simple, simulation-based model of translocation is presented, which provides a comprehensive description of this process and allows one to estimate experimentally observable quantities such as the dwell time of a protein inside the pore and the frequency of translocation events. Limitations of this model are further described and possible strategies to overcome them are outlined. Recent simulation studies are beginning to provide insights into the physical mechanisms that drive protein translocation in living systems, which are also discussed here.

  16. 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.

  17. Are proteins translocated through pores? An historical overview.

    PubMed

    Henry, Jean-Pierre

    2014-03-01

    Most proteinaceous pores are characterized as ionic channels. However, some are also involved in protein translocation through phospholipidic membranes. This concept has evolved slowly in cell biology and in biophysics, requiring the development of adapted electrical and biochemical methods. Protein translocation in mitochondria biogenesis, secretion by endoplasmic reticulum or bacteria, and bacterial toxins internalization are the main fields where proteinconducting pores have been described. The concept is now well established and progress at the molecular and atomic levels have shown how different this paradigm is from ionic channels involved in neurobiology. Protein-conducting pores are often parts of large complexes and electrical analysis gives on-line information at the single-molecule level. They have a large conductance that, in certain membranes, should be highly regulated to prevent ionic leaking through the membrane. Finally, they are involved not only in protein translocation, but also in membrane protein insertion (α-helix and β-barrel types).

  18. 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

  19. 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).

  20. Protein translocation by the Sec61/SecY channel.

    PubMed

    Osborne, Andrew R; Rapoport, Tom A; van den Berg, Bert

    2005-01-01

    The conserved protein-conducting channel, referred to as the Sec61 channel in eukaryotes or the SecY channel in eubacteria and archaea, translocates proteins across cellular membranes and integrates proteins containing hydrophobic transmembrane segments into lipid bilayers. Structural studies illustrate how the protein-conducting channel accomplishes these tasks. Three different mechanisms, each requiring a different set of channel binding partners, are employed to move polypeptide substrates: The ribosome feeds the polypeptide chain directly into the channel, a ratcheting mechanism is used by the eukaryotic endoplasmic reticulum chaperone BiP, and a pushing mechanism is utilized by the bacterial ATPase SecA. We review these translocation mechanisms, relating biochemical and genetic observations to the structures of the protein-conducting channel and its binding partners.

  1. Plastid Protein Targeting: Preprotein Recognition and Translocation.

    PubMed

    Chotewutmontri, P; Holbrook, K; Bruce, B D

    2017-01-01

    Eukaryotic organisms are defined by their endomembrane system and various organelles. The membranes that define these organelles require complex protein sorting and molecular machines that selectively mediate the import of proteins from the cytosol to their functional location inside the organelle. The plastid possibly represents the most complex system of protein sorting, requiring many different translocons located in the three membranes found in this organelle. Despite having a small genome of its own, the vast majority of plastid-localized proteins is nuclear encoded and must be posttranslationally imported from the cytosol. These proteins are encoded as a larger molecular weight precursor that contains a special "zip code," a targeting sequence specific to the intended final destination of a given protein. The "zip code" is located at the precursor N-terminus, appropriately called a transit peptide (TP). We aim to provide an overview of plastid trafficking with a focus on the mechanism and regulation of the general import pathway, which serves as a central import hub for thousands of proteins that function in the plastid. We extend comparative analysis of plant proteomes to develop a better understanding of the evolution of TPs and differential TP recognition. We also review alternate import pathways, including vesicle-mediated trafficking, dual targeting, and import of signal-anchored and tail-anchored proteins. © 2017 Elsevier Inc. All rights reserved.

  2. Twin-arginine-dependent translocation of folded proteins.

    PubMed

    Fröbel, Julia; Rose, Patrick; Müller, Matthias

    2012-04-19

    Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF.

  3. Twin-arginine-dependent translocation of folded proteins

    PubMed Central

    Fröbel, Julia; Rose, Patrick; Müller, Matthias

    2012-01-01

    Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF. PMID:22411976

  4. 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

  5. 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.

  6. 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

  7. Ratcheting in post-translational protein translocation: a mathematical model.

    PubMed

    Liebermeister, W; Rapoport, T A; Heinrich, R

    2001-01-19

    We have developed a non-steady-state mathematical model describing post-translational protein translocation across the endoplasmic reticulum membrane. Movement of the polypeptide chain through the channel in the endoplasmic reticulum membrane is considered to be a stochastic process which is biased at the lumenal side of the channel by the binding of BiP (Kar2p), a member of the Hsp70 family of ATPases (ratcheting model). Assuming that movement of the chain through the channel is caused by passive diffusion (Brownian ratchet), the model describes all available experimental data. The optimum set of model parameters indicates that the ratcheting mechanism functions at near-maximum rate, being relatively insensitive to variations of the association or dissociation rate constants of BiP or its concentration. The estimated rate constant for diffusion of a polypeptide inside the channel indicates that the chain makes contact with the walls of the channel. Since fitting of the model to the data required that the backward rate constant be larger than the forward constant during early diffusion steps, translocation must occur against a force. The latter may arise, for example, from the unfolding of the polypeptide chain in the cytosol. Our results indicate that the ratchet can transport polypeptides against a free energy of about 25 kJ/mol without significant retardation of translocation. The modeling also suggests that the BiP ratchet is optimized, allowing fast translocation to be coupled with minimum consumption of ATP and rapid dissociation of BiP in the lumen of the ER. Finally, we have estimated the maximum hydrophobicity of a polypeptide segment up to which lateral partitioning from the channel into the lipid phase does not result in significant retardation of translocation. Copyright 2001 Academic Press.

  8. TRANSLOCATOR PROTEIN-MEDIATED PHARMACOLOGY OF CHOLESTEROL TRANSPORT AND STEROIDOGENESIS

    PubMed Central

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

    2015-01-01

    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. PMID:25818881

  9. 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.

  10. Biophysical Characterization of the Type III Secretion System Translocator Proteins and the Translocator Proteins Attached to Bacterium-Like Particles.

    PubMed

    Chen, Xiaotong; Choudhari, Shyamal P; Kumar, Prashant; Toth, Ronald T; Kim, Jae Hyun; Van Roosmalen, Maarten L; Leenhouts, Kees; Middaugh, C Russell; Picking, Wendy L; Picking, William D

    2015-12-01

    Diarrhea caused by Shigella, Salmonella, and Yersinia is an important public health problem, but development of safe and effective vaccines against such diseases is challenging. A new antigen delivery platform called bacterium-like particles (BLPs) was explored as a means for delivering protective antigens from the type III secretion systems (T3SS) of these pathogens. BLPs are peptidoglycan skeletons derived from Lactococcus lactis that are safe for newborns and can carry multiple antigens. Hydrophobic T3SS translocator proteins were fused to a peptidoglycan anchor (PA) for BLP attachment. The proteins and protein-BLP complexes associated with BLPs were characterized and the resulting data used to create three-index empirical phase diagrams (EPDs). On the basis of these EPDs, IpaB (Shigella) and SipB (Salmonella) behave distinctly from YopB (Yersinia) under different environmental stresses. Adding the PA domain appears to enhance the stability of both the PA and translocator proteins, which was confirmed using differential scanning calorimetry, and although the particles dominated the spectroscopic signals in the protein-loaded BLPs, structural changes in the proteins were still detected. The protein-BLPs were most stable near neutral pH, but these proteins' hydrophobicity made them sensitive to environmental stresses. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  11. Coliphage HK022 Nun protein inhibits RNA polymerase translocation

    PubMed Central

    Vitiello, Christal L.; Kireeva, Maria L.; Lubkowska, Lucyna; Kashlev, Mikhail; Gottesman, Max

    2014-01-01

    The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA–DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β′ subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5′ edge of the RNA–DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors. PMID:24853501

  12. Enigmatic Translocator protein (TSPO) and cellular stress regulation.

    PubMed

    Batoko, Henri; Veljanovski, Vasko; Jurkiewicz, Pawel

    2015-09-01

    Translocator proteins (TSPOs) are conserved, ubiquitous membrane proteins identified initially as benzodiazepine-binding proteins in mammalian cells. Recent genetic and biochemical studies have challenged the accepted model that TSPOs are essential and required for steroidogenesis in animal cells. Instead, evidence from different kingdoms of life suggests that TSPOs are encoded by nonessential genes that are temporally upregulated in cells encountering conditions of oxidative stress, including inflammation and tissue injury. Here we discuss how TSPOs may be involved in complex homeostasis signaling mechanisms. We suggest that the main physiological role of TSPOs may be to modulate oxidative stress, irrespective of the cell type or subcellular localization, in part through the subtle regulation of tetrapyrrole metabolism. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. 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.

  14. Mechanisms of Sec61/SecY-mediated protein translocation across membranes.

    PubMed

    Park, Eunyong; Rapoport, Tom A

    2012-01-01

    The Sec61 or SecY channel, a universally conserved protein-conducting channel, translocates proteins across and integrates proteins into the eukaryotic endoplasmic reticulum (ER) membrane and the prokaryotic plasma membrane. Depending on channel-binding partners, polypeptides are moved by different mechanisms. In cotranslational translocation, the ribosome feeds the polypeptide chain directly into the channel. In posttranslational translocation, a ratcheting mechanism is used by the ER-lumenal chaperone BiP in eukaryotes, and a pushing mechanism is utilized by the SecA ATPase in bacteria. In prokaryotes, posttranslational translocation is facilitated through the function of the SecD/F protein. Recent structural and biochemical data show how the channel opens during translocation, translocates soluble proteins, releases hydrophobic segments of membrane proteins into the lipid phase, and maintains the barrier for small molecules.

  15. Minireview: Translocator Protein (TSPO) and Steroidogenesis: A Reappraisal

    PubMed Central

    Stocco, Douglas M.; Tu, Lan N.

    2015-01-01

    The 18-kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a transmembrane protein in the outer mitochondrial membrane. TSPO has long been described as being indispensable for mitochondrial cholesterol import that is essential for steroid hormone production. In contrast to this initial proposition, recent experiments reexamining TSPO function have demonstrated that it is not involved in steroidogenesis. This fundamental change has forced a reexamination of the functional interpretations made for TSPO that broadly impacts both basic and clinical research across multiple fields. In this minireview, we recapitulate the key studies from 25 years of TSPO research and concurrently examine their limitations that perhaps led towards the incorrect association of TSPO and steroid hormone production. Although this shift in understanding raises new questions regarding the molecular function of TSPO, these recent developments are poised to have a significant positive impact for research progress in steroid endocrinology. PMID:25730708

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

    PubMed

    Szymczak, Piotr

    2016-03-21

    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.

  17. 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

  18. 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.

  19. 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

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

    PubMed

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

    2016-04-01

    The mitochondrial translocator protein (TSPO) is a high affinity cholesterol binding protein which is primarily located in the outer mitochondrial membrane where it has been shown to interact with proteins implicated in mitochondrial permeability transition pore (mPTP) formation. TSPO is found in different species and is expressed at high levels in tissues that synthesize steroids but is also present in other peripheral tissues especially in the heart. TSPO has been involved in the import of cholesterol into mitochondria, a key step in steroidogenesis. This constitutes the main established function of the protein which was recently challenged by genetic studies. TSPO has also been associated directly or indirectly with a wide range of cellular functions such as apoptosis, cell proliferation, differentiation, regulation of mitochondrial function or porphyrin transport. In the heart the role of TSPO remains undefined but a growing body of evidence suggests that TSPO plays a critical role in regulating physiological cardiac function and that TSPO ligands may represent interesting drugs to protect the heart under pathological conditions. This article briefly reviews current knowledge regarding TSPO and discusses its role in the cardiovascular system under physiological and pathologic conditions. More particularly, it provides evidence that TSPO can represent an alternative strategy to develop new pharmacological agents to protect the myocardium against ischemia-reperfusion injury. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-09-01

    The translocator protein 18 kDa (TSPO) has been the focus of intense research by the biomedical community and the pharmaceutical industry because of its apparent involvement in many disease-related processes. These include steroidogenesis, apoptosis, inflammation, neurological disease and cancer, resulting in the use of TSPO as a biomarker and its potential as a drug target. Despite more than 30 years of study, the precise function of TSPO remains elusive. A recent breakthrough in determining the high-resolution crystal structures of bacterial homologs of mitochondrial TSPO provides new insight into the structural and functional properties at a molecular level and new opportunities for investigating the significance of this ancient and highly conserved protein family. The availability of atomic level structural information from different species also provides a platform for structure-based drug development. Here we briefly review current knowledge regarding TSPO and the implications of the new structures with respect to hypotheses and controversies in the field. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. Controlling the translocation of proteins through nanopores with bioinspired fluid walls

    PubMed Central

    Yusko, Erik C.; Johnson, Jay M.; Majd, Sheereen; Prangkio, Panchika; Rollings, Ryan C.; Li, Jiali; Yang, Jerry; Mayer, Michael

    2011-01-01

    Synthetic nanopores have been used to study individual biomolecules in high thoroughput but their performance as sensors does not match biological ion channels. Controlling the translocation times of single-molecule analytes and their non-specific interaction with pore walls remain a challenge. Inspired by the olfactory sensilla of the insect antenna, here we show that coating nanopores with fluid bilayer lipids allows the pore diameters to be fine-tuned in sub-nanometre increments. Incorporation of mobile ligands in the lipid conferred specificity and slowed down the translocation of targeted proteins sufficiently to time-resolve translocation events of individual proteins. The lipid coatings also prevented pores from clogging, eliminated non-specific binding and enabled the translocation of amyloid-beta (Aβ) oligomers and fibrils. Through combined analysis of translocation time, volume, charge, shape and ligand affinity, different proteins were identified. PMID:21336266

  8. 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.

  9. 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

  10. 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.

  11. Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein.

    PubMed

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

    2013-08-09

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

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

    PubMed Central

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

    2013-01-01

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

  13. Translocator Protein PET Imaging in a Preclinical Prostate Cancer Model.

    PubMed

    Tantawy, Mohammed N; Charles Manning, H; Peterson, Todd E; Colvin, Daniel C; Gore, John C; Lu, Wenfu; Chen, Zhenbang; Chad Quarles, C

    2017-08-18

    The identification and targeting of biomarkers specific to prostate cancer (PCa) could improve its detection. Given the high expression of translocator protein (TSPO) in PCa, we investigated the use of [(18)F]VUIIS1008 (a novel TSPO-targeting radioligand) coupled with positron emission tomography (PET) to identify PCa in mice and to characterize their TSPO uptake. Pten(pc-/-), Trp53(pc-/-) prostate cancer-bearing mice (n = 9, 4-6 months old) were imaged in a 7T MRI scanner for lesion localization. Within 24 h, the mice were imaged using a microPET scanner for 60 min in dynamic mode following a retro-orbital injection of ~ 18 MBq [(18)F]VUIIS1008. Following imaging, tumors were harvested and stained with a TSPO antibody. Regions of interest (ROIs) were drawn around the tumor and muscle (hind limb) in the PET images. Time-activity curves (TACs) were recorded over the duration of the scan for each ROI. The mean activity concentrations between 40 and 60 min post radiotracer administration between tumor and muscle were compared. Tumor presence was confirmed by visual inspection of the MR images. The uptake of [(18)F]VUIIS1008 in the tumors was significantly higher (p < 0.05) than that in the muscle, where the percent injected dose per unit volume for tumor was 7.1 ± 1.6 % ID/ml and that of muscle was < 1 % ID/ml. In addition, positive TSPO expression was observed in tumor tissue analysis. The foregoing preliminary data suggest that TSPO may be a useful biomarker of PCa. Therefore, using TSPO-targeting PET ligands, such as [(18)F]VUIIS1008, may improve PCa detectability and characterization.

  14. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Measurement of Effector Protein Translocation Using Phosphorylatable Epitope Tags and Phospho-Specific Antibodies.

    PubMed

    Bartra, Sara Schesser; Plano, Gregory V

    2017-01-01

    Numerous bacterial pathogens employ specialized protein secretion machineries to directly inject anti-host proteins, termed effector proteins, into eukaryotic cells. Effector proteins carrying small phosphorylatable tags can be used to detect and quantify effector protein injection. Here, we describe the use of the ELK- and GSK-tags to detect the translocation of the Y. pestis YopE effector protein into RAW 264.7 macrophage-like cells using immunoblot analysis with phospho-specific antibodies.

  1. 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.

  2. Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast

    PubMed Central

    1990-01-01

    BiP/GRP78 is an essential member of the HSP70 family that resides in the lumen of the endoplasmic reticulum. In yeast, BiP/GRP78 is encoded by the KAR2 gene. A temperature sensitive mutation was isolated in KAR2 and found to cause a rapid block in protein secretion. Secretory precursors of a number of proteins (invertase, carboxypeptidase Y, alpha-factor, and BiP) accumulated that were characteristic of a block in translocation into the lumen of the ER. Protease protection experiments confirmed that the precursors accumulated on the cytoplasmic side of the ER membrane. Moreover, depletion of wild-type KAR2 protein also resulted in a block in translocation of secretory proteins. These results implicate BiP/GRP78 function in the continued translocation of proteins into the lumen of the ER. PMID:2190988

  3. Twin-arginine translocation-arresting protein regions contact TatA and TatB.

    PubMed

    Taubert, Johannes; Brüser, Thomas

    2014-07-01

    Tat systems translocate folded proteins across biological membranes of prokaryotes and plant plastids. TatBC complexes recognize N-terminal Tat signal peptides that contain a sequence motif with two conserved arginines (RR-motif), and transport takes place after a recruitment of TatA. Unfolded Tat substrate domains lower translocation efficiency and too long linkers lead to translocation arrest. To identify the components that interact with transported proteins during their passage through the translocon, we used a Tat substrate that arrests translocation at a long unfolded linker region, and we chose in vivo site-directed photo cross-linking to specifically detect the interactions of this linker region. For comparison, we included the interactions of the signal peptide and of the folded domain at the C-terminus of this construct. The data show that the linker contacts only two, structurally similar Tat components, namely TatA and TatB. These contacts depend on the recognition of the Tat-specific signal peptide. Only when membrane translocation of the globular domain was allowed--i.e., in the absence of the linker--we observed the same TatAB-contacts also to the globular domain. The data thus suggest that mature protein domains are translocated through a TatAB environment.

  4. 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.

  5. 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…

  6. 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…

  7. Translocator protein (18 kD) as target for anxiolytics without benzodiazepine-like side effects.

    PubMed

    Rupprecht, Rainer; Rammes, Gerhard; Eser, Daniela; Baghai, Thomas C; Schüle, Cornelius; Nothdurfter, Caroline; Troxler, Thomas; Gentsch, Conrad; Kalkman, Hans O; Chaperon, Frederique; Uzunov, Veska; McAllister, Kevin H; Bertaina-Anglade, Valerie; La Rochelle, Christophe Drieu; Tuerck, Dietrich; Floesser, Annette; Kiese, Beate; Schumacher, Michael; Landgraf, Rainer; Holsboer, Florian; Kucher, Klaus

    2009-07-24

    Most antianxiety drugs (anxiolytics) work by modulating neurotransmitters in the brain. Benzodiazepines are fast and effective anxiolytic drugs; however, their long-term use is limited by the development of tolerance and withdrawal symptoms. Ligands of the translocator protein [18 kilodaltons (kD)] may promote the synthesis of endogenous neurosteroids, which also exert anxiolytic effects in animal models. Here, we found that the translocator protein (18 kD) ligand XBD173 enhanced gamma-aminobutyric acid-mediated neurotransmission and counteracted induced panic attacks in rodents in the absence of sedation and tolerance development. XBD173 also exerted antipanic activity in humans and, in contrast to benzodiazepines, did not cause sedation or withdrawal symptoms. Thus, translocator protein (18 kD) ligands are promising candidates for fast-acting anxiolytic drugs with less severe side effects than benzodiazepines.

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

    NASA Astrophysics Data System (ADS)

    Rodriguez-Larrea, David; Bayley, Hagan

    2014-09-01

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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. Exocyst Sec10 is involved in basolateral protein translation and translocation in the endoplasmic reticulum.

    PubMed

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

    2012-01-01

    Protein translation and translocation at the rough endoplasmic reticulum (RER) are the first steps in the secretory pathway. The translocon through which newly made proteins are translocated into or across the RER membrane consists of three main subunits: Sec61α, -β, and -γ. Sec61β facilitates translocation, and we and others have shown that the highly conserved eight-protein exocyst complex interacts with Sec61β. We have also shown that the exocyst is involved in basolateral, not apical, protein synthesis and delivery. Recently, however, exocyst involvement in apical protein delivery has been reported. Furthermore, we have shown that the exocyst is necessary for formation of primary cilia, organelles found on the apical surface. GST pulldown was performed on lysate of renal tubule cells to investigate biochemical interactions. Cell-free assays consisting of cell-free extracts from rabbit reticulocytes, pancreatic endoplasmic reticulum (ER) microsomal membranes, transcripts of cDNA from apical and basolateral proteins, ATP/GTP, amino acids, and (35)S-methionine for protein detection were used to investigate the role of the exocyst in synthesis of polarized proteins. P(32)-orthophosphate and immunoprecipitation with antibody against Sec61β was used to investigate Sec61β phosphorylation in exocyst Sec10-overexpressing cells. Sec10 biochemically interacts with Sec61β using GST pulldown. Using cell-free assays, there is enhanced exocyst recruitment to endoplasmic reticulum membranes following exocyst depletion and basolateral G protein of vesicular stomatitis virus protein translation, compared to apical hemagglutinin of influenza virus protein translation. Finally, Sec10 overexpression increases Sec61β phosphorylation. These data confirm that the exocyst is preferentially involved in basolateral protein translation and translocation, and may well act through the phosphorylation of Sec61β. Copyright © 2012 S. Karger AG, Basel.

  15. 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. © 2015. Published by The Company of Biologists Ltd.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-08-06

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

  18. 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.

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

    PubMed Central

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

    2013-01-01

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

  20. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.

    PubMed

    Rapoport, Tom A

    2007-11-29

    A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.

  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

    2017-05-12

    The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum 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 endoplasmic reticulum 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-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Sheep pancreatic microsomes as an alternative to the dog source for studying protein translocation.

    PubMed Central

    Kaderbhai, M A; Harding, V J; Karim, A; Austen, B M; Kaderbhai, N N

    1995-01-01

    A procedure is described for the preparation of rough membrane vesicles of endoplasmic-reticular origin from the pancreas of sheep. These isolated membranes translocate, process and glycosylate in vitro-translated heterologous proteins in a manner comparable with that exhibited by dog pancreatic microsomes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7864829

  3. 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

  4. Early Contacts between Substrate Proteins and TatA Translocase Component in Twin-arginine Translocation*

    PubMed Central

    Fröbel, Julia; Rose, Patrick; Müller, Matthias

    2011-01-01

    Twin-arginine translocation (Tat) is a unique protein transport pathway in bacteria, archaea, and plastids. It mediates the transmembrane transport of fully folded proteins, which harbor a consensus twin-arginine motif in their signal sequences. In Gram-negative bacteria and plant chloroplasts, three membrane proteins, named TatA, TatB, and TatC, are required to enable Tat translocation. Available data suggest that TatA assembles into oligomeric pore-like structures that might function as the protein conduit across the lipid bilayer. Using site-specific photo-cross-linking, we have investigated the molecular environment of TatA under resting and translocating conditions. We find that monomeric TatA is an early interacting partner of functionally targeted Tat substrates. This interaction with TatA likely precedes translocation of Tat substrates and is influenced by the proton-motive force. It strictly depends on the presence of TatB and TatC, the latter of which is shown to make contacts with the transmembrane helix of TatA. PMID:22041896

  5. Early contacts between substrate proteins and TatA translocase component in twin-arginine translocation.

    PubMed

    Fröbel, Julia; Rose, Patrick; Müller, Matthias

    2011-12-23

    Twin-arginine translocation (Tat) is a unique protein transport pathway in bacteria, archaea, and plastids. It mediates the transmembrane transport of fully folded proteins, which harbor a consensus twin-arginine motif in their signal sequences. In Gram-negative bacteria and plant chloroplasts, three membrane proteins, named TatA, TatB, and TatC, are required to enable Tat translocation. Available data suggest that TatA assembles into oligomeric pore-like structures that might function as the protein conduit across the lipid bilayer. Using site-specific photo-cross-linking, we have investigated the molecular environment of TatA under resting and translocating conditions. We find that monomeric TatA is an early interacting partner of functionally targeted Tat substrates. This interaction with TatA likely precedes translocation of Tat substrates and is influenced by the proton-motive force. It strictly depends on the presence of TatB and TatC, the latter of which is shown to make contacts with the transmembrane helix of TatA.

  6. 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.

  7. Structural basis of chaperone recognition of type III secretion system minor translocator proteins.

    PubMed

    Job, Viviana; Matteï, Pierre-Jean; Lemaire, David; Attree, Ina; Dessen, Andréa

    2010-07-23

    The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the "translocators") are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.

  8. 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

  9. 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.

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

    PubMed

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

    2017-03-30

    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.

  11. 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

  12. 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.

  13. Cotranslational Partitioning of Nascent Prion Protein into Multiple Populations at the Translocation Channel

    PubMed Central

    Kim, Soo Jung; Hegde, Ramanujan S.

    2002-01-01

    The decisive events that direct a single polypeptide such as the prion protein (PrP) to be synthesized at the endoplasmic reticulum in both fully translocated and transmembrane forms are poorly understood. In this study, we demonstrate that the topological heterogeneity of PrP is determined cotranslationally, while at the translocation channel. By evaluating sequential intermediates during PrP topogenesis, we find that signal sequence-mediated initiation of translocation results in an interaction between nascent PrP and endoplasmic reticulum chaperones, committing the N terminus to the lumen. Synthesis of the transmembrane domain before completion of this step allows it to direct the generation of CtmPrP, a transmembrane form with its N terminus in the cytosol. Thus, segregation of nascent PrP into different topological configurations is critically dependent on the precise timing of signal-mediated initiation of N-terminus translocation. Consequently, this step could be experimentally tuned to modify PrP topogenesis, including complete reversal of the elevated CtmPrP caused by disease-associated mutations in the transmembrane domain. These results delineate the sequence of events involved in PrP biogenesis, explain the mechanism of action of CtmPrP-favoring mutations associated with neurodegenerative disease, and more generally, reveal that translocation substrates can be cotranslationally partitioned into multiple populations at the translocon. PMID:12429823

  14. Cooperation of TOM and TIM23 complexes during translocation of proteins into mitochondria.

    PubMed

    Waegemann, Karin; Popov-Čeleketić, Dušan; Neupert, Walter; Azem, Abdussalam; Mokranjac, Dejana

    2015-03-13

    Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence that at least two contacts from the side of the TIM23 complex play an important role in TOM-TIM23 cooperation in vivo. Tim50, likely through its very C-terminal segment, interacts with Tom22. This interaction is stimulated by translocating proteins and is independent of any other TOM-TIM23 contact known so far. Furthermore, the exposure of Tim23 on the mitochondrial surface depends not only on its interaction with Tim50 but also on the dynamics of the TOM complex. Destabilization of the individual contacts reduces the efficiency of import of proteins into mitochondria and destabilization of both contacts simultaneously is not tolerated by yeast cells. We conclude that an intricate and coordinated network of protein-protein interactions involving primarily Tim50 and also Tim23 is required for efficient translocation of proteins across both mitochondrial membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. 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

  16. Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles.

    PubMed Central

    Rhoads, D B; Tai, P C; Davis, B D

    1984-01-01

    In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energized membrane and not simply from ATP. Treatment of the vesicles with protease, under conditions that did not interfere with subsequent protein synthesis, also inactivated them for subsequent translocation. We conclude that export of some proteins requires protein-containing machinery in the cytoplasmic membrane that derives energy from the proton motive force. Images PMID:6203892

  17. 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.

  18. A Brownian ratchet for protein translocation including dissociation of ratcheting sites.

    PubMed

    Depperschmidt, A; Ketterer, N; Pfaffelhuber, P

    2013-02-01

    We study a model for the translocation of proteins across membranes through a nanopore using a ratcheting mechanism. When the protein enters the nanopore it diffuses in and out of the pore according to a Brownian motion. Moreover, it is bound by ratcheting molecules which hinder the diffusion of the protein out of the nanopore, i.e. the Brownian motion is reflected such that no ratcheting molecule exits the pore. New ratcheting molecules bind at rate γ. Extending our previous approach (Depperschmidt and Pfaffelhuber in Stoch Processes Appl 120:901-925, 2010) we allow the ratcheting molecules to dissociate (at rate δ) from the protein (Model I). We also provide an approximate model (Model II) which assumes a Poisson equilibrium of ratcheting molecules on one side of the current reflection boundary. Using analytical methods and simulations we show that the speeds of both models are approximately the same. Our analytical results on Model II give the speed of translocation by means of a solution of an ordinary differential equation. This speed gives an approximation for the time it takes to translocate a protein of given length.

  19. 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.

  20. 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. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Structural Basis of Chaperone Recognition of Type III Secretion System Minor Translocator Proteins*

    PubMed Central

    Job, Viviana; Matteï, Pierre-Jean; Lemaire, David; Attree, Ina; Dessen, Andréa

    2010-01-01

    The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the “translocators”) are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development. PMID:20385547

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

    PubMed

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

    2015-08-13

    Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  3. 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

  4. 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.

  5. 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.

  6. Common ground for protein translocation: access control for mitochondria and chloroplasts.

    PubMed

    Schleiff, Enrico; Becker, Thomas

    2011-01-01

    Mitochondria and chloroplasts import the vast majority of their proteins across two membranes, and use translocases of the outer membrane as an entry gate. These translocases interact with the incoming precursor protein and guiding chaperone factors. Within the translocon, precursor-protein receptors dock to a central component that mediates both transfer through a cation-selective channel and initial sorting towards internal subcompartments. Despite these similarities, the mode of translocation differs between the two organelles: in chloroplasts, GTP-binding and hydrolysis by the receptors is required for transport, whereas in mitochondria passage of the preprotein is driven by its increasing affinity for the translocase subunits.

  7. LMO2 at 25 years: a paradigm of chromosomal translocation proteins

    PubMed Central

    Chambers, Jennifer; Rabbitts, Terence H.

    2015-01-01

    LMO2 was first discovered through proximity to frequently occurring chromosomal translocations in T cell acute lymphoblastic leukaemia (T-ALL). Subsequent studies on its role in tumours and in normal settings have highlighted LMO2 as an archetypical chromosomal translocation oncogene, activated by association with antigen receptor gene loci and a paradigm for translocation gene activation in T-ALL. The normal function of LMO2 in haematopoietic cell fate and angiogenesis suggests it is a master gene regulator exerting a dysfunctional control on differentiation following chromosomal translocations. Its importance in T cell neoplasia has been further emphasized by the recurrent findings of interstitial deletions of chromosome 11 near LMO2 and of LMO2 as a target of retroviral insertion gene activation during gene therapy trials for X chromosome-linked severe combined immuno-deficiency syndrome, both types of event leading to similar T cell leukaemia. The discovery of LMO2 in some B cell neoplasias and in some epithelial cancers suggests a more ubiquitous function as an oncogenic protein, and that the current development of novel inhibitors will be of great value in future cancer treatment. Further, the role of LMO2 in angiogenesis and in haematopoietic stem cells (HSCs) bodes well for targeting LMO2 in angiogenic disorders and in generating autologous induced HSCs for application in various clinical indications. PMID:26108219

  8. Phenylalanine-427 of anthrax protective antigen functions in both pore formation and protein translocation

    PubMed Central

    Sun, Jianjun; Lang, Alexander E.; Aktories, Klaus; Collier, R. John

    2008-01-01

    The protective antigen (PA) moiety of anthrax toxin forms a heptameric pore in endosomal membranes of mammalian cells and translocates the enzymatic moieties of the toxin to the cytosol of these cells. Phenylalanine-427 (F427), a solvent-exposed residue in the lumen of the pore, was identified earlier as being crucial for the transport function of PA. The seven F427 residues were shown in electrophysiological studies to form a clamp that catalyzes protein translocation through the pore. Here, we demonstrate by a variety of tests that certain F427 mutations also profoundly inhibit the conformational transition of the heptameric PA prepore to the pore and thereby block pore formation in membranes. Lysine, arginine, aspartic acid, or glycine at position 427 strongly inhibited this acidic pH-induced conformational transition, whereas histidine, serine, and threonine had virtually no effect on this step, but inhibited translocation instead. Thus, it is possible to inhibit pore formation or translocation selectively, depending on the choice of the side chain at position 427; and the net inhibition of the PA transport function by any given F427 mutation is the product of its effects on both steps. Mutations inhibiting either or both steps elicited a strong dominant-negative phenotype. These findings demonstrate the dual functions of F427 and underline its central role in transporting the enzymatic moieties of anthrax toxin across membranes. PMID:18334631

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

    PubMed Central

    Simons, Michelle; Szczelkun, Mark D.

    2011-01-01

    The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5′-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can ‘turnover’ in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase–nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed. PMID:21712244

  10. LMO2 at 25 years: a paradigm of chromosomal translocation proteins.

    PubMed

    Chambers, Jennifer; Rabbitts, Terence H

    2015-06-01

    LMO2 was first discovered through proximity to frequently occurring chromosomal translocations in T cell acute lymphoblastic leukaemia (T-ALL). Subsequent studies on its role in tumours and in normal settings have highlighted LMO2 as an archetypical chromosomal translocation oncogene, activated by association with antigen receptor gene loci and a paradigm for translocation gene activation in T-ALL. The normal function of LMO2 in haematopoietic cell fate and angiogenesis suggests it is a master gene regulator exerting a dysfunctional control on differentiation following chromosomal translocations. Its importance in T cell neoplasia has been further emphasized by the recurrent findings of interstitial deletions of chromosome 11 near LMO2 and of LMO2 as a target of retroviral insertion gene activation during gene therapy trials for X chromosome-linked severe combined immuno-deficiency syndrome, both types of event leading to similar T cell leukaemia. The discovery of LMO2 in some B cell neoplasias and in some epithelial cancers suggests a more ubiquitous function as an oncogenic protein, and that the current development of novel inhibitors will be of great value in future cancer treatment. Further, the role of LMO2 in angiogenesis and in haematopoietic stem cells (HSCs) bodes well for targeting LMO2 in angiogenic disorders and in generating autologous induced HSCs for application in various clinical indications.

  11. New iodinated quinoline-2-carboxamides for SPECT imaging of the translocator protein.

    PubMed

    Stevenson, Louise; Tavares, Adriana A S; Brunet, Aurélie; McGonagle, Fiona I; Dewar, Deborah; Pimlott, Sally L; Sutherland, Andrew

    2010-02-01

    With the aim of developing new SPECT imaging agents for the translocator protein (TSPO), a small library of iodinated quinoline-2-carboxamides have been prepared and tested for binding affinity with TSPO. N,N-Diethyl-3-iodomethyl-4-phenylquinoline-2-carboxamide was found to have excellent affinity (K(i) 12.0 nM), comparable to that of the widely used TSPO imaging agent PK11195. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  12. 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. Published by Elsevier Inc.

  13. 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

  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. Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links*

    PubMed Central

    Nakano, Toshiaki; Miyamoto-Matsubara, Mayumi; Shoulkamy, Mahmoud I.; Salem, Amir M. H.; Pack, Seung Pil; Ishimi, Yukio; Ide, Hiroshi

    2013-01-01

    DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks. PMID:23283980

  18. Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier.

    PubMed

    Kim, Hyochin; Bhunia, Arun K

    2013-06-24

    Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown. Amounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells. Western blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates. This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic

  19. Secreted Listeria adhesion protein (Lap) influences Lap-mediated Listeria monocytogenes paracellular translocation through epithelial barrier

    PubMed Central

    2013-01-01

    Background Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown. Methods Amounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells. Results Western blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates. Conclusion This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may

  20. SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

    PubMed Central

    Deng, Wanyin; Yu, Hong B.; Li, Yuling

    2015-01-01

    ABSTRACT The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. IMPORTANCE Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into

  1. Ethanol-induced translocation of protein kinase A occurs in two phases: control by different molecular mechanisms.

    PubMed

    Dohrman, Douglas P; Chen, Hui-min; Gordon, Adrienne S; Diamond, Ivan

    2002-03-01

    Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) regulates cellular functions. The specificity of PKA-mediated phosphorylation is determined primarily by PKA localization to sub-cellular sites. Chronic exposure to ethanol causes sustained translocation of the PKA catalytic subunit (C) from the Golgi to the nucleus in NG108-15 cells. Here we find that this is preceded by a transient short-term ethanol-induced translocation of PKA C. Different molecular mechanisms appear to underlie early and late phases of ethanol-induced translocation of PKA subunits. The time course and localization of PKA C and regulatory (RII) subunits was assessed by immunocytochemistry in NG108-15 cells in the presence of ethanol, adenosine receptor (A2) blockade, and inhibitors of PKA activity and RNA and protein synthesis. Ethanol induces an early phase (<30 min) of C translocation to the cytoplasm and nucleus. This requires cAMP via adenosine A2 receptor activation. C then returns to the Golgi area after 60 min. A second phase of C translocation occurs during continuing exposure to ethanol (>12 hr). Re-accumulation of nuclear C no longer requires A2 or cAMP. RII also translocates to the nucleus during chronic treatment with ethanol. Both C and RII remain in the nucleus as long as ethanol is present. Unlike the early phase of ethanol induced translocation, the second phase of PKA subunit translocation requires protein and RNA synthesis. We identify two distinct phases of ethanol-induced PKA translocation which appear to be regulated by different molecular mechanisms. The first requires A2 signaling and cAMP; the later phase requires RNA and protein synthesis. The two phases of ethanol-induced PKA translocation observed in cell lines may contribute to changes in PKA signaling, cAMP-dependent gene expression, and the initiation and maintenance of sustained drinking behavior in experimental animals.

  2. Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus.

    PubMed

    Dohrman, D P; Diamond, I; Gordon, A S

    1996-09-17

    Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.

  3. 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

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

    PubMed

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

    2015-01-01

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

  5. Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

    PubMed Central

    Kostecki, Jan S.; Li, Haiming; Turner, Raymond J.; DeLisa, Matthew P.

    2010-01-01

    The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways. PMID:20169075

  6. 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.

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

    PubMed Central

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

    2016-01-01

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

  8. 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

  9. 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.

  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. Novel Phenotypes Detectable with PET in Mood Disorders: Elevated Monoamine Oxidase A and Translocator Protein Level.

    PubMed

    Meyer, Jeffrey

    2017-07-01

    As a result of high prevalence and high rates of treatment resistance, major depressive disorder has become the leading cause of death and disability in moderate-income to high-income nations. Poor targeting of phenotypes is a plausible reason for treatment resistance and PET imaging offers a unique role to identify phenotypes. Both increased monoamine oxidase A binding and greater translocator protein 18 kDa binding occur throughout the gray matter during major depressive episodes, including affect-modulating brain regions such as the prefrontal and anterior cingulate cortex, and are detectable with advanced radioligand technology for both of these targets. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. A Role for Timely Nuclear Translocation of Clock Repressor Proteins in Setting Circadian Clock Speed

    PubMed Central

    Lee, Euna

    2014-01-01

    By means of a circadian clock system, all the living organisms on earth including human beings can anticipate the environmental rhythmic changes such as light/dark and warm/cold periods in a daily as well as in a yearly manner. Anticipating such environmental changes provide organisms with survival benefits via manifesting behavior and physiology at an advantageous time of the day and year. Cell-autonomous circadian oscillators, governed by transcriptional feedback loop composed of positive and negative elements, are organized into a hierarchical system throughout the organisms and generate an oscillatory expression of a clock gene by itself as well as clock controlled genes (ccgs) with a 24 hr periodicity. In the feedback loop, hetero-dimeric transcription factor complex induces the expression of negative regulatory proteins, which in turn represses the activity of transcription factors to inhibit their own transcription. Thus, for robust oscillatory rhythms of the expression of clock genes as well as ccgs, the precise control of subcellular localization and/or timely translocation of core clock protein are crucial. Here, we discuss how sub-cellular localization and nuclear translocation are controlled in a time-specific manner focusing on the negative regulatory clock proteins. PMID:25258565

  13. 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.

  14. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25759509

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

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

    SciTech Connect

    Kern, Stephanie M.; Feig, Andrew L.

    2011-02-25

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

  2. Distinct patterns of increased translocator protein in posterior cortical atrophy and amnestic Alzheimer's disease.

    PubMed

    Kreisl, William C; Lyoo, Chul Hyoung; Liow, Jeih-San; Snow, Joseph; Page, Emily; Jenko, Kimberly J; Morse, Cheryl L; Zoghbi, Sami S; Pike, Victor W; Turner, R Scott; Innis, Robert B

    2017-03-01

    We sought to determine whether patients with posterior cortical atrophy (PCA) demonstrate a pattern of binding to translocator protein 18 kDa, a marker of microglial activation, that is distinct from that in patients with amnestic presentation of Alzheimer's disease (AD). Eleven PCA patients, 11 amnestic AD patients, and 15 age-matched controls underwent positron emission tomography with (11)C-PBR28 to measure translocator protein 18 kDa. PCA patients showed greater (11)C-PBR28 binding than controls in occipital, posterior parietal, and temporal regions. In contrast, amnestic AD patients showed greater (11)C-PBR28 binding in inferior and medial temporal cortex. Increased (11)C-PBR28 binding overlapped with reduced cortical volume for both PCA and amnestic AD patients, and with areas of reduced glucose metabolism in PCA patients. While both patient groups showed diffuse amyloid binding, PCA patients showed greater binding than amnestic AD patients in bilateral occipital cortex. These results suggest that microglial activation is closely associated with neurodegeneration across different subtypes of AD. Published by Elsevier Inc.

  3. 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.

  4. 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. © 2016 American Institute of Chemical Engineers.

  5. 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

  6. 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

  7. 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.

  8. SepD/SepL-dependent secretion signals of the type III secretion system translocator proteins in enteropathogenic Escherichia coli.

    PubMed

    Deng, Wanyin; Yu, Hong B; Li, Yuling; Finlay, B Brett

    2015-04-01

    The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into the host cells to

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. The 18-kDa mitochondrial translocator protein in gliomas: from the bench to bedside.

    PubMed

    Janczar, Karolina; Su, Zhangjie; Raccagni, Isabella; Anfosso, Andrea; Kelly, Charlotte; Durrenberger, Pascal F; Gerhard, Alexander; Roncaroli, Federico

    2015-08-01

    The 18-kDa mitochondrial translocator protein (TSPO) is known to be highly expressed in several types of cancer, including gliomas, whereas expression in normal brain is low. TSPO functions in glioma are still incompletely understood. The TSPO can be quantified pre-operatively with molecular imaging making it an ideal candidate for personalized treatment of patient with glioma. Studies have proposed to exploit the TSPO as a transporter of chemotherapics to selectively target tumour cells in the brain. Our studies proved that positron emission tomography (PET)-imaging can contribute to predict progression of patients with glioma and that molecular imaging with TSPO-specific ligands is suitable to stratify patients in view of TSPO-targeted treatment. Finally, we proved that TSPO in gliomas is predominantly expressed by tumour cells. © 2015 Authors; published by Portland Press Limited.

  15. Ether analogues of DPA-714 with subnanomolar affinity for the translocator protein (TSPO).

    PubMed

    Banister, Samuel D; Beinat, Corinne; Wilkinson, Shane M; Shen, Bin; Bartoli, Cecilia; Selleri, Silvia; Da Pozzo, Eleonora; Martini, Claudia; Chin, Frederick T; Kassiou, Michael

    2015-03-26

    Sixteen new phenyl alkyl ether derivatives (12, 14-28) of the 5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-ylacetamide (DPA) class were synthesized and evaluated in a competition binding assay against [(3)H]PK11195 using 18 kDa translocator protein (TSPO) derived from rat kidney mitochondrial fractions. All analogues showed superior binding affinities for TSPO compared to DPA-713 (5) and DPA-714 (6). Picomolar affinities were observed for this class of TSPO ligands in this assay for the first time, with phenethyl ether 28 showing the greatest affinity (Ki = 0.13 nM). Additionally, all analogues increased pregnenolone biosynthesis (134-331% above baseline) in a rat C6 glioma cell steroidogenesis assay. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  16. Targeting the 18-kDa translocator protein: recent perspectives for neuroprotection.

    PubMed

    Da Pozzo, Eleonora; Giacomelli, Chiara; Barresi, Elisabetta; Costa, Barbara; Taliani, Sabrina; Passetti, Federico Da Settimo; Martini, Claudia

    2015-08-01

    The translocator protein (TSPO, 18 kDa), mainly localized in the outer mitochondrial membrane of steroidogenic tissues, is involved in several cellular functions. TSPO level alterations have been reported in a number of human disorders, particularly in cancer, psychiatric and neurological diseases. In the central nervous system (CNS), TSPO is usually expressed in glial cells, but also in some neuronal cell types. Interestingly, the expression of TSPO on glial cells rises after brain injury and increased TSPO expression is often observed in neurological disorders, gliomas, encephalitis and traumatic injury. Since TSPO is up-regulated in brain diseases, several structurally different classes of ligands targeting TSPO have been described as potential diagnostic or therapeutic agents. Recent researches have reported that TSPO ligands might be valuable in the treatment of brain diseases. This review focuses on currently available TSPO ligands, as useful tools for the treatment of neurodegeneration, neuro-inflammation and neurotrauma. © 2015 Authors; published by Portland Press Limited.

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

    SciTech Connect

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

    1990-01-01

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

  18. 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.

  19. Determinants of pH-Dependent Modulation of Translocation in Dermonecrotic G-Protein-Deamidating Toxins

    PubMed Central

    Repella, Tana L.; Ho, Mengfei; Wilson, Brenda A.

    2013-01-01

    Cytotoxic necrotizing factors from E. coli (CNF1, CNF2) and Yersinia (CNFy) share N-terminal sequence similarity with Pasteurella multocida toxin (PMT). This common N-terminal region harbors the receptor-binding and translocation domains that mediate uptake and delivery of the C-terminal catalytic cargo domains into the host cytosol. Subtle variations in the N-terminal ~500 amino acids of CNFs and PMT could allow for selective recognition of cellular receptors and thus, selective target cell specificity. Through studies with cellular inhibitors, we have identified an additional novel function for this region in modulating responses of these toxin proteins to changes in pH during intoxication and delivery of the catalytic cargo domain into the cytosol. PMID:23888517

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

    NASA Astrophysics Data System (ADS)

    Zhang, Zhenzhen; Wu, Shengnan; Feng, Jie

    2011-01-01

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

  1. 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

  2. The translocator protein gene is associated with symptom severity and cerebral pain processing in fibromyalgia.

    PubMed

    Kosek, Eva; Martinsen, Sofia; Gerdle, Björn; Mannerkorpi, Kaisa; Löfgren, Monika; Bileviciute-Ljungar, Indre; Fransson, Peter; Schalling, Martin; Ingvar, Martin; Ernberg, Malin; Jensen, Karin B

    2016-11-01

    The translocator protein (TSPO) is upregulated during glia activation in chronic pain patients. TSPO constitutes the rate-limiting step in neurosteroid synthesis, thus modulating synaptic transmission. Related serotonergic mechanisms influence if pro- or anti-nociceptive neurosteroids are produced. This study investigated the effects of a functional genetic polymorphism regulating the binding affinity to the TSPO, thus affecting symptom severity and cerebral pain processing in fibromyalgia patients. Gene-to-gene interactions with a functional polymorphism of the serotonin transporter gene were assessed. Fibromyalgia patients (n=126) were genotyped regarding the polymorphisms of the TSPO (rs6971) and the serotonin transporter (5-HTTLPR/rs25531). Functional magnetic resonance imaging (n=24) was used to study brain activation during individually calibrated pressure pain. Compared to mixed/low TSPO affinity binders, the high TSPO affinity binders rated more severe pain (p=0.016) and fibromyalgia symptoms (p=0.02). A significant interaction was found between the TSPO and the serotonin transporter polymorphisms regarding pain severity (p<0.0001). Functional connectivity analyses revealed that the TSPO high affinity binding group had more pronounced pain-evoked functional connectivity in the right frontoparietal network, between the dorsolateral prefrontal area and the parietal cortex. In conclusion, fibromyalgia patients with the TSPO high affinity binding genotype reported a higher pain intensity and more severe fibromyalgia symptoms compared to mixed/low affinity binders, and this was modulated by interaction with the serotonin transporter gene. To our knowledge this is the first evidence of functional genetic polymorphisms affecting pain severity in FM and our findings are in line with proposed glia-related mechanisms. Furthermore, the functional magnetic resonance findings indicated an effect of translocator protein on the affective-motivational components of pain

  3. 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.

  4. Chlamydia pneumoniae CopD Translocator Protein Plays a Critical Role in Type III Secretion (T3S) and Infection

    PubMed Central

    Bulir, David C.; Waltho, Daniel A.; Stone, Christopher B.; Mwawasi, Kenneth A.; Nelson, Jordan C.; Mahony, James B.

    2014-01-01

    Pathogenic Gram-negative bacteria use type III secretion (T3S) to inject effector proteins into the host cell to create appropriate conditions for infection and intracellular replication. Chlamydia spp. are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. Chlamydia pneumoniae contains genes encoding two sets of translocator proteins; CopB and CopD, and CopB2 and CopD2. In this study, we identified novel interactions between CopD and three type III secretion proteins; namely, CopN, CdsN, and CdsF. We identified a CopD putative chaperone binding motif, PxLxxP, within the N-terminal region (CopD amino acids 120–125), which was necessary for interaction with its putative chaperone LcrH_1. Using size exclusion chromatography, we showed that CopD and LcrH_1 formed higher order structures in solution with CopD and LcrH_1 binding in a ratio of 1∶1, which is unique for T3SS translocator proteins. Lastly, we showed that antibodies to CopD reduced C. pneumoniae infectivity by >95%. Collectively, this data suggests that CopD plays a critical role in pathogenesis and likely functions as a hydrophobic translocator of the type III secretion system in Chlamydia pneumoniae. PMID:24959658

  5. Chlamydia pneumoniae CopD translocator protein plays a critical role in type III secretion (T3S) and infection.

    PubMed

    Bulir, David C; Waltho, Daniel A; Stone, Christopher B; Mwawasi, Kenneth A; Nelson, Jordan C; Mahony, James B

    2014-01-01

    Pathogenic Gram-negative bacteria use type III secretion (T3S) to inject effector proteins into the host cell to create appropriate conditions for infection and intracellular replication. Chlamydia spp. are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. Chlamydia pneumoniae contains genes encoding two sets of translocator proteins; CopB and CopD, and CopB2 and CopD2. In this study, we identified novel interactions between CopD and three type III secretion proteins; namely, CopN, CdsN, and CdsF. We identified a CopD putative chaperone binding motif, PxLxxP, within the N-terminal region (CopD amino acids 120-125), which was necessary for interaction with its putative chaperone LcrH_1. Using size exclusion chromatography, we showed that CopD and LcrH_1 formed higher order structures in solution with CopD and LcrH_1 binding in a ratio of 1∶1, which is unique for T3SS translocator proteins. Lastly, we showed that antibodies to CopD reduced C. pneumoniae infectivity by >95%. Collectively, this data suggests that CopD plays a critical role in pathogenesis and likely functions as a hydrophobic translocator of the type III secretion system in Chlamydia pneumoniae.

  6. Listeria monocytogenes uses Listeria adhesion protein (LAP) to promote bacterial transepithelial translocation and induces expression of LAP receptor Hsp60.

    PubMed

    Burkholder, Kristin M; Bhunia, Arun K

    2010-12-01

    Listeria monocytogenes interaction with the intestinal epithelium is a key step in the infection process. We demonstrated that Listeria adhesion protein (LAP) promotes adhesion to intestinal epithelial cells and facilitates extraintestinal dissemination in vivo. The LAP receptor is a stress response protein, Hsp60, but the precise role for the LAP-Hsp60 interaction during Listeria infection is unknown. Here we investigated the influence of physiological stressors and Listeria infection on host Hsp60 expression and LAP-mediated bacterial adhesion, invasion, and transepithelial translocation in an enterocyte-like Caco-2 cell model. Stressors such as heat (41°C), tumor necrosis factor alpha (TNF-α) (100 U), and L. monocytogenes infection (10(4) to 10(6) CFU/ml) significantly (P < 0.05) increased plasma membrane and intracellular Hsp60 levels in Caco-2 cells and consequently enhanced LAP-mediated L. monocytogenes adhesion but not invasion of Caco-2 cells. In transepithelial translocation experiments, the wild type (WT) exhibited 2.7-fold more translocation through Caco-2 monolayers than a lap mutant, suggesting that LAP is involved in transepithelial translocation, potentially via a paracellular route. Short hairpin RNA (shRNA) suppression of Hsp60 in Caco-2 cells reduced WT adhesion and translocation 4.5- and 3-fold, respectively, while adhesion remained unchanged for the lap mutant. Conversely, overexpression of Hsp60 in Caco-2 cells enhanced WT adhesion and transepithelial translocation, but not those of the lap mutant. Furthermore, initial infection with a low dosage (10(6) CFU/ml) of L. monocytogenes increased plasma membrane and intracellular expression of Hsp60 significantly, which rendered Caco-2 cells more susceptible to subsequent LAP-mediated adhesion and translocation. These data provide insight into the role of LAP as a virulence factor during intestinal epithelial infection and pose new questions regarding the dynamics between the host stress response

  7. 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

  8. 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.

  9. 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

  10. Sec-independent protein translocation in Escherichia coli. A distinct and pivotal role for the TatB protein.

    PubMed

    Sargent, F; Stanley, N R; Berks, B C; Palmer, T

    1999-12-17

    In Escherichia coli, transmembrane translocation of proteins can proceed by a number of routes. A subset of periplasmic proteins are exported via the Tat pathway to which proteins are directed by N-terminal "transfer peptides" bearing the consensus (S/T)RRXFLK "twin-arginine" motif. The Tat system involves the integral membrane proteins TatA, TatB, TatC, and TatE. Of these, TatA, TatB, and TatE are homologues of the Hcf106 component of the DeltapH-dependent protein import system of plant thylakoids. Deletion of the tatB gene alone is sufficient to block the export of seven endogenous Tat substrates, including hydrogenase-2. Complementation analysis indicates that while TatA and TatE are functionally interchangeable, the TatB protein is functionally distinct. This conclusion is supported by the observation that Helicobacter pylori tatA will complement an E. coli tatA mutant, but not a tatB mutant. Analysis of Tat component stability in various tat deletion backgrounds shows that TatC is rapidly degraded in the absence of TatB suggesting that TatC complexes, and is stabilized by, TatB.

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

    PubMed

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

    2014-01-01

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

  12. Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein.

    PubMed

    Teplyakov, Alexey; Lim, Kap; Zhu, Peng-Peng; Kapadia, Geeta; Chen, Celia C H; Schwartz, Jennifer; Howard, Andrew; Reddy, Prasad T; Peterkofsky, Alan; Herzberg, Osnat

    2006-10-31

    Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg(2+)-dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg(2+) and PEP, followed by oxalate, an EI inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg(2+) and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189 approximately P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an alpha-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189 approximately P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.

  13. 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.

  14. Requirements for the Nuclear-Cytoplasmic Translocation of Infected-Cell Protein 0 of Herpes Simplex Virus 1

    PubMed Central

    Lopez, Pascal; Van Sant, Charles; Roizman, Bernard

    2001-01-01

    Earlier studies have shown that wild-type infected-cell protein 0 (ICP0), a key herpes simplex virus regulatory protein, translocates from the nucleus to the cytoplasm of human embryonic lung (HEL) fibroblasts within several hours after infection (Y. Kawaguchi, R. Bruni, and B. Roizman, J. Virol. 71:1019–1024, 1997). Translocation of ICP0 was also observed in cells infected with the d120 mutant, in which both copies of the gene encoding ICP4, the major regulatory protein, had been deleted (V. Galvan, R. Brandimarti, J. Munger, and B. Roizman, J. Virol. 74:1931–1938, 2000). Furthermore, a mutant (R7914) carrying the D199A substitution in ICP0 does not bind or stabilize cyclin D3 and is retained in the nucleus (C. Van Sant, P. Lopez, S. J. Advani, and B. Roizman, J. Virol. 75:1888–1898, 2001). Studies designed to elucidate the requirements for the translocation of ICP0 between cellular compartments revealed the following. (i) Translocation of ICP0 to the cytoplasm in productive infection maps to the D199 amino acid, inasmuch as wild-type ICP0 delivered in trans to cells infected with an ICP0 null mutant was translocated to the cytoplasm whereas the D199A-substituted mutant ICP0 was not. (ii) Translocation of wild-type ICP0 requires a function expressed late in infection, inasmuch as phosphonoacetate blocked the translocation of ICP0 in wild-type virus-infected cells but not in d120 mutant-infected cells. Moreover, whereas in d120 mutant-infected cells ICP0 was translocated rapidly from the cytoplasm to the nucleus at approximately 5 h after infection, the translocation of ICP0 in wild-type virus-infected cells extended from 5 to at least 9 h after infection. (iii) In wild-type virus-infected cells, the MG132 proteasomal inhibitor blocked the translocation of ICP0 to the cytoplasm early in infection, but when added late in infection, it caused ICP0 to be relocated back to the nucleus from the cytoplasm. (iv) MG132 blocked the translocation of ICP0 in d120 mutant

  15. 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.

  16. Dynamin-related protein Drp1 is required for Bax translocation to mitochondria in response to irradiation-induced apoptosis.

    PubMed

    Wang, Ping; Wang, Peiguo; Liu, Becky; Zhao, Jing; Pang, Qingsong; Agrawal, Samir G; Jia, Li; Liu, Feng-Ting

    2015-09-08

    Translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondria is a crucial step in DNA damage-mediated apoptosis, and is also found to be involved in mitochondrial fragmentation. Irradiation-induced cytochrome c release and apoptosis was associated with Bax activation, but not mitochondrial fragmentation. Both Bax and Drp1 translocated from the cytosol to the mitochondria in response to irradiation. However, Drp1 mitochondrial translocation and oligomerization did not require Bax, and failed to induce apoptosis in Bax deficient diffuse large B-cell lymphoma (DLBCL) cells. Using fluorescent microscopy and the intensity correlation analysis, we demonstrated that Bax and Drp1 were colocalized and the levels of colocalization were increased by UV irradiation. Using co-immuno-precipitation, we confirmed that Bax and Drp1 were binding partners. Irradiation induced a time-associated increase in the interaction between active Bax and Drp1. Knocking down Drp1 using siRNA blocked UV irradiation-mediated Bax mitochondrial translocation. In conclusion, our findings demonstrate for the first time, that Drp1 is required for Bax mitochondrial translocation, but Drp1-induced mitochondrial fragmentation alone is not sufficient to induce apoptosis in DLBCL cells.

  17. Perspective: Evolving understanding of translocator protein 18 kD (TSPO)

    PubMed Central

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

    2015-01-01

    The translocator protein 18 kD (TSPO) has been the focus of intense research by the biomedical community and the pharmaceutical industry because of its apparent involvement in many disease-related processes. These include steroidogenesis, apoptosis, inflammation, neurological disease and cancer, resulting in the use of TSPO as a biomarker and its potential as a drug target. Despite more than 30 years of study, the precise function of TSPO remains elusive. A recent breakthrough in determining the high-resolution crystal structures of bacterial homologs of mitochondrial TSPO provides new insight into the structural and functional properties at a molecular level and new opportunities for investigating the significance of this ancient and highly conserved protein family. The availability of atomic level structural information from different species also provides a platform for structure-based drug development. Here we briefly review current knowledge regarding TSPO and the implications of the new structures with respect to hypotheses and controversies in the field. PMID:25882248

  18. Translocator protein (18 kDa) TSPO: an emerging therapeutic target in neurotrauma

    PubMed Central

    Papadopoulos, Vassilios; Lecanu, Laurent

    2009-01-01

    Traumatic brain injury (TBI) induces physical, cognitive, and psychosocial deficits that affect millions of patients. TBI activates numerous cellular mechanisms and molecular cascades that produce detrimental outcomes, including neuronal death and loss of function. The mitochondrion is one of the major targets of TBI, as seen by increased mitochondrial activity in activated and proliferating microglia (due to high energy requirements and/or calcium overload) as well as increased reactive oxygen species, changes in mitochondrial permeability transition, release of cytochrome c, caspase activation, reduced ATP levels, and cell death in neurons. Translocator protein (TSPO) is an 18-kDa outer mitochondrial membrane protein that interacts with the mitochondria permeability transition pore and binds with high affinity to cholesterol and various classes of drug ligands, including some benzodiazepines such as 4′-chlorodiazepam (Ro5-4864). Although TSPO levels in the brain are low, they are increased after brain injury and inflammation. This finding has led to the proposed use of TSPO expression as a marker of brain injury and repair. TSPO drug ligands have been shown to participate in the control of mitochondrial respiration and function, mitochondrial steroid and neurosteroid formation, as well as apoptosis. This review and commentary will outline our current knowledge of the benefits of targeting TSPO for TBI treatment and the mechanisms underlying the neuroprotective effects of TSPO drug ligands in neurotrauma. PMID:19409385

  19. 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

  20. 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

  1. Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts[C][W][OA

    PubMed Central

    Su, Pai-Hsiang; Li, Hsou-min

    2010-01-01

    Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system. PMID:20484004

  2. 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

  3. 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.

  4. Tumor mitochondria-targeted photodynamic therapy with a translocator protein (TSPO)-specific photosensitizer.

    PubMed

    Zhang, Shaojuan; Yang, Ling; Ling, Xiaoxi; Shao, Pin; Wang, Xiaolei; Edwards, W Barry; Bai, Mingfeng

    2015-12-01

    Photodynamic therapy (PDT) has been proven to be a minimally invasive and effective therapeutic strategy for cancer treatment. It can be used alone or as a complement to conventional cancer treatments, such as surgical debulking and chemotherapy. The mitochondrion is an attractive target for developing novel PDT agents, as it produces energy for cells and regulates apoptosis. Current strategy of mitochondria targeting is mainly focused on utilizing cationic photosensitizers that bind to the negatively charged mitochondria membrane. However, such an approach is lack of selectivity of tumor cells. To minimize the damage on healthy tissues and improve therapeutic efficacy, an alternative targeting strategy with high tumor specificity is in critical need. Herein, we report a tumor mitochondria-specific PDT agent, IR700DX-6T, which targets the 18kDa mitochondrial translocator protein (TSPO). IR700DX-6T induced apoptotic cell death in TSPO-positive breast cancer cells (MDA-MB-231) but not TSPO-negative breast cancer cells (MCF-7). In vivo PDT study suggested that IR700DX-6T-mediated PDT significantly inhibited the growth of MDA-MB-231 tumors in a target-specific manner. These combined data suggest that this new TSPO-targeted photosensitizer has great potential in cancer treatment. Photodynamic therapy (PDT) is an effective and minimally invasive therapeutic technique for treating cancers. Mitochondrion is an attractive target for developing novel PDT agents, as it produces energy to cells and regulates apoptosis. Current mitochondria targeted photosensitizers (PSs) are based on cationic molecules, which interact with the negatively charged mitochondria membrane. However, such PSs are not specific for cancerous cells, which may result in unwanted side effects. In this study, we developed a tumor mitochondria-targeted PS, IR700DX-6T, which binds to translocator protein (TSPO). This agent effectively induced apoptosis in TSPO-positive cancer cells and significantly

  5. Increased Translocator Protein Distribution Volume, A Marker of Neuroinflammation, in the Brain During Major Depressive Episodes

    PubMed Central

    Setiawan, Elaine; Wilson, Alan A.; Mizrahi, Romina; Rusjan, Pablo M.; Miler, Laura; Rajkowska, Grazyna; Suridjan, Ivonne; Kennedy, James L.; Rekkas, P. Vivien; Houle, Sylvain; Meyer, Jeffrey H.

    2016-01-01

    Importance The neuroinflammatory hypothesis of major depressive disorder (MDD) is supported by several main findings: First, in humans and animals, activation of the immune system causes sickness behaviors that present during a major depressive episode (MDE) such as low mood, anhedonia, anorexia and weight loss. Second, peripheral markers of inflammation are frequently reported in MDD. Third, neuroinflammatory illnesses are associated with high rates of MDE. However, a fundamental limitation of the neuroinflammatory hypothesis is a paucity of evidence for brain inflammation during MDE. To investigate whether microglial activation, an important aspect of neuroinflammation, is present during MDE, [18F]FEPPA positron emission tomography (PET) was applied to measure translocator protein total distribution volume (TSPO VT), an index of TSPO density. Translocator protein density is elevated in activated microglia. Objective To determine whether TSPO VT, is elevated in the prefrontal cortex, anterior cingulate cortex (ACC) and insula in MDE secondary to MDD. Design Case-control study. Setting Tertiary care psychiatric hospital. Participants 20 subjects with MDE secondary to MDD and 20 healthy controls, underwent an [18F]FEPPA PET scan. MDE subjects were medication-free for at least 6 weeks. All participants were otherwise healthy, and non-smoking. Main Outcome Measure TSPO VT was measured in the prefrontal cortex, ACC, and insula. Results In MDE, TSPO VT was significantly elevated in all brain regions examined (multivariate analysis of variance, F15,23=4.46, P=0.001).TSPO VT was increased, on average, by 30% in the prefrontal cortex, ACC and insula. In MDE, greater TSPO VT in the ACC correlated with greater depression severity (ACC: r=0.628, P=0.005). Conclusions and Relevance This finding provides the most compelling evidence to date for brain inflammation, and more specifically microglial activation, in MDE. This is important for improving treatment since it implies

  6. Effects of altered TatC proteins on protein secretion efficiency via the twin-arginine translocation pathway of Bacillus subtilis.

    PubMed

    Eijlander, Robyn T; Kolbusz, Magdalena A; Berendsen, Erwin M; Kuipers, Oscar P

    2009-06-01

    Protein translocation via the Tat machinery in thylakoids and bacteria occurs through a cooperation between the TatA, TatB and TatC subunits, of which the TatC protein forms the initial Tat substrate-binding site. The Bacillus subtilis Tat machinery lacks TatB and comprises two separate TatAC complexes with distinct substrate specificities: PhoD is secreted by the TatAdCd complex, whereas YwbN is secreted by the TatAyCy complex. To study the role of the Gram-positive TatC proteins in Tat-dependent protein secretion efficiency, we applied several genetic engineering approaches to modify and analyse the B. subtilis TatCd and TatCy proteins. Cytoplasmic and transmembrane domain exchange between TatCd and TatCy resulted in stable chimeric proteins that were unable to secrete both known substrates of the B. subtilis Tat system. Site-directed mutagenesis of conserved residues in the N-terminal part of both TatC proteins revealed significant differences in the degree of importance of these residues between TatCd, TatCy and Escherichia coli TatC. In addition, two small C-terminal deletions in TatCy completely abolished YwbN translocation, indicating that this terminus is essential for Tat translocation activity. Important differences from previous observations for E. coli TatC and implications for substrate binding and translocation are discussed.

  7. Chlamydia Outer Protein (Cop) B from Chlamydia pneumoniae possesses characteristic features of a type III secretion (T3S) translocator protein.

    PubMed

    Bulir, David C; Waltho, Daniel A; Stone, Christopher B; Liang, Steven; Chiang, Christopher K W; Mwawasi, Kenneth A; Nelson, Jordan C; Zhang, Steven W; Mihalco, Samantha P; Scinocca, Zachariah C; Mahony, James B

    2015-08-14

    Chlamydia spp. are believed to use a conserved virulence factor called type III secretion (T3S) to facilitate the delivery of effector proteins from the bacterial pathogen to the host cell. Important early effector proteins of the type III secretion system (T3SS) are a class of proteins called the translocators. The translocator proteins insert into the host cell membrane to form a pore, allowing the injectisome to dock onto the host cell to facilitate translocation of effectors. CopB is a predicted hydrophobic translocator protein within the chlamydial T3SS. In this study, we identified a novel interaction between the hydrophobic translocator, CopB, and the putative filament protein, CdsF. Furthermore, we identified a conserved PxLxxP motif in CopB (amino acid residues 166-171), which is required for interaction with its cognate chaperone, LcrH_1. Using a synthetic peptide derived from the chaperone binding motif of CopB, we were able to block the LcrH_1 interaction with either CopB or CopD; this CopB peptide was capable of inhibiting C. pneumoniae infection of HeLa cells at micromolar concentrations. An antibody raised against the N-terminus of CopB was able to inhibit C. pneumoniae infection of HeLa cells. The inhibition of the LcrH_1:CopB interaction with a cognate peptide and subsequent inhibition of host cell infection provides strong evidence that T3S is an essential virulence factor for chlamydial infection and pathogenesis. Together, these results support that CopB plays the role of a hydrophobic translocator.

  8. Translocator Protein/Peripheral Benzodiazepine Receptor Is Not Required for Steroid Hormone Biosynthesis

    PubMed Central

    Morohaku, Kanako; Pelton, Susanne H.; Daugherty, Daniel J.; Butler, W. Ronald

    2014-01-01

    Molecular events that regulate cellular biosynthesis of steroid hormones have been a topic of intense research for more than half a century. It has been established that transport of cholesterol into the mitochondria forms the rate-limiting step in steroid hormone production. In current models, both the steroidogenic acute regulatory protein (StAR) and the translocator protein (TSPO) have been implicated to have a concerted and indispensable effort in this cholesterol transport. Deletion of StAR in mice resulted in a critical failure of steroid hormone production, but deletion of TSPO in mice was found to be embryonic lethal. As a result, the role of TSPO in cholesterol transport has been established only using pharmacologic and genetic tools in vitro. To allow us to explore in more detail the function of TSPO in cell type-specific experimental manipulations in vivo, we generated mice carrying TSPO floxed alleles (TSPOfl/fl). In this study we made conditional knockout mice (TSPOcΔ/Δ) with TSPO deletion in testicular Leydig cells by crossing with an anti-Mullerian hormone receptor type II cre/+ mouse line. Genetic ablation of TSPO in steroidogenic Leydig cells in mice did not affect testosterone production, gametogenesis, and reproduction. Expression of StAR, cytochrome P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase type I, and TSPO2 in TSPOcΔ/Δ testis was unaffected. These results challenge the prevailing dogma that claims an essential role for TSPO in steroid hormone biosynthesis and force reexamination of functional interpretations made for this protein. This is the first study examining conditional TSPO gene deletion in mice. The results show that TSPO function is not essential for steroid hormone biosynthesis. PMID:24174323

  9. Binding mode analysis of a major T3SS translocator protein PopB with its chaperone PcrH from Pseudomonas aeruginosa.

    PubMed

    Banerjee, Anindyajit; Dey, Supratim; Chakraborty, Abhijit; Datta, Aohona; Basu, Abhishek; Chakrabarti, Saikat; Datta, Saumen

    2014-12-01

    Pseudomonas aeruginosa, a Gram-negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein-chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N-terminal domain. However, disordered and flexible residues located both at the N- and C-terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a "disorder-to-order" transition of PopB protein might take place upon PcrH binding. The long helical coiled-coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism. © 2014 Wiley Periodicals, Inc.

  10. 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

  11. 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.

  12. Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae

    SciTech Connect

    Hirst, T.R.; Holmgren, J.

    1987-11-01

    The secretion of enterotoxin by Vibrio cholerae is punctuated by the transient entry of the toxin subunits into the periplasm. In this paper, the authors show that the subunits oligomerize into an assembled holotoxin within the periplasm prior to their secretion across the outer membrane. The rate of toxin assembly was studied by pulse-labeling cells with (/sup 35/S)-methionine and then monitoring the turnover of radiolabeled subunits as they assembled within the periplasm. The subunits entered the periplasm as monomers and assembled into oligomers with a half-time of approx. = 1 min. Since assembly was a rapid event compared to the rate of toxin efflux from the periplasm, which had a half-time of approx. = 13 min, they conclude that all of the subunits that pass through the periplasm assemble before they traverse the outer membrane. The average concentration of subunit monomers and assembled holotoxin within the periplasm was calculated to be approx. = 20 and approx. = 260 ..mu..g/ml, respectively. This indicates that the periplasm is a suitably concentrated milieu where spontaneous toxin assembly can occur. These findings suggest that protein movement across bacterial outer membranes, in apparent contrast to export across other biological membranes, involves translocation of polypeptides that have already folded into tertiary and even quaternary conformations.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

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

    PubMed

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

    2011-01-01

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

  1. Spinal translocator protein (TSPO) modulates pain behavior in rats with CFA-induced monoarthritis.

    PubMed

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

    2009-08-25

    Translocator protein 18 kDa (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 Days 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 laminae I-II on all experimental days. Iba-1 expression in the ipsilateral dorsal horn was also increased on Days 7 and 14. Moreover, TSPO was colocalized 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.

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

    NASA Astrophysics Data System (ADS)

    Muthukumar, M.

    2001-03-01

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

  3. 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.

  4. 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.

  5. A dietary nucleoside-nucleotide mixture inhibits endotoxin-induced bacterial translocation in mice fed protein-free diet.

    PubMed

    Adjei, A A; Yamamoto, S

    1995-01-01

    Nucleosides and nucleotides are important substrates utilized by the intestinal mucosa. To determine the relative effect of dietary nucleosides and nucleotides on the gut, we investigated the effects of these compounds on endotoxin-induced bacterial translocation, cecal bacterial populations and ileal histology in protein-malnourished mice. There was an inhibition of gram-negative enteric bacteria in the mesenteric lymph node and spleen of the surviving mice fed the protein-free diet supplemented with a nucleoside-nucleotide mixture compared with the nonsupplemented group. Histologically, the damage to the gut mucosal barrier was more pronounced in the nonsupplemented group than in the nucleoside-nucleotide supplemented group. However, the cecal bacterial populations in the groups were not different. The villous height, crypt depth and total wall thickness were more developed in the supplemented group compared with the nonsupplemented group, indicating that the nucleoside-nucleotide mixture blocked bacterial translocation by preventing endotoxin-induced mucosal or epithelial damage. These results suggest that the nucleoside-nucleotide mixture could be used to inhibit or reduce the incidence of bacterial translocation, decrease intestinal injury and improve survival in a lethal model of protein deficiency and endotoxemia.

  6. 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

  7. 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.

  8. Brain inflammation in a chronic epilepsy model: Evolving pattern of the translocator protein during epileptogenesis.

    PubMed

    Amhaoul, Halima; Hamaide, Julie; Bertoglio, Daniele; Reichel, Stephanie Nadine; Verhaeghe, Jeroen; Geerts, Elly; Van Dam, Debby; De Deyn, Peter Paul; Kumar-Singh, Samir; Katsifis, Andrew; Van Der Linden, Annemie; Staelens, Steven; Dedeurwaerdere, Stefanie

    2015-10-01

    A hallmark in the neuropathology of temporal lobe epilepsy is brain inflammation which has been suggested as both a biomarker and a new mechanistic target for treatments. The translocator protein (TSPO), due to its high upregulation under neuroinflammatory conditions and the availability of selective PET tracers, is a candidate target. An important step to exploit this target is a thorough characterisation of the spatiotemporal profile of TSPO during epileptogenesis. TSPO expression, microglial activation, astrocyte reactivity and cell loss in several brain regions were evaluated at five time points during epileptogenesis, including the chronic epilepsy phase in the kainic acid-induced status epilepticus (KASE) model (n = 52) and control Wistar Han rats (n = 33). Seizure burden was also determined in the chronic phase. Furthermore, ¹⁸F-PBR111 PET/MRI scans were acquired longitudinally in an additional four KASE animals. TSPO expression measured with in vitro and in vivo techniques was significantly increased at each time point and peaked two weeks post-SE in the limbic system. A prominent association between TSPO expression and activated microglia (p < 0.001; r = 0.7), as well as cell loss (p < 0.001; r = -0.8) could be demonstrated. There was a significant positive correlation between spontaneous seizures and TSPO upregulation in several brain regions with increased TSPO expression. TSPO expression was dynamically upregulated during epileptogenesis, persisted in the chronic phase and correlated with microglia activation rather than reactive astrocytes. TSPO expression was correlating with spontaneous seizures and its high expression during the latent phase might possibly suggest being an important switching point in disease ontogenesis which could be further investigated by PET imaging.

  9. 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

  10. 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.

  11. 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.

  12. BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity.

    PubMed

    Vander Broek, Charles W; Zainal Abidin, Nurhamimah; Stevens, Joanne M

    2017-01-01

    Burkholderia pseudomallei is an intracellular bacterial pathogen and the causative agent of melioidosis, a severe disease of humans and animals. Like other clinically important Gram-negative bacteria, fundamental to B. pseudomallei pathogenesis is the Bsa Type III Secretion System. The Bsa system injects bacterial effector proteins into the cytoplasm of target host cells subverting cellular pathways for the benefit of the bacteria. It is required for invasion of non-phagocytic host cells, escape from the endocytic compartment into the host cell cytoplasm, and for virulence in murine models of melioidosis. We have recently described the repertoire of effector proteins secreted by the B. pseudomallei Bsa system, however the functions of many of these effector proteins remain an enigma. One such protein is BipC, a homolog of the translocator/effector proteins SipC and IpaC from Salmonella spp. and Shigella flexneri respectively. SipC and IpaC each have separate and distinct roles acting both as translocators, involved in creating a pore in the eukaryotic cell membrane through which effector proteins can transit, and as effectors by interacting with and polymerizing host cell actin. In this study, pull-down assays demonstrate an interaction between BipC and actin. Furthermore, we show that BipC directly interacts with actin, preferentially with actin polymers (F-actin) and has the ability to polymerize actin in a similar manner as that described for SipC. Yet unlike SipC, BipC does not stabilize F-actin filaments, indicating a functionally distinct interaction with actin. Expression of Myc-tagged BipC in HeLa cells induces the formation of pseudopodia similar to that seen for IpaC. This study explores the effector function of BipC and reveals that actin interaction is conserved within the BipC/SipC/IpaC family of translocator/effector proteins.

  13. BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity

    PubMed Central

    Vander Broek, Charles W.; Zainal Abidin, Nurhamimah; Stevens, Joanne M.

    2017-01-01

    Burkholderia pseudomallei is an intracellular bacterial pathogen and the causative agent of melioidosis, a severe disease of humans and animals. Like other clinically important Gram-negative bacteria, fundamental to B. pseudomallei pathogenesis is the Bsa Type III Secretion System. The Bsa system injects bacterial effector proteins into the cytoplasm of target host cells subverting cellular pathways for the benefit of the bacteria. It is required for invasion of non-phagocytic host cells, escape from the endocytic compartment into the host cell cytoplasm, and for virulence in murine models of melioidosis. We have recently described the repertoire of effector proteins secreted by the B. pseudomallei Bsa system, however the functions of many of these effector proteins remain an enigma. One such protein is BipC, a homolog of the translocator/effector proteins SipC and IpaC from Salmonella spp. and Shigella flexneri respectively. SipC and IpaC each have separate and distinct roles acting both as translocators, involved in creating a pore in the eukaryotic cell membrane through which effector proteins can transit, and as effectors by interacting with and polymerizing host cell actin. In this study, pull-down assays demonstrate an interaction between BipC and actin. Furthermore, we show that BipC directly interacts with actin, preferentially with actin polymers (F-actin) and has the ability to polymerize actin in a similar manner as that described for SipC. Yet unlike SipC, BipC does not stabilize F-actin filaments, indicating a functionally distinct interaction with actin. Expression of Myc-tagged BipC in HeLa cells induces the formation of pseudopodia similar to that seen for IpaC. This study explores the effector function of BipC and reveals that actin interaction is conserved within the BipC/SipC/IpaC family of translocator/effector proteins. PMID:28770177

  14. Importin α-importin β complex mediated nuclear translocation of insulin-like growth factor binding protein-5.

    PubMed

    Sun, Min; Long, Juan; Yi, Yuxin; Xia, Wei

    2017-08-23

    Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions, as well as regulates cell proliferation, migration, and apoptosis independent of IGF. Proper cellular localization is critical for the effective function of most signaling molecules. In previous studies, we have shown that the nuclear IGFBP-5 comes from ER-cytosol retro-translocation. In this study, we further investigated the pathway mediating IGFBP-5 nuclear import after it retro-translocation. Importin-α5 was identified as an IGFBP-5-interacting protein with a yeast two-hybrid system, and its interaction with IGFBP-5 was further confirmed by GST pull down and co-immunoprecipitation. Binding affinity of IGFBP-5 and importins were determined by surface plasmon resonance (IGFBP-5/importin-β: KD=2.44e-7, IGFBP-5/importin-α5: KD=3.4e-7). Blocking the importin-α5/importin-β nuclear import pathway using SiRNA or dominant negative impotin-β dramatically inhibited IGFBP-5-EGFP nuclear import, though importin-α5 overexpress does not affect IGFBP-5 nuclear import. Furthermore, nuclear IGFBP-5 was quantified using luciferase report assay. When deleted the IGFBP-5 nuclear localization sequence (NLS), IGFBP-5ΔNLS loss the ability to translocate into the nucleus and accumulation of IGFBP-5ΔNLS was visualized in the cytosol. Altogether, our findings provide a substantially evidence showed that the IGFBP-5 nuclear import is mediated by importin-α/importin-β complex, and NLS is critical domain in IGFBP-5 nuclear translocation.

  15. Truncation Analysis of TatA and TatB Defines the Minimal Functional Units Required for Protein Translocation

    PubMed Central

    Lee, Philip A.; Buchanan, Grant; Stanley, Nicola R.; Berks, Ben C.; Palmer, Tracy

    2002-01-01

    The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli. C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport. These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted. PMID:12374820

  16. Truncation analysis of TatA and TatB defines the minimal functional units required for protein translocation.

    PubMed

    Lee, Philip A; Buchanan, Grant; Stanley, Nicola R; Berks, Ben C; Palmer, Tracy

    2002-11-01

    The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli. C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport. These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted.

  17. Ethanol-induced translocation of cAMP-dependent protein kinase to the nucleus. Mechanism and functional consequences.

    PubMed

    Constantinescu, A; Diamond, I; Gordon, A S

    1999-09-17

    Ethanol induces translocation of the catalytic subunit (Calpha) of cAMP-dependent protein kinase (PKA) from the Golgi area to the nucleus in NG108-15 cells. Ethanol also induces translocation of the RIIbeta regulatory subunit of PKA to the nucleus; RI and Cbeta are not translocated. Nuclear PKA activity in ethanol-treated cells is no longer regulated by cAMP. Gel filtration and immunoprecipitation analysis confirm that ethanol blocks the reassociation of Calpha with RII but does not induce dissociation of these subunits. Ethanol also reduces inhibition of Calpha by the PKA inhibitor PKI. Pre-incubation of Calpha with ethanol decreases phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and casein but has no effect on the phosphorylation of highly charged molecules such as histone H1 or protamine. cAMP-response element-binding protein (CREB) phosphorylation by Calpha is also increased in ethanol-treated cells. This increase in CREB phosphorylation is inhibited by the PKA antagonist (R(p))-cAMPS and by an adenosine receptor antagonist. These results suggest that ethanol affects a cascade of events allowing for sustained nuclear localization of Calpha and prolonged CREB phosphorylation. These events may account for ethanol-induced changes in cAMP-dependent gene expression.

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

    PubMed

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

    2016-04-07

    Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: only the myristoylated form translocates into the lipid bilayer.

    PubMed

    Harishchandran, Avaronnan; Nagaraj, Ramakrishnan

    2005-07-30

    Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.

  4. Role of translocator protein density, a marker of neuroinflammation, in the brain during major depressive episodes.

    PubMed

    Setiawan, Elaine; Wilson, Alan A; Mizrahi, Romina; Rusjan, Pablo M; Miler, Laura; Rajkowska, Grazyna; Suridjan, Ivonne; Kennedy, James L; Rekkas, P Vivien; Houle, Sylvain; Meyer, Jeffrey H

    2015-03-01

    The neuroinflammatory hypothesis of major depressive disorder is supported by several main findings. First, in humans and animals, activation of the immune system causes sickness behaviors that present during a major depressive episode (MDE), such as low mood, anhedonia, anorexia, and weight loss. Second, peripheral markers of inflammation are frequently reported in major depressive disorder. Third, neuroinflammatory illnesses are associated with high rates of MDEs. However, a fundamental limitation of the neuroinflammatory hypothesis is a paucity of evidence of brain inflammation during MDE. Translocator protein density measured by distribution volume (TSPO VT) is increased in activated microglia, an important aspect of neuroinflammation. To determine whether TSPO VT is elevated in the prefrontal cortex, anterior cingulate cortex (ACC), and insula in patients with MDE secondary to major depressive disorder. Case-control study in a tertiary care psychiatric hospital from May 1, 2010, through February 1, 2014. Twenty patients with MDE secondary to major depressive disorder and 20 healthy control participants underwent positron emission tomography with fluorine F 18-labeled N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide ([18F]FEPPA). Patients with MDE were medication free for at least 6 weeks. All participants were otherwise healthy and nonsmokers. Values of TSPO VT in the prefrontal cortex, ACC, and insula. In MDE, TSPO VT was significantly elevated in all brain regions examined (multivariate analysis of variance, F15,23 = 4.5 [P = .001]). The magnitude of TSPO VT elevation was 26% in the prefrontal cortex (mean [SD] TSPO VT, 12.5 [3.6] in patients with MDE and 10.0 [2.4] in controls), 32% in the ACC (mean [SD] TSPO VT, 12.3 [3.5] in patients with MDE and 9.3 [2.2] in controls), and 33% in the insula (mean [SD] TSPO VT, 12.9 [3.7] in patients with MDE and 9.7 [2.3] in controls). In MDE, greater TSPO VT in the ACC correlated with greater

  5. Mixed-Affinity Binding in Humans with 18-kDa Translocator Protein Ligands

    PubMed Central

    Owen, David R.J.; Gunn, Roger N.; Rabiner, Eugenii A.; Bennacef, Idriss; Fujita, Masahiro; Kreisl, William C.; Innis, Robert B.; Pike, Victor W.; Reynolds, Richard; Matthews, Paul M.; Parker, Christine A.

    2011-01-01

    11C-PBR28 PET can detect the 18-kDa translocator protein (TSPO) expressed within macrophages. However, quantitative evaluation of the signal in brain tissue from donors with multiple sclerosis (MS) shows that PBR28 binds the TSPO with high affinity (binding affinity [Ki], ~4 nM), low affinity (Ki, ~200 nM), or mixed affinity (2 sites with Ki, ~4 nM and ~300 nM). Our study tested whether similar binding behavior could be detected in brain tissue from donors with no history of neurologic disease, with TSPO-binding PET ligands other than 11C-PBR28, for TSPO present in peripheral blood, and with human brain PET data acquired in vivo with 11C-PBR28. Methods The affinity of TSPO ligands was measured in the human brain post-mortem from donors with a history of MS (n = 13), donors without any history of neurologic disease (n = 20), and in platelets from healthy volunteers (n = 13). Binding potential estimates from thirty-five 11C-PBR28 PET scans from an independent sample of healthy volunteers were analyzed using a gaussian mixture model. Results Three binding affinity patterns were found in brains from subjects without neurologic disease in similar proportions to those reported previously from studies of MS brains. TSPO ligands showed substantial differences in affinity between subjects classified as high-affinity binders (HABs) and low-affinity binders (LABs). Differences in affinity between HABs and LABs are approximately 50-fold with PBR28, approximately 17-fold with PBR06, and approximately 4-fold with DAA1106, DPA713, and PBR111. Where differences in affinity between HABs and LABs were low (~4-fold), distinct affinities were not resolvable in binding curves for mixed-affinity binders (MABs), which appeared to express 1 class of sites with an affinity approximately equal to the mean of those for HABs and LABs. Mixed-affinity binding was detected in platelets from an independent sample (HAB, 69%; MAB, 31%), although LABs were not detected. Analysis of 11C-PBR28 PET

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

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

    PubMed

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

    2005-10-01

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

  14. The Role of T-Cell Leukemia Translocation-Associated Gene Protein in Human Tumorigenesis and Osteoclastogenesis

    PubMed Central

    Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki

    2012-01-01

    Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-κB ligand (RANKL), TNF-α, IL-6, IL-17, and IFN-γ. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in regulating bone resorption. In 2009, we demonstrated that novel peptides from T-cell leukemia translocation-associated gene (TCTA) protein expressed in synovial tissues from patients with RA inhibit human osteoclastogenesis, preventing cellular fusion via the interaction between TCTA protein and a putative counterpart molecule. Only a few studies on the role of TCTA protein have been reported. Genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines, suggesting the loss of one of the two copies of the gene. In the current paper, we reviewed the roles of TCTA protein in lung cancer cell lines and human osteoclastogenesis. PMID:22174563

  15. 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.

  16. 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.

  17. 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.

  18. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  20. 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

  1. 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

  2. The structure of the extracellular domain of the jumping translocation breakpoint protein reveals a variation of the midkine fold.

    PubMed

    Rousseau, Francois; Pan, Borlan; Fairbrother, Wayne J; Bazan, J Fernando; Lingel, Andreas

    2012-01-06

    Jumping Translocation Breakpoint (JTB) is an orphan receptor that is conserved from nematodes to humans and whose gene expression in humans is strikingly upregulated in diverse types of cancers. Translocations occur frequently at the hJTB genomic locus, leading to multiple copies of a truncated JTB gene, which potentially encodes a soluble secreted ectodomain. In addition, JTB and its orthologs likely represent a unique and ancient protein family since homologs could not be identified by direct sequence comparison. In the present study, we have determined the NMR solution structure of the N-terminal ectodomain of human JTB, showing that its fold architecture is a new variant of a three-β-strand antiparallel β-meander. The JTB structure has a distant relationship to the midkine/pleiotrophin fold, particularly in the conservation of distinctive disulfide bridge patterns. The structure of this newly characterized small cysteine-rich domain suggests potential involvement of JTB in interactions with proteins or extracellular matrix and may help to uncover the elusive biological functions of this protein. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Isolation and characterization of bifunctional Escherichia coli TatA mutant proteins that allow efficient tat-dependent protein translocation in the absence of TatB.

    PubMed

    Blaudeck, Natascha; Kreutzenbeck, Peter; Müller, Matthias; Sprenger, Georg A; Freudl, Roland

    2005-02-04

    In Escherichia coli, the Tat system promotes the membrane translocation of a subset of exported proteins across the cytoplasmic membrane. Four genes (tatA, tatB, tatC, and tatE) have been identified that encode the components of the E. coli Tat translocation apparatus. Whereas TatA and TatE can functionally substitute for each other, the TatB and the TatC proteins have been shown to perform distinct functions. In contrast to Tat systems of the ABC(E) type found in E. coli and many other bacteria, some microorganisms possess a TatAC-type translocase that consists of TatA and TatC only, suggesting that, in these systems, TatB is not required or that one of the remaining components (TatA or TatC) additionally takes over the TatB function. We have addressed the molecular basis for the difference in subunit composition between TatABC(E) and TatAC-type systems by using a genetic approach. A plasmid-encoded E. coli minimal Tat translocase consisting solely of TatA and TatC was shown to mediate a low level translocation of a sensitive Tat-dependent reporter protein. Suppressor mutations in the minimal Tat translocase were isolated that compensate for the absence of TatB and that showed substantial increases in translocation activities. All of the mutations mapped to the extreme amino-terminal domain of TatA. No mutations affecting TatC were identified. These results suggest that in TatAC-type systems, the TatA protein represents a bifunctional component fulfilling both the TatA and TatB functions. Furthermore, our results indicate that the structure of the amino-terminal domain of TatA is decisive for whether or not TatB is required.

  4. 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.

  5. 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

  6. Neuroinflammation in Temporal Lobe Epilepsy Measured Using Positron Emission Tomographic Imaging of Translocator Protein.

    PubMed

    Gershen, Leah D; Zanotti-Fregonara, Paolo; Dustin, Irene H; Liow, Jeih-San; Hirvonen, Jussi; Kreisl, William C; Jenko, Kimberly J; Inati, Sara K; Fujita, Masahiro; Morse, Cheryl L; Brouwer, Chad; Hong, Jinsoo S; Pike, Victor W; Zoghbi, Sami S; Innis, Robert B; Theodore, William H

    2015-08-01

    Neuroinflammation may play a role in epilepsy. Translocator protein 18 kDa (TSPO), a biomarker of neuroinflammation, is overexpressed on activated microglia and reactive astrocytes. A preliminary positron emission tomographic (PET) imaging study using carbon 11 ([11C])-labeled PBR28 in patients with temporal lobe epilepsy (TLE) found increased TSPO ipsilateral to seizure foci. Full quantitation of TSPO in vivo is needed to detect widespread inflammation in the epileptic brain. To determine whether patients with TLE have widespread TSPO overexpression using [11C]PBR28 PET imaging, and to replicate relative ipsilateral TSPO increases in patients with TLE using [11C]PBR28 and another TSPO radioligand, [11C]DPA-713. In a cohort study from March 2009 through September 2013 at the Clinical Epilepsy Section of the National Institute of Neurological Disorders and Stroke, participants underwent brain PET and a subset had concurrent arterial sampling. Twenty-three patients with TLE and 11 age-matched controls were scanned with [11C]PBR28, and 8 patients and 7 controls were scanned with [11C]DPA-713. Patients with TLE had unilateral temporal seizure foci based on ictal electroencephalography and structural magnetic resonance imaging. Participants with homozygous low-affinity TSPO binding were excluded. The [11C]PBR28 distribution volume (VT) corrected for free fraction (fP) was measured in patients with TLE and controls using FreeSurfer software and T1-weighted magnetic resonance imaging for anatomical localization of bilateral temporal and extratemporal regions. Side-to-side asymmetry in patients with TLE was calculated as the ratio of ipsilateral to contralateral [11C]PBR28 and [11C]DPA-713 standardized uptake values from temporal regions. The [11C]PBR28 VT to fp ratio was higher in patients with TLE than in controls for all ipsilateral temporal regions (27%-42%; P < .05) and in contralateral hippocampus, amygdala, and temporal pole (approximately 30%-32%; P < .05

  7. 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

  8. 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

  9. 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. © 2015. Published by The Company of Biologists Ltd.

  10. PPP1R16A, the membrane subunit of protein phosphatase 1beta, signals nuclear translocation of the nuclear receptor constitutive active/androstane receptor.

    PubMed

    Sueyoshi, Tatsuya; Moore, Rick; Sugatani, Junko; Matsumura, Yonehiro; Negishi, Masahiko

    2008-04-01

    Constitutive active/androstane receptor (CAR), a member of the nuclear steroid/thyroid hormone receptor family, activates transcription of numerous hepatic genes upon exposure to therapeutic drugs and environmental pollutants. Sequestered in the cytoplasm, this receptor signals xenobiotic exposure, such as phenobarbital (PB), by translocating into the nucleus. Unlike other hormone receptors, translocation can be triggered indirectly without binding to xenobiotics. We have now identified a membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A) as a novel CAR-binding protein. When CAR and R16A are coexpressed in mouse liver, CAR translocates into the nucleus. Close association of R16A and CAR molecule on liver membrane was shown by fluorescence resonance energy transfer (FRET) analysis using expressed yellow fluorescent protein (YFP)-CAR and CFP-R16A fusion proteins. R16A can form dimer through its middle region, where protein kinase A phosphorylation sites are recently identified. Translocation of CAR by R16A correlates with the ability of R16A to form an intermolecular interaction via the middle region. Moreover, this interaction is enhanced by PB treatment in mouse liver. R16A specifically interacted with PP1beta in HepG2 cells despite the highly conserved structure of PP1 family molecules. PP1beta activity was inhibited by R16A in vitro and coexpression of PP1beta in liver can prevent YFP-CAR translocation into mouse liver. Taken together, R16A at the membrane may mediate the PB signal to initiate CAR nuclear translocation, through a mechanism including its dimerization and inhibition of PP1beta activity, providing a novel model for the translocation of nuclear receptors in which direct interaction of ligands and the receptors may not be crucial.

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

    PubMed

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

    2012-06-22

    AIRE (Autoimmune Regulator) has a central role in the transcriptional regulation of self-antigens in medullary thymic epithelial cells, which is necessary for negative selection of autoreactive T cells. Recent data have shown that AIRE can also induce apoptosis, which may be linked to cross-presentation of these self-antigens. Here we studied AIRE-induced apoptosis using AIRE over-expression in a thymic epithelial cell line as well as doxycycline-inducible HEK293 cells. We show that the HSR/CARD domain in AIRE together with a nuclear localization signal is sufficient to induce apoptosis. In the nuclei of AIRE-positive cells, we also found an increased accumulation of a glycolytic enzyme, glyceraldehyde-3-phosphate (GAPDH) reflecting cellular stress and apoptosis. Additionally, AIRE-induced apoptosis was inhibited with an anti-apoptotic agent deprenyl that blocks GAPDH nitrosylation and nuclear translocation. We propose that the AIRE-induced apoptosis pathway is associated with GAPDH nuclear translocation and induction of NO-induced cellular stress in AIRE-expressing cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Mesenteric fat as a source of C reactive protein and as a target for bacterial translocation in Crohn's disease

    PubMed Central

    Peyrin-Biroulet, Laurent; Gonzalez, Florent; Dubuquoy, Laurent; Rousseaux, Christel; Dubuquoy, Caroline; Decourcelle, Cécilia; Saudemont, Alain; Tachon, Mickael; Béclin, Elodie; Odou, Marie-Françoise; Neut, Christel; Colombel, Jean-Frédéric

    2011-01-01

    Objective Mesenteric fat hyperplasia is a hallmark of Crohn's disease (CD), and C reactive protein (CRP) is correlated with disease activity. The authors investigated whether mesenteric adipocytes may be a source of CRP in CD and whether inflammatory and bacterial triggers may stimulate its production by adipocytes. Design CRP expression in the mesenteric and subcutaneous fats of patients with CD and the correlation between CRP plasma concentrations and mesenteric messenger RNA (mRNA) levels were assessed. The impact of inflammatory and bacterial challenges on CRP synthesis was tested using an adipocyte cell line. Bacterial translocation to mesenteric fat was studied in experimental models of colitis and ileitis and in patients with CD. Results CRP expression was increased in the mesenteric fat of patients with CD, with mRNA levels being 80±40 (p<0.05) and 140±65 (p=0.04) times higher than in the mesenteric fat of patients with ulcerative colitis and in the subcutaneous fat of the same CD subjects, respectively, and correlated with plasma levels. Escherichia coli (1230±175-fold, p<0.01), lipopolysaccharide (26±0.5-fold, p<0.01), tumour necrosis factor α (15±0.3-fold, p<0.01) and interleukin-6 (10±0.7-fold, p<0.05) increased CRP mRNA levels in adipocyte 3T3-L1 cells. Bacterial translocation to mesenteric fat occurred in 13% and 27% of healthy and CD subjects, respectively, and was increased in experimental colitis and ileitis. Human mesenteric adipocytes constitutively expressed mRNA for TLR2, TLR4, NOD1 and NOD2. Conclusion Mesenteric fat is an important source of CRP in CD. CRP production by mesenteric adipocytes may be triggered by local inflammation and bacterial translocation to mesenteric fat, providing a mechanism whereby mesenteric fat hyperplasia may contribute to inflammatory response in CD. PMID:21940721

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

    PubMed

    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

    2016-11-03

    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.

  14. 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

  15. SecA alone can promote protein translocation and ion channel activity: SecYEG increases efficiency and signal peptide specificity.

    PubMed

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

    2011-12-30

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

  16. 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.

  17. 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

  18. 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.

  19. Fluctuations in polymer translocation

    NASA Astrophysics Data System (ADS)

    Krapivsky, P. L.; Mallick, K.

    2010-07-01

    We investigate a model of chaperone-assisted polymer translocation through a nanopore in a membrane. Translocation is driven by irreversible random sequential absorption of chaperone proteins that bind to the polymer on one side of the membrane. The proteins are larger than the pore and hence the backward motion of the polymer is inhibited. This mechanism rectifies Brownian fluctuations and results in an effective force that drags the polymer in a preferred direction. The translocated polymer undergoes an effective biased random walk and we compute the corresponding diffusion constant. Our methods allow us to determine the large deviation function which, in addition to velocity and diffusion constant, contains the entire statistics of the translocated length.

  20. 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.

  1. 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

  2. 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

  3. Rice C2-domain proteins are induced and translocated to the plasma membrane in response to a fungal elicitor.

    PubMed

    Kim, Cha Young; Koo, Yoon Duck; Jin, Jing Bo; Moon, Byeong Cheol; Kang, Chang Ho; Kim, Sun Tae; Park, Byung Ouk; Lee, So Young; Kim, Man Lyang; Hwang, Inhwan; Kang, Kyu Young; Bahk, Jeong Dong; Lee, Sang Yeol; Cho, Moo Je

    2003-10-14

    Hundreds of proteins involved in signaling pathways contain a Ca(2+)-dependent membrane-binding motif called the C2-domain. However, no small C2-domain proteins consisting of a single C2-domain have been reported in animal cells. We have isolated two cDNA clones, OsERG1a and OsERG1b, that encode two small C2-domain proteins of 156 and 159 amino acids, respectively, from a fungal elicitor-treated rice cDNA library. The clones are believed to have originated from a single gene by alternative splicing. Transcript levels of the OsERG1 gene are dramatically elevated by a fungal elicitor prepared from Magnaporthe grisea or by Ca(2+) ions. The OsERG1 protein produced in Escherichia coli binds to phospholipid vesicles in a Ca(2+)-dependent manner and is translocated to the plasma membrane of plant cells by treatment with either a fungal elicitor or a Ca(2+) ionophore. These results suggest that OsERG1 proteins containing a single C2-domain are involved in plant defense signaling systems.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. Long Residence Time at the Neurosteroidogenic 18 kDa Translocator Protein Characterizes the Anxiolytic Ligand XBD173.

    PubMed

    Costa, Barbara; Da Pozzo, Eleonora; Cavallini, Chiara; Taliani, Sabrina; Da Settimo, Federico; Martini, Claudia

    2016-08-17

    Recent data have demonstrated a positive correlation between the residence time (RT) and neurosteroidogenic efficacy of a ligand at the translocator protein (TSPO), an attractive anxyolitic target. To explore the potential impact of RT on TSPO ligand anxiolytic activity, the RT and the steroidogenic activity of XBD173, a ligand exerting anxiolytic activity in humans, were retrospectively evaluated. To this aim, XBD173 association and dissociation rate constants were measured (1.23 × 10(7) M(-1) min(-1) and 0.0079 min(-1), respectively). XBD173 resulted to have a long RT (127 min) and to stimulate efficaciously neurosteroidogenesis, in terms of pregnenolone production. The present findings corroborate the importance of TSPO ligand RT to predict their effective neurosteroidogenic activity and promising anxiolytic action. These positive results prompted us to set up a fast and high-throughput kinetic method to improve the efficiency of RT-based TSPO drug-discovery process.

  10. Voxel-based imaging of translocator protein 18kDa (TSPO) in high-resolution PET

    PubMed Central

    Ko, Ji Hyun; Koshimori, Yuko; Mizrahi, Romina; Rusjan, Pablo; Wilson, Alan A; Lang, Anthony E; Houle, Sylvain; Strafella, Antonio P

    2013-01-01

    In vivo imaging of translocator protein 18 kDa (TSPO) has received significant attention as potential biomarker of microglia activation. Several radioligands have been designed with improved properties. Our group recently developed an 18F-labeled TSPO ligand, [18F]-FEPPA, and confirmed its reliability with a 2-tissue compartment model. Here, we extended, in a group of healthy subjects, its suitability for use in voxel-based analysis with the newly proposed graphical analysis approach, Relative-Equilibrium-Gjedde-Patlak (REGP) plot. The REGP plot successfully replicated the total distribution volumes estimated by the 2-tissue compartment model. We also showed its proof-of-concept in a patient with possible meningioma showing increased [18F]-FEPPA total distribution volume. PMID:23281426

  11. Voxel-based imaging of translocator protein 18 kDa (TSPO) in high-resolution PET.

    PubMed

    Ko, Ji Hyun; Koshimori, Yuko; Mizrahi, Romina; Rusjan, Pablo; Wilson, Alan A; Lang, Anthony E; Houle, Sylvain; Strafella, Antonio P

    2013-03-01

    In vivo imaging of translocator protein 18 kDa (TSPO) has received significant attention as potential biomarker of microglia activation. Several radioligands have been designed with improved properties. Our group recently developed an (18)F-labeled TSPO ligand, [(18)F]-FEPPA, and confirmed its reliability with a 2-tissue compartment model. Here, we extended, in a group of healthy subjects, its suitability for use in voxel-based analysis with the newly proposed graphical analysis approach, Relative-Equilibrium-Gjedde-Patlak (REGP) plot. The REGP plot successfully replicated the total distribution volumes estimated by the 2-tissue compartment model. We also showed its proof-of-concept in a patient with possible meningioma showing increased [(18)F]-FEPPA total distribution volume.

  12. 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

  13. Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration

    PubMed Central

    Miller, Thomas F.

    2017-01-01

    We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency. PMID:28328943

  14. 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

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

    PubMed

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

    2011-01-01

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

  16. 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

  17. Listeria monocytogenes Uses Listeria Adhesion Protein (LAP) To Promote Bacterial Transepithelial Translocation and Induces Expression of LAP Receptor Hsp60▿

    PubMed Central

    Burkholder, Kristin M.; Bhunia, Arun K.

    2010-01-01

    Listeria monocytogenes interaction with the intestinal epithelium is a key step in the infection process. We demonstrated that Listeria adhesion protein (LAP) promotes adhesion to intestinal epithelial cells and facilitates extraintestinal dissemination in vivo. The LAP receptor is a stress response protein, Hsp60, but the precise role for the LAP-Hsp60 interaction during Listeria infection is unknown. Here we investigated the influence of physiological stressors and Listeria infection on host Hsp60 expression and LAP-mediated bacterial adhesion, invasion, and transepithelial translocation in an enterocyte-like Caco-2 cell model. Stressors such as heat (41°C), tumor necrosis factor alpha (TNF-α) (100 U), and L. monocytogenes infection (104 to 106 CFU/ml) significantly (P < 0.05) increased plasma membrane and intracellular Hsp60 levels in Caco-2 cells and consequently enhanced LAP-mediated L. monocytogenes adhesion but not invasion of Caco-2 cells. In transepithelial translocation experiments, the wild type (WT) exhibited 2.7-fold more translocation through Caco-2 monolayers than a lap mutant, suggesting that LAP is involved in transepithelial translocation, potentially via a paracellular route. Short hairpin RNA (shRNA) suppression of Hsp60 in Caco-2 cells reduced WT adhesion and translocation 4.5- and 3-fold, respectively, while adhesion remained unchanged for the lap mutant. Conversely, overexpression of Hsp60 in Caco-2 cells enhanced WT adhesion and transepithelial translocation, but not those of the lap mutant. Furthermore, initial infection with a low dosage (106 CFU/ml) of L. monocytogenes increased plasma membrane and intracellular expression of Hsp60 significantly, which rendered Caco-2 cells more susceptible to subsequent LAP-mediated adhesion and translocation. These data provide insight into the role of LAP as a virulence factor during intestinal epithelial infection and pose new questions regarding the dynamics between the host stress response and

  18. 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

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

    NASA Astrophysics Data System (ADS)

    Lopez, Benjamin; Valentine, Megan

    2014-03-01

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

  20. 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

  1. GFP tagging sheds light on protein translocation: implications for key methods in cell biology.

    PubMed

    Deponte, Marcel

    2012-04-01

    Green fluorescent protein (GFP) is a powerful tool for studying gene expression, protein localization, protein-protein interactions, calcium concentrations, and redox potentials owing to its intrinsic fluorescence. However, GFP not only contains a chromophore but is also tightly folded in a temperature-dependent manner. The latter property of GFP has recently been exploited (1) to characterize the translocase of the outer mitochondrial membrane and (2) to discriminate between protein transport across and into biomembranes in vivo. I therefore suggest that GFP could be a valuable tool for the general analysis of protein transport machineries and pathways in a variety of organisms. Moreover, results from such studies could be important for the interpretation and optimization of classical experiments using GFP tagging.

  2. 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

  3. 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.

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

    PubMed

    Daniele, Simona; Barresi, Elisabetta; Zappelli, Elisa; Marinelli, Luciana; Novellino, Ettore; Da Settimo, Federico; Taliani, Sabrina; Trincavelli, Maria L; Martini, Claudia

    2016-02-16

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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  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. 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.

  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. 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.

  14. Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.

    PubMed Central

    Meyers, S; Downing, J R; Hiebert, S W

    1993-01-01

    The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) translocation associated with acute myelogenous leukemia and encodes a protein with a central 118-amino-acid domain with 69% homology to the Drosophila pair-rule gene, runt. We demonstrate that AML-1 is a DNA-binding protein which specifically interacts with a sequence belonging to the group of enhancer core motifs, TGT/cGGT. Electrophoretic mobility shift analysis of cell extracts identified two AML-1-containing protein-DNA complexes whose electrophoretic mobilities were slower than those of complexes formed with AML-1 produced in vitro. Mixing of in vitro-produced AML-1 with cell extracts prior to gel mobility shift analysis resulted in the formation of higher-order complexes. Deletion mutagenesis of AML-1 revealed that the runt homology domain mediates both sequence-specific DNA binding and protein-protein interactions. The hybrid product, AML-1/ETO, which results from the (8;21) translocation and retains the runt homology domain, both recognizes the AML-1 consensus sequence and interacts with other cellular proteins. Images PMID:8413232

  15. Specific Molecular Chaperone Interactions and an ATP-dependent Conformational Change Are Required during Posttranslational Protein Translocation into the Yeast ER

    PubMed Central

    McClellan, Amie J.; Endres, James B.; Vogel, Joseph P.; Palazzi, Debra; Rose, Mark D.; Brodsky, Jeffrey L.

    1998-01-01

    The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST–63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST–63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST–63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners. PMID:9843586

  16. The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation

    PubMed Central

    Hausner, Jens; Hartmann, Nadine; Jordan, Michael

    2016-01-01

    ABSTRACT The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system. PMID:27895129

  17. The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation.

    PubMed

    Hausner, Jens; Hartmann, Nadine; Jordan, Michael; Büttner, Daniela

    2017-02-01

    The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system. Copyright © 2017 American Society for Microbiology.

  18. 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

  19. 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.

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

    PubMed Central

    Davis, Alison J.; Mecsas, Joan

    2007-01-01

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

  1. Mutations in the Yersinia pseudotuberculosis type III secretion system needle protein, YscF, that specifically abrogate effector translocation into host cells.

    PubMed

    Davis, Alison J; Mecsas, Joan

    2007-01-01

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

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

    PubMed

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

    2003-06-06

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

  3. Control of Protein Activity and Cell Fate Specification via Light-Mediated Nuclear Translocation

    PubMed Central

    Zimmerman, Seth P.; Bear, James E.; Goldstein, Bob; Hahn, Klaus; Kuhlman, Brian

    2015-01-01

    Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), functions in the C. elegans embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor. We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo. PMID:26083500

  4. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.

    PubMed

    Rodriguez-Sinovas, Antonio; Boengler, Kerstin; Cabestrero, Alberto; Gres, Petra; Morente, Miriam; Ruiz-Meana, Marisol; Konietzka, Ina; Miró, Elisabet; Totzeck, Andreas; Heusch, Gerd; Schulz, Rainer; Garcia-Dorado, David

    2006-07-07

    We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

  5. Prognostic role of translocator protein and oxidative stress markers in chronic lymphocytic leukemia patients treated with bendamustine plus rituximab

    PubMed Central

    DE ROSA, ANIELLO; ZAPPAVIGNA, SILVIA; VILLA, MARIA ROSARIA; IMPROTA, SALVATORE; CESARIO, ELENA; MASTRULLO, LUCIA; CARAGLIA, MICHELE; STIUSO, PAOLA

    2015-01-01

    Principally located in the outer mitochondrial membrane, the translocator protein (TSPO) is an 18-kDa transmembrane protein that is a key component of the mitochondrial permeability transition pore. TSPO is associated with a number of biological processes, including apoptosis, the regulation of cellular proliferation, porphyrin transport and heme biosynthesis, immunomodulation, anion transport and the regulation of steroidogenesis. Thus, numerous studies have proposed TSPO as a promising target for novel therapeutic agents, particularly for the treatment of cancer. In the present study, the response of 30 consecutive chronic lymphocytic leukemia (CLL) patients to bendamustine and rituximab treatment was evaluated according to TSPO expression levels. Furthermore, thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) levels, as well as caspase-3 activity were determined. Compared with the lymphocytes of healthy donors, the 30 consecutive CLL patients exhibited increased TSPO expression levels, decreased TBARS and NO levels and reduced caspase-3 activity. Six months after the treatment commenced, the TSPO/mitochondria ratio resembled that of the healthy controls in 24/30 CLL patients. In addition, an increase in TBARS and NO levels, two markers of oxidative stress, and a potentiation of caspase-3 activity in all responder patients was observed. Notably, the six patients who appeared to be resistant to treatment also displayed higher TSPO levels, and lower caspase-3 activity and TBARS levels. These data indicate that TSPO expression may be a molecular prognostic factor in CLL patients. PMID:25663907

  6. Prognostic role of translocator protein and oxidative stress markers in chronic lymphocytic leukemia patients treated with bendamustine plus rituximab.

    PubMed

    DE Rosa, Aniello; Zappavigna, Silvia; Villa, Maria Rosaria; Improta, Salvatore; Cesario, Elena; Mastrullo, Lucia; Caraglia, Michele; Stiuso, Paola

    2015-03-01

    Principally located in the outer mitochondrial membrane, the translocator protein (TSPO) is an 18-kDa transmembrane protein that is a key component of the mitochondrial permeability transition pore. TSPO is associated with a number of biological processes, including apoptosis, the regulation of cellular proliferation, porphyrin transport and heme biosynthesis, immunomodulation, anion transport and the regulation of steroidogenesis. Thus, numerous studies have proposed TSPO as a promising target for novel therapeutic agents, particularly for the treatment of cancer. In the present study, the response of 30 consecutive chronic lymphocytic leukemia (CLL) patients to bendamustine and rituximab treatment was evaluated according to TSPO expression levels. Furthermore, thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) levels, as well as caspase-3 activity were determined. Compared with the lymphocytes of healthy donors, the 30 consecutive CLL patients exhibited increased TSPO expression levels, decreased TBARS and NO levels and reduced caspase-3 activity. Six months after the treatment commenced, the TSPO/mitochondria ratio resembled that of the healthy controls in 24/30 CLL patients. In addition, an increase in TBARS and NO levels, two markers of oxidative stress, and a potentiation of caspase-3 activity in all responder patients was observed. Notably, the six patients who appeared to be resistant to treatment also displayed higher TSPO levels, and lower caspase-3 activity and TBARS levels. These data indicate that TSPO expression may be a molecular prognostic factor in CLL patients.

  7. 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.

  8. 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.

  9. Translocator Protein-18 kDa (TSPO) Positron Emission Tomography (PET) Imaging and Its Clinical Impact in Neurodegenerative Diseases

    PubMed Central

    Dupont, Anne-Claire; Largeau, Bérenger; Santiago Ribeiro, Maria Joao; Guilloteau, Denis; Tronel, Claire; Arlicot, Nicolas

    2017-01-01

    In vivo exploration of activated microglia in neurodegenerative diseases is achievable by Positron Emission Tomography (PET) imaging, using dedicated radiopharmaceuticals targeting the translocator protein-18 kDa (TSPO). In this review, we emphasized the major advances made over the last 20 years, thanks to TSPO PET imaging, to define the pathophysiological implication of microglia activation and neuroinflammation in neurodegenerative diseases, including Parkinson’s disease, Huntington’s disease, dementia, amyotrophic lateral sclerosis, multiple sclerosis, and also in psychiatric disorders. The extent and upregulation of TSPO as a molecular biomarker of activated microglia in the human brain is now widely documented in these pathologies, but its significance, and especially its protective or deleterious action regarding the disease’s stage, remains under debate. Thus, we exposed new and plausible suggestions to enhance the contribution of TSPO PET imaging for biomedical research by exploring microglia’s role and interactions with other cells in brain parenchyma. Multiplex approaches, associating TSPO PET radiopharmaceuticals with other biomarkers (PET imaging of cellular metabolism, neurotransmission or abnormal protein aggregates, but also other imaging modalities, and peripheral cytokine levels measurement and/or metabolomics analysis) was considered. Finally, the actual clinical impact of TSPO PET imaging as a routine biomarker of neuroinflammation was put into perspective regarding the current development of diagnostic and therapeutic strategies for neurodegenerative diseases. PMID:28387722

  10. Transcriptional regulation of translocator protein (Tspo) via a SINE B2-mediated natural antisense transcript in MA-10 Leydig cells.

    PubMed

    Fan, Jinjiang; Papadopoulos, Vassilios

    2012-05-01

    Translocator protein (18 kDa; TSPO) is a mitochondrial cholesterol- and drug-binding protein involved in cholesterol import into mitochondria, the rate-limiting step in steroidogenesis. TSPO is expressed at high levels in Leydig cells of the testis, and its expression levels dictate the ability of the cells to form androgen. In search of mechanisms that regulate Tspo expression, a number of transcription factors acting on its promoter region have been identified. We report herein the presence of a mechanism of regulation of Tspo expression via complementation with a natural antisense transcript (NAT). At the Tspo locus, a short interspersed repetitive element (SINE) of the SINE B2 family has the potential for high transcriptional activity. The extension of the SINE B2 element-mediated transcript overlapped with exon 3 of the Tspo gene and formed a NAT specific for Tspo (Tspo-NAT) in MA-10 mouse tumor Leydig cells. The identified Tspo-NAT was also found in testis and kidney tissues. Overexpression of the Tspo-NAT regulated Tspo gene expression and its function in steroid formation in MA-10 cells. Time-course studies have indicated that Tspo-NAT expression is regulated by cAMP and could regulate TSPO levels to maintain optimal steroid production by MA-10 Leydig cells. Taken together, these results suggest a new micro-transcriptional mechanism that regulates Tspo expression and thus steroidogenesis via an intron-based SINE B2-driven NAT specific for the Tspo gene.

  11. 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.

  12. Translocator Protein-18 kDa (TSPO) Positron Emission Tomography (PET) Imaging and Its Clinical Impact in Neurodegenerative Diseases.

    PubMed

    Dupont, Anne-Claire; Largeau, Bérenger; Santiago Ribeiro, Maria Joao; Guilloteau, Denis; Tronel, Claire; Arlicot, Nicolas

    2017-04-07

    In vivo exploration of activated microglia in neurodegenerative diseases is achievable by Positron Emission Tomography (PET) imaging, using dedicated radiopharmaceuticals targeting the translocator protein-18 kDa (TSPO). In this review, we emphasized the major advances made over the last 20 years, thanks to TSPO PET imaging, to define the pathophysiological implication of microglia activation and neuroinflammation in neurodegenerative diseases, including Parkinson's disease, Huntington's disease, dementia, amyotrophic lateral sclerosis, multiple sclerosis, and also in psychiatric disorders. The extent and upregulation of TSPO as a molecular biomarker of activated microglia in the human brain is now widely documented in these pathologies, but its significance, and especially its protective or deleterious action regarding the disease's stage, remains under debate. Thus, we exposed new and plausible suggestions to enhance the contribution of TSPO PET imaging for biomedical research by exploring microglia's role and interactions with other cells in brain parenchyma. Multiplex approaches, associating TSPO PET radiopharmaceuticals with other biomarkers (PET imaging of cellular metabolism, neurotransmission or abnormal protein aggregates, but also other imaging modalities, and peripheral cytokine levels measurement and/or metabolomics analysis) was considered. Finally, the actual clinical impact of TSPO PET imaging as a routine biomarker of neuroinflammation was put into perspective regarding the current development of diagnostic and therapeutic strategies for neurodegenerative diseases.

  13. 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.

  14. 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

  15. 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.

  16. Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins.

    PubMed

    Zheng, Yujuan; Xie, Jinghang; Huang, Xin; Dong, Jin; Park, Miki S; Chan, William K

    2016-06-01

    The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR of other species, since it is relatively unstable and less sensitive to some ligands in vitro. Overexpression of human AHR has been limited to the baculovirus expression, which is costly and tedious due to the need of repetitive baculovirus production. Here we explored whether we could generate abundant amounts of human AHR and ARNT in a better overexpression system for functional study. We observed that human AHR and ARNT can be expressed in Pichia pastoris with yields that are comparable to the baculovirus system only if their cDNAs are optimized for Pichia expression. Fusion with a c-myc tag at their C-termini seems to increase the expression yield. These Pichia expressed proteins can effectively heterodimerize and form the ternary AHR/ARNT/enhancer complex in the presence of β-naphthoflavone or kynurenine. Limited proteolysis using thermolysin can be used to study the heterodimerization of these human AHR and ARNT proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. 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.

  18. 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.

  19. 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.

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

    PubMed Central

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

    2017-01-01

    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 PMID:26546155

  1. 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

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

    PubMed

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

    2015-12-07

    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. 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

  3. Pseudonegative BCL2 protein expression in a t(14;18) translocation positive lymphoma cell line: a need for an alternative BCL2 antibody.

    PubMed

    Masir, Noraidah; Campbell, Lisa J; Jones, Margaret; Mason, David Y

    2010-04-01

    The t(14;18)(q32;q21) chromosomal translocation induces BCL2 protein expression in most follicular lymphomas. However, a small number of cases lack BCL2 expression despite carrying the t(14;18)(q32;q21) translocation. This study aims to explore the mechanism accounting for the lack of BCL2 protein expression when the t(14;18) translocation is present. BCL2 expression in the t(14;18) positive cell lines FL18, Karpas-422, SU-DHL-4 and SU-DHL-6, was analysed by Western blotting and by immunohistochemistry using two different antibodies. FISH analysis was performed to confirm the cytogenetic changes in the cell lines and real time quantitative PCR was used to evaluate the BCL2 mRNA level. Sequence analysis of translocated BCL2 was performed on FL18, Karpas-422, SU-DHL-4 and SU-DHL-6 cell lines. In FL18, Karpas-422, and SU-DHL-4, the BCL2 mRNA level correlated with the BCL2 protein expression. In contrast, BCL2 protein was not detected in SU-DHL-6 line using standard anti-BCL2 antibody (BCL2/124), despite the presence of the t(14;18) translocation and high level of mRNA. cDNA sequencing of translocated BCL2 showed three mutations in the SU-DHL-6 cell line, one of which resulted in an amino acid substitution (I48F) in the region recognised by the standard BCL2 antibody, whereas the other two were silent mutations at aa71 and aa72. Interestingly, when BCL2 expression was tested with an alternative antibody, E17, the protein was detected in SU-DHL-6, suggesting that the 'negativity' of SU-DHL-6 line for BCL2 using the standard antibody is spurious. Amino acid changes were found in Karpas-422 (G47D, P59L) and SU-DHL-4 (P59T, S117R) but these did not affect BCL2 detection. This study suggests that some somatic mutations of the translocated BCL2 gene may prevent epitope recognition by BCL2 antibodies, and hence cause false negative expression using the standard antibody. It is recommended that in practice all BCL2 negative cases should routinely be stained with an alternative

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  5. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. A novel transposon construct expressing PhoA with potential for studying protein expression and translocation in Mycoplasma gallisepticum.

    PubMed

    Panicker, Indu S; Kanci, Anna; Chiu, Chien-Ju; Veith, Paul D; Glew, Michelle D; Browning, Glenn F; Markham, Philip F

    2012-07-08

    identified by mass-spectrometry after separation by two-dimensional electrophoresis. This is the first study to express PhoA as a lipoprotein in mycoplasmas. The pTAP plasmid will facilitate investigations of lipoproteins and protein translocation across the cell membrane in mycoplasmas, and the ease of detection of these transformants makes this vector system suitable for the simultaneous screening and detection of cloned genes expressed as membrane proteins in mycoplasmas.

  8. A novel transposon construct expressing PhoA with potential for studying protein expression and translocation in Mycoplasma gallisepticum

    PubMed Central

    2012-01-01

    C]palmitate. PhoA could be identified by mass-spectrometry after separation by two-dimensional electrophoresis. Conclusion This is the first study to express PhoA as a lipoprotein in mycoplasmas. The pTAP plasmid will facilitate investigations of lipoproteins and protein translocation across the cell membrane in mycoplasmas, and the ease of detection of these transformants makes this vector system suitable for the simultaneous screening and detection of cloned genes expressed as membrane proteins in mycoplasmas. PMID:22770122

  9. Bacterial twin-arginine signal peptide-dependent protein translocation pathway: evolution and mechanism.

    PubMed

    Wu, L F; Ize, B; Chanal, A; Quentin, Y; Fichant, G

    2000-04-01

    The recently identified bacterial Tat pathway is capable of exporting proteins with a peculiar twin-arginine signal peptide in folded conformation independently of the Sec machinery. It is structurally and mechanistically similar to the delta pH-dependent pathway used for importing chloroplast proteins into the thylakoid. The tat genes are not ubiquitously present and are absent from half of the completely sequenced bacterial genomes. The presence of the tat genes seems to correlate with genome size and with the presence of important enzymes with a twin-arginine signal peptide. A minimal Tat system requires a copy of tatA and a copy of tatC. The composition and gene order of a tat locus are generally conserved within the same taxonomy group but vary considerably to other groups, which would exclude an acquisition of the Tat system by recent horizontal gene transfer. The tat genes are also found in the genomes of chloroplasts and plant mitochondria but are absent from animal mitochondrial genomes. The topology of evolution trees suggests a bacterial origin of the Tat system. In general, the twin-arginine signal peptide is capable of targeting any passenger protein to the Tat pathway. However, a structural signal carried by the mature part of a passenger protein can override targeting information in a signal peptide under certain circumstances. Tat systems show a substrate-Tat component specificity and a species specificity. The pore size of the Tat channel is estimated as being between 5 and 9 nm. Operational models of the Tat system are proposed.

  10. 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.

  11. 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.

  12. Expression, purification, and characterization of Sss1p, an essential component of the yeast Sec61p protein translocation complex.

    PubMed

    Beswick, V; Brodsky, J L; Képès, F; Neumann, J M; Sanson, A; Garrigos, M

    1998-08-01

    Sss1p, a 8.9-kDa membrane protein, is an essential component of the protein translocation complex involved in the transport of secretory proteins across the Saccharomyces cerevisiae endoplasmic reticulum membrane. In order to determine the high resolution structure of Sss1p by NMR, we have undertaken its overexpression and purification. We first inserted the yeast SSS1 gene into the pGEX-2T plasmid expression vector. Sss1p was expressed as fusions with Schistosoma japonica glutathione S-transferase (GST-Sss1p) in MC1061 Escherichia coli cells. Maximum yield of GST-Sss1p was obtained from cells harvested 2 h after induction at 37 degreesC in Luria broth medium. GST-Sss1p was found associated predominantly with the membrane pool and was readily extracted with Triton X-100. Detergent-solubilized GST-Sss1p was isolated by adsorption on glutathione-agarose beads. Sss1p was released from its GST carrier by cleavage with thrombin and its recovery was maximized by addition of dodecyl maltoside. Desorbed Sss1p was loaded on a high-performance liquid chromatography hydroxyapatite column equilibrated in phosphate buffer supplemented with dodecyl maltoside and the fractions containing Sss1p were subsequently purified to homogeneity by reverse-phase chromatography on a C4 column. The entire purification protocol can be completed in 5-6 h and yields about 0.4 mg of Sss1p per gram of transformed cells. CD and preliminary 1H NMR experiments show that purified Sss1p solubilized in SDS micelles is very stable and adopts a helical secondary structure. Copyright 1998 Academic Press.

  13. 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.

  14. 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

  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. 11C-PBR28 binding to translocator protein increases with progression of Alzheimer’s disease

    PubMed Central

    Kreisl, William C.; Lyoo, Chul Hyoung; Liow, Jeih-San; Wei, Monica; Snow, Joseph; Page, Emily; Jenko, Kimberly J.; Morse, Cheryl L.; Zoghbi, Sami S.; Pike, Victor W.; Turner, R. Scott; Innis, Robert B.

    2016-01-01

    This longitudinal study sought to determine whether the 18 kDa translocator protein (TSPO), a marker of neuroinflammation, increases over time in Alzheimer’s disease. Positron emission tomography (PET) imaging with the TSPO radioligand 11C-PBR28 imaging was performed at baseline and after a median follow-up of 2.7 years in 14 amyloid-positive patients and eight amyloid-negative controls. Patients had a greater increase in TSPO binding than controls in inferior parietal lobule, precuneus, occipital cortex, hippocampus, entorhinal cortex, and combined middle and inferior temporal cortex. TSPO binding in temporo-parietal regions increased 3.9 – 6.3% per annum in patients, but ranged from −0.5 – 1% per annum in controls. The change in TSPO binding correlated with cognitive worsening on Clinical Dementia Rating scale – Sum of Boxes and with reduced cortical volume. The annual rate of increased TSPO binding in temporo-parietal regions was about five-fold higher in patients with clinical progression (n = 9) compared to those who did not progress (n = 5). TSPO may serve as a biomarker of Alzheimer’s progression and response to anti-inflammatory therapies. PMID:27318133

  17. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Early upregulation of 18-kDa translocator protein in response to acute neurodegenerative damage in TREM2-deficient mice.

    PubMed

    Belloli, Sara; Pannese, Maria; Buonsanti, Cecilia; Maiorino, Chiara; Di Grigoli, Giuseppe; Carpinelli, Assunta; Monterisi, Cristina; Moresco, Rosa Maria; Panina-Bordignon, Paola

    2017-05-01

    Mutations in the TREM2 gene confer risk for Alzheimer's disease and susceptibility for Parkinson's disease (PD). We evaluated the effect of TREM2 deletion in a 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model, measuring neurodegeneration and microglia activation using a combined in vivo imaging and postmortem molecular approach. In wild-type mice, MPTP administration induced a progressive decrease of [(11)C]FECIT uptake, culminating at day 7. Neuronal loss was accompanied by an increase of TREM2, IL-1β, and translocator protein (TSPO) transcript levels, [(11)C]PK11195 binding and GFAP staining (from day 2), and an early and transient increase of TNF-α, Galectin-3, and Iba-1 (from day 1). In TREM2 null (TREM2(-/-)) mice, MPTP similarly affected neuron viability and microglial cells, as shown by the lower level of Iba-1 staining in basal condition, and reduced increment of Iba-1, TNF-α, and IL-1β in response to MPTP. Likely to compensate for TREM2 absence, TREM2(-/-) mice showed an earlier increment of [(11)C]PK11195 binding and a significant increase of IL-4. Taken together, our data demonstrate a central role of TREM2 in the regulation of microglia response to acute neurotoxic insults and suggest a potential modulatory role of TSPO in response to immune system deficit. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Translocator protein ligands based on N-methyl-(quinolin-4-yl)oxypropanamides with properties suitable for PET radioligand development.

    PubMed

    Brouwer, Chad; Jenko, Kimberly J; Zoghbi, Sami S; Morse, Cheryl L; Innis, Robert B; Pike, Victor W

    2016-11-29

    Modifications to an N-methyl-(quinolin-4-yl)oxypropanamide scaffold were explored to discover leads for developing new radioligands for PET imaging of brain TSPO (translocator protein), a biomarker of neuroinflammation. Whereas contraction of the quinolinyl portion of the scaffold or cyclization of the tertiary amido group abolished high TSPO affinity, insertion of an extra nitrogen atom into the 2-arylquinolinyl portion was effective in retaining sub-nanomolar affinity for rat TSPO, while also decreasing lipophilicity to within the moderate range deemed preferable for a PET radioligand. Replacement of a phenyl group on the amido nitrogen with an isopropyl group was similarly effective. Among others, compound 20 (N-methyl-N-phenyl-2-[2-(pyridin-2-yl)-1,8-naphthyridin-4-yloxy]propanamide) appears especially appealing for PET radioligand development, based on high selectivity and high affinity (Ki = 0.5 nM) for rat TSPO, moderate lipophilicity (logD = 2.48), and demonstrated amenability to labeling with carbon-11. Published by Elsevier Masson SAS.

  20. A novel translocator protein 18 kDa ligand, ZBD-2, exerts neuroprotective effects against acute spinal cord injury.

    PubMed

    Cheng, Qiang; Sun, Guo-Jing; Liu, Shui-Bing; Yang, Qi; Li, Xiao-Ming; Li, Xu-Bo; Liu, Gang; Zhao, Jian-Ning; Zhao, Ming-Gao

    2016-10-01

    Traumatic spinal cord injury (SCI) happens accidently and often leads to motor dysfunction due to a series of biochemical and pathological events and damage, either temporarily or permanently. Translocator protein 18 (TSPO) has been found to be involved in the synthesis of endogenous neurosteroids which have multiple effects on neurons, but the internal mechanisms are not clear. N-benzyl-N-ethyl-2-(7,8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2), a newly reported ligand of TSPO, shows some neuroprotective effect against focal cerebral ischemia in vivo and NMDA-induced neurotoxicity in vitro. The present study aims to examine the role of ZBD-2 in SCI mice and elucidate the underlying molecular mechanisms. The SCI model was established by crushing spinal cord. ZBD-2 (10 mg/kg) significantly enhanced the hindlimb locomotor functions after SCI and decreased the tissue damage and conserved the white matter of the spinal cord. High-dose ZBD-2 alleviated the oxidative stress induced by SCI and regulated the imbalance between NR2B-containing NMDA and GABA receptors by increasing the levels of GAD67 in the spinal cord of SCI mice. Additionally, ZBD-2 (10 mg/kg) increased phosphorylated Akt (p-Akt) and decreased the ratio of Bax/Bcl-2. These results demonstrate that ZBD-2 performs neuroprotection against SCI through regulating the synaptic transmission and the PI3K/AKT signaling pathway. © 2016 John Wiley & Sons Australia, Ltd.

  1. 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

  2. Quantification of ONO-2952 Occupancy of 18-kDaTranslocator Protein in Conscious Monkey Brains using Positron Emission Tomography.

    PubMed

    Mitsui, Katsukuni; Morimoto, Noriko; Niwa, Tomohiro; Yamaura, Yoshiyuki; Ohba, Hiroyuki; Tsukada, Hideo; Katsumata, Seishi

    2017-03-01

    We have previously shown that ONO-2952, a novel 18-kDa translocator protein (TSPO) antagonist, inhibits stress-induced accumulation of neurosteroids and noradrenaline release in the rat brain and alleviates the subsequent symptomatic responses with a brain TSPO occupancy of 50% or more. In this study, we measured ONO-2952 brain TSPO occupancy in conscious rhesus monkeys using positron emission tomography (PET) with (11)C-PBR28 as ligand for translational research to clinical application. PET scans were performed after single and repeated oral administration of ONO-2952 at several dose levels for each animal, with sequential arterial blood sampling. In vitro binding studies showed that ONO-2952 potently binds to brain TSPO in monkeys with an affinity equivalent to that in rats. ONO-2952, given orally before PET scans, dose dependently decreased (11)C-PBR28 uptake without marked brain region specificity. Results of the quantitative analysis using arterial input function revealed that TSPO occupancy after ONO-2952 single and repeated oral administration tended to increase in parallel with its plasma concentration, reaching the highest level of 100%. These findings indicate that ONO-2952 has sufficient brain distribution in primates and that ONO-2952 TSPO occupancy in humans can also be determined using PET. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Quantitation of translocator protein binding in human brain with the novel radioligand [18F]-FEPPA and positron emission tomography.

    PubMed

    Rusjan, Pablo M; Wilson, Alan A; Bloomfield, Peter M; Vitcu, Irina; Meyer, Jeffrey H; Houle, Sylvain; Mizrahi, Romina

    2011-08-01

    This article describes the kinetic modeling of [(18)F]-FEPPA binding to translocator protein 18 kDa in the human brain using high-resolution research tomograph (HRRT) positron emission tomography. Positron emission tomography scans were performed in 12 healthy volunteers for 180 minutes. A two-tissue compartment model (2-CM) provided, with no exception, better fits to the data than a one-tissue model. Estimates of total distribution volume (V(T)), specific distribution volume (V(S)), and binding potential (BP(ND)) demonstrated very good identifiability (based on coefficient of variation (COV)) for all the regions of interest (ROIs) in the gray matter (COV V(T)<7%, COV V(S)<8%, COV BP(ND)<11%). Reduction of the length of the scan to 2 hours is feasible as V(S) and V(T) showed only a small bias (6% and 7.5%, respectively). Monte Carlo simulations showed that, even under conditions of a 500% increase in specific binding, the identifiability of V(T) and V(S) was still very good with COV<10%, across high-uptake ROIs. The excellent identifiability of V(T) values obtained from an unconstrained 2-CM with data from a 2-hour scan support the use of V(T) as an appropriate and feasible outcome measure for [(18)F]-FEPPA.

  4. Glyphosate inhibits the translocation of green fluorescent protein and sucrose from a transgenic tobacco host to Cuscuta campestris Yunk.

    PubMed

    Nadler-Hassar, Talia; Goldshmidt, Alexander; Rubin, Baruch; Wolf, Shmuel

    2004-09-01

    The parasitic plant Cuscuta campestris is dependent on its host for water, assimilates and amino acids. It can be controlled by the herbicide glyphosate, which inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), resulting in shikimate accumulation. In this study, C. campestris was parasitic on transgenic tobacco plants expressing green fluorescent protein (GFP) in the phloem. Changes in [14C]sucrose and GFP accumulation in the parasite were used as indicators of the herbicide's effect on translocation between the host and parasite. Host plants were treated with glyphosate 22 days after sowing. Shikimate accumulation in the parasite 1 day after glyphosate treatment (DAGT) confirmed EPSPS inhibition in C. campestris. No damage was visible in the host plants for the first 3 DAGT, while during that same time, a significant reduction in [14C]sucrose and GFP accumulation was observed in the parasite. Thus, we propose that the parallel reduction in GFP and sucrose accumulation in C. campestris is a result of a glyphosate effect on the parasite's ability to withdraw assimilates from the host.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Translocator protein (18 kDa) mediates the pro-growth effects of diazepam on Ehrlich tumor cells in vivo.

    PubMed

    Sakai, M; Ferraz-de-Paula, V; Pinheiro, M L; Ribeiro, A; Quinteiro-Filho, W M; Rone, M B; Martinez-Arguelles, D B; Dagli, M L Z; Papadopoulos, V; Palermo-Neto, J

    2010-01-25

    The Translocator Protein (TSPO), previously known as the peripheral-type benzodiazepine receptor, is a ubiquitous drug- and cholesterol-binding protein that is up regulated in several types of cancer cells. TSPO drug ligands (e.g., diazepam) induce or inhibit tumor cell proliferation, depending on the dose and tissue origin. We have previously shown that TSPO is expressed in Ehrlich tumor cells and that diazepam increases proliferation of these cells in vitro. Here, we investigated the in vivo effects of diazepam on Ehrlich tumor growth and the role of TSPO in mediating this process. Oral administration of diazepam to mice (3.0mg/kg/day for 7 days) produced plasma and ascitic fluid drug concentrations of 83.83 and 54.12 nM, respectively. Diazepam increased Ehrlich tumor growth, likely due to its ability to increase tumor cell proliferation and Reactive Oxygen Species production. Radioligand binding assays and nucleotide sequencing revealed that Ehrlich tumor cell TSPO had the same pharmacological and biochemical properties as TSPO described in other tumor cells. The estimated K(d) for PK 11195 in Ehrlich tumor cells was 0.44 nM and 8.70 nM (low and high binding site, respectively). Structurally diverse TSPO drug ligands with exclusive affinity for TSPO (i.e., 4-chlordiazepam, Ro5-4864, and isoquinoline-carboxamide PK 11195) also increased Ehrlich tumor growth. However, clonazepam, a GABA(A)-specific ligand with no affinity for TSPO, failed to do so. Taken together, these data suggest that diazepam induces in vivo Ehrlich tumor growth in a TSPO-dependent manner.

  10. 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.

  11. 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.

  12. Protein Polymorphism and Genic Heterozygosity in a Population of the Permanent Translocation Heterozygote, Oenothera biennis

    PubMed Central

    Levin, Donald A.; Howland, Gary P.; Steiner, Erich

    1972-01-01

    Genic allozyme polymorphism and heterozygosity was studied in a large population of the evening primrose, Oenothera biennis, growing in North Haven, Connecticut. This species is a permanent structural heterozygote for the entire complement of 14 chromosomes, and is thus capable of accumulating and maintaining genic heterozygosity. A total of 11 protein species were examined, and these were judged to be controlled by 19 loci. Polymorphism occurs at 26% of the loci, with only two alleles being present at each locus. The proportion of polymorphic loci per population in organisms with normal meiotic systems is no less than in Oe. biennis, indicating that structural heterozygosity is not necessarily accompanied by a greater proportion of polymorphic loci. On the other hand, the average Oe. biennis was heterozygous at 26% of its loci, an amount considerably greater than in most other organisms. With few exceptions, all plants in the North Haven population had the same genotype. Since the species is a colonizer, it is likely that the population was founded by a single individual and retained this genotype by virtue of the genetic system. PMID:4504363

  13. Heat shock-induced HIKESHI protects cell viability via nuclear translocation of heat shock protein 70.

    PubMed

    Yanoma, Toru; Ogata, Kyoichi; Yokobori, Takehiko; Ide, Munenori; Mochiki, Erito; Toyomasu, Yoshitaka; Yanai, Mitsuhiro; Kogure, Norimichi; Kimura, Akiharu; Suzuki, Masaki; Nakazawa, Nobuhiro; Bai, Tuya; Oyama, Tetsunari; Asao, Takayuki; Shirabe, Ken; Kuwano, Hiroyuki

    2017-09-01

    Heat shock proteins (HSPs), particularly HSP70, help restore normal cellular function following damage caused by stressors. HSP expression in tumor tissues indicates cancer progression, and while the development of HSP inhibitors is progressing, these substances are not widely used to treat cancer. HIKESHI (C11orf73) does not control the intracellular movement of HSP70 at normal temperatures; however, it does regulate the function and movements of HSP70 during heat shock. In this study, we examined the intracellular movement of HSP70 during heat shock to investigate the significance of HIKESHI expression in gastric cancer (GC) and determine if HIKESHI inhibition has cytotoxic effects. We examined HIKESHI using GC cell lines and immunostaining in 207 GC tissue samples. HIKESHI expression in GC tissues was associated with the progression of lymphatic invasion. Suppressing HIKESHI using siRNA did not affect cell viability at normal temperatures. However, suppressing HIKESHI during heat shock inhibited HSP70 nuclear transport and suppressed cell viability. Our results suggest that HIKESHI is a marker of cancer progression and that the combination of HIKESHI inhibition and hyperthermia is a therapeutic tool for refractory GC.

  14. Translocator protein (18 kDa) (TSPO) is expressed in reactive retinal microglia and modulates microglial inflammation and phagocytosis

    PubMed Central

    2014-01-01

    Background The translocator protein (18 kDa) (TSPO) is a mitochondrial protein expressed on reactive glial cells and a biomarker for gliosis in the brain. TSPO ligands have been shown to reduce neuroinflammation in several mouse models of neurodegeneration. Here, we analyzed TSPO expression in mouse and human retinal microglia and studied the effects of the TSPO ligand XBD173 on microglial functions. Methods TSPO protein analyses were performed in retinoschisin-deficient mouse retinas and human retinas. Lipopolysaccharide (LPS)-challenged BV-2 microglial cells were treated with XBD173 and TSPO shRNAs in vitro and pro-inflammatory markers were determined by qRT-PCR. The migration potential of microglia was determined with wound healing assays and the proliferation was studied with Fluorescence Activated Cell Sorting (FACS) analysis. Microglial neurotoxicity was estimated by nitrite measurement and quantification of caspase 3/7 levels in 661 W photoreceptors cultured in the presence of microglia-conditioned medium. The effects of XBD173 on filopodia formation and phagocytosis were analyzed in BV-2 cells and human induced pluripotent stem (iPS) cell-derived microglia (iPSdM). The morphology of microglia was quantified in mouse retinal explants treated with XBD173. Results TSPO was strongly up-regulated in microglial cells of the dystrophic mouse retina and also co-localized with microglia in human retinas. Constitutive TSPO expression was high in the early postnatal Day 3 mouse retina and declined to low levels in the adult tissue. TSPO mRNA and protein were also strongly induced in LPS-challenged BV-2 microglia while the TSPO ligand XBD173 efficiently suppressed transcription of the pro-inflammatory marker genes chemokine (C-C motif) ligand 2 (CCL2), interleukin 6 (IL6) and inducible nitric oxide (NO)-synthase (iNOS). Moreover, treatment with XBD173 significantly reduced the migratory capacity and proliferation of microglia, their level of NO secretion and their

  15. Prolonged glucose infusion into conscious rats inhibits early steps in insulin signalling and induces translocation of GLUT4 and protein kinase C in skeletal muscle.

    PubMed

    Houdali, B; Nguyen, V; Ammon, H P T; Haap, M; Schechinger, W; Machicao, F; Rett, K; Häring, H-U; Schleicher, E D

    2002-03-01

    Previous studies on diabetic patients have shown that hyperglycaemia increases glucose uptake in an apparently insulin-independent manner. However, the molecular mechanism has not been clarified. We studied rats receiving continuous glucose infusion to address this question. In this animal model, rats accommodate systemic glucose oversupply and rapidly develop insulin resistance. Glucose infusion increased both plasma glucose and insulin concentrations to peak after one day. In spite of continuous glucose infusion normoglycaemia was reached after 5 days while insulin concentrations remained higher. Focusing our studies in day 2 (hyperglycaemia/hyperinsulinaemia) and day 5 (normoglycaemia/hyperinsulinaemia) we found, particularly in day 5, that the early steps of the insulin signalling cascade in skeletal muscle of glucose-infused rats were not more activated when compared to control animals as assessed by a comparable phosphorylation of the insulin receptor, IRS-1 and PKB and by an unaltered IRS-1-associated Ptd(Ins) 3' kinase activity. Continuous glucose infusion induced GLUT4 protein expression and translocation to the plasma membrane while neither expression nor translocation of GLUT1 was affected. Translocation of PKC- betaI, - betaII (> threefold) and -alpha, -theta (to a lesser extent) to the plasma membrane was significantly induced after 2 days but not after 5 days of glucose infusion when normoglycaemia was reached. Our data support the hypothesis that continuous glucose infusion induces translocation of GLUT4 while the early steps of the insulin signalling cascade were not increased. These effects could be mediated by activation of PKC.

  16. 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

  17. 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.

  18. 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.

  19. Effects of DEHP and its metabolite MEHP on insulin signalling and proteins involved in GLUT4 translocation in cultured L6 myotubes.

    PubMed

    Viswanathan, Mangala Priya; Mullainadhan, Vigneswari; Chinnaiyan, Mayilvanan; Karundevi, Balasubramanian

    2017-07-01

    Di-(2-ethyl hexyl) phthalate (DEHP) is the plasticizer used in variety of medical and consumer products to impart structural flexibility. DEHP and its primary metabolite mono-(2-ethyl hexyl)phthalate (MEHP) posed a considerable interest because of their contribution to insulin resistance, type-2 diabetes and obesity. Experimental and epidemiological data have shown that DEHP affects blood glucose homeostasis. However, direct effect of DEHP and its metabolite MEHP on insulin signal transduction and glucose transporter 4 (GLUT4) translocation remain obscure. The present study was delineated to decipher the direct effects of DEHP and MEHP on insulin signal transduction and proteins involved in GLUT4 translocation in cultured L6 myotubes, the rat skeletal muscle model. For this study we have exposed cells with 50 and 100μM DEHP and MEHP for 24h followed by insulin stimulation for 20min. GLUT4 level in both cytosol and plasma membrane fractions were analysed by western blot analysis and found to be significantly decreased. Further, DEHP and MEHP treatment significantly altered the insulin signalling molecules and proteins involved in GLUT4 translocation (Rab8A (Ras related proteins in skeletal muscle), insulin - regulated amino peptidase (IRAP), synaptosomal - associated protein 23 (SNAP23), Syntaxin4, Munc18c) from cytosol to plasma membrane. Impaired GLUT4 in the plasma membrane resulted in decreased (14)C-deoxy glucose uptake. (14)C-glucose oxidation and glycogen content were also significantly decreased in treated groups. In essence, the present study is first of its kind to show the direct adverse effects of DEHP and MEHP on insulin signal transduction and GLUT4 translocation in cultured L6 myotubes. Further, MEHP is found to be more effective than DEHP as a result of its differential structure and physico-chemical properties. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. The aryl hydrocarbon receptor nuclear translocator (ARNT) family of proteins: transcriptional modifiers with multi-functional protein interfaces.

    PubMed

    Labrecque, M P; Prefontaine, G G; Beischlag, T V

    2013-08-01

    The basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH-PAS) domain family of transcription factors mediates cellular responses to a variety of internal and external stimuli. As functional transcription factors, these proteins act as bHLH-PAS heterodimers and can be further sub-classified into sensory/activated subunits and regulatory or ARNT-like proteins. This class of proteins act as master regulators of the bHLH-PAS superfamily of transcription factors that mediate circadian rhythm gene programs, innate and adaptive immune responses, oxygen-sensing mechanisms and compensate for deleterious environmental exposures. Some contribute to the etiology of human pathologies including cancer because of their effects on cell growth and metabolism. We will review the canonical roles of ARNT and ARNT-like proteins with an emphasis on coactivator selectivity and recruitment. We will also discuss recent advances in our understanding of noncanonical DNA-binding independent or off-target roles of ARNT that are uncoupled from its classic heterodimeric bHLH-PAS binding partners. Understanding the DNA binding-independent functions of ARNT may identify novel therapeutic options for the treatment of a large spectrum of disease states.

  1. Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry.

    PubMed Central

    Brunet, A; Roux, D; Lenormand, P; Dowd, S; Keyse, S; Pouysségur, J

    1999-01-01

    Mitogen-activated protein kinase (MAPK) modules, composed of three protein kinases activated by successive phosphorylation, are involved in the signal transduction of a wide range of extracellular agents. In mammalian cells, mitogenic stimulation triggers the translocation of p42/p44MAPK from the cytoplasm to the nucleus, whereas the other protein kinases of the module remain cytosolic. Since MAPK has been shown to phosphorylate and activate nuclear targets, such as the transcription factor Elk1, it has been proposed, but not yet demonstrated, that MAPK nuclear translocation could represent a critical step in signal transduction. In this study, we sequestered p42/p44MAPK in the cytoplasm by the expression of a catalytically inactive form of cytoplasmic MAP kinase phosphatase (MKP-3/Pyst-1). Sequestering MAPK in the cytoplasm did not alter its activation or its ability to phosphorylate cytoplasmic substrates of MAPK (p90RSK1 or an engineered cytoplasmic form of Elk1). In contrast, prevention of MAPK nuclear translocation strongly inhibited Elk1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response to growth factors. Thus the relocalization of MAPK to the nucleus appears to be an important regulatory step for mitogen-induced gene expression and cell cycle re-entry. PMID:9927426

  2. Differential translocation of green fluorescent protein fused to signal sequences of Ruminococcus albus cellulases by the Tat and Sec pathways of Escherichia coli.

    PubMed

    Esbelin, Julia; Martin, Christine; Forano, Evelyne; Mosoni, Pascale

    2009-05-01

    Ruminococcus albus is a Gram-positive bacterium that degrades plant cell walls in the rumen of herbivores. It was described to synthesize two major glycoside-hydrolases (Cel9B and Cel48A), which are exported and anchored at the cell surface. In bacteria, proteins destined to cross the cytoplasmic membrane are synthesized as precursors and possess a signal sequence (SS) directing them to the 'Sec' (general secretory) or 'Tat' (twin arginine translocation) pathway. SS composition of Cel9B and Cel48A suggests that these two enzymes translocate using different secretory pathways. In order to confirm this hypothesis, the SSs of Cel9B and Cel48A were fused to the green fluorescent protein (GFP) and expressed in wild-type Escherichia coli and in its Tat and Sec isogenic mutants. The SS cleavage and the formation of the mature protein were then followed by Western blot and fluorescence microscopy. This study shows that the SS of Cel9B directs the preprotein to the 'Tat' translocation pathway while the GFP fused to the SS of Cel48A is exported through the 'Sec' machinery. These observations suggest that R. albus possess a Tat pathway, in addition to the general secretory pathway.

  3. 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.

  4. 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

  5. Balanced translocations in mental retardation.

    PubMed

    Vandeweyer, Geert; Kooy, R Frank

    2009-07-01

    Over the past few decades, the knowledge on genetic defects causing mental retardation has dramatically increased. In this review, we discuss the importance of balanced chromosomal translocations in the identification of genes responsible for mental retardation. We present a database-search guided overview of balanced translocations identified in patients with mental retardation. We divide those in four categories: (1) balanced translocations that helped to identify a causative gene within a contiguous gene syndrome, (2) balanced translocations that led to the identification of a mental retardation gene confirmed by independent methods, (3) balanced translocations disrupting candidate genes that have not been confirmed by independent methods and (4) balanced translocations not reported to disrupt protein coding sequences. It can safely be concluded that balanced translocations have been instrumental in the identification of multiple genes that are involved in mental retardation. In addition, many more candidate genes were identified with a suspected but (as yet?) unconfirmed role in mental retardation. Some balanced translocations do not disrupt a protein coding gene and it can be speculated that in the light of recent findings concerning ncRNA's and ultra-conserved regions, such findings are worth further investigation as these potentially may lead us to the discovery of novel disease mechanisms.

  6. 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

  7. Inactivation of the endoplasmic reticulum protein translocation factor, Sec61p, or its homolog, Ssh1p, does not affect peroxisome biogenesis

    PubMed Central

    South, Sarah T.; Baumgart, Eveline; Gould, Stephen J.

    2001-01-01

    Peroxisomes are single membrane-bound organelles present in virtually all eukaryotes. These organelles participate in several important metabolic processes, and defects in peroxisome function and biogenesis are a significant contributor to human disease. Several models propose that peroxisomes arise from the endoplasmic reticulum (ER) in a process that involves the translocation of “group I” peroxisomal membrane proteins into the ER, the exit of these group I peroxisomal membrane proteins from the ER by vesicle budding, and the formation of nascent peroxisomes from vesicles containing the group I peroxisomal membrane proteins. A central prediction of these models is that the formation of nascent peroxisomes requires protein translocation into the ER. Sec61p is an essential component of the ER translocon, and we show here that loss of Sec61p activity has no effect on peroxisome biogenesis. In addition, loss of the SEC61-related gene, SSH1, also has no effect on peroxisome biogenesis. Although some proteins may enter the ER independently of Sec61p or Ssh1p, none are known, leading us to propose that peroxisome biogenesis may not require protein import into the ER, and by extension, transfer of proteins from the ER to the peroxisome. PMID:11593013

  8. 18-kDa translocator protein ligand (18)F-FEMPA: Biodistribution and uptake into atherosclerotic plaques in mice.

    PubMed

    Hellberg, Sanna; Silvola, Johanna M U; Kiugel, Max; Liljenbäck, Heidi; Savisto, Nina; Li, Xiang-Guo; Thiele, Andrea; Lehmann, Lutz; Heinrich, Tobias; Vollmer, Sonja; Hakovirta, Harri; Laine, V Jukka O; Ylä-Herttuala, Seppo; Knuuti, Juhani; Roivainen, Anne; Saraste, Antti

    2017-06-01

    Radioligands of 18-kDa translocator protein (TSPO) expressed on activated macrophages are a potential approach for imaging of inflammation in atherosclerosis. We evaluated a novel TSPO-targeted tracer (18)F-FEMPA for the detection of atherosclerotic plaque inflammation in mice. The distribution kinetics of (18)F-FEMPA was evaluated by in vivo PET/CT imaging. (18)F-FEMPA uptake was compared in atherosclerotic (LDLR(-/-)ApoB(100/100), n = 10) and healthy mice (C57BL/6 N, n = 7) ex vivo at twenty minutes post-injection. Biodistribution was analyzed from harvested tissue samples, and aortas were sectioned for autoradiography. Aortas of LDLR(-/-)ApoB(100/100) mice showed large, macrophage-rich atherosclerotic plaques. In vivo, (18)F-FEMPA showed rapid blood clearance but no difference in aortic uptake between atherosclerotic and healthy mice. In the mice studied ex vivo at 20 minutes post-injection, quantification of radioactivity in the whole aorta showed 1.3-fold higher (18)F-FEMPA accumulation in atherosclerotic than healthy mice (P = .028). Autoradiography showed higher tracer uptake in plaque areas with high macrophage content as compared with areas of no macrophages (count densities 190 ± 54 vs 40 ± 13 PSL/mm(2), P < .001), but the uptake in the plaques was not higher than in the normal vessel wall (230 ± 78 PSL/mm(2)). In vitro blocking showed specific accumulation in mouse and human atherosclerotic plaques. Immunohistochemistry confirmed co-localization of TSPO and macrophages. (18)F-FEMPA shows rapid blood clearance and uptake in the mouse aorta. Uptake in atherosclerotic plaques correlated with the amount of macrophages, but did not exceed that in the normal vessel wall.

  9. 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. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. 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.

  11. 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.

  12. Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish.

    PubMed

    Lu, Jin Fang; Wang, Wei Na; Wang, Gai Ling; Zhang, He; Zhou, Ying; Gao, Zhi Peng; Nie, Pin; Xie, Hai Xia

    2015-10-12

    The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Folding kinetics and thermodynamics of Pseudomonas syringae effector protein AvrPto provide insight into translocation via the type III secretion system.

    PubMed

    Dawson, Jennifer E; Nicholson, Linda K

    2008-07-01

    In order to infect their hosts, many Gram-negative bacteria translocate agents of infection, called effector proteins, through the type III secretion system (TTSS) into the host cytoplasm. This process is thought to require at least partial unfolding of these agents, raising the question of how an effector protein might unfold to enable its translocation and then refold once it reaches the host cytoplasm. AvrPto is a well-studied effector protein of Pseudomonas syringae pv tomato. The presence of a readily observed unfolded population of AvrPto in aqueous solution and the lack of a known secretion chaperone make it ideal for studying the kinetic and thermodynamic characteristics that facilitate translocation. Application of Nzz exchange spectroscopy revealed a global, two-state folding equilibrium with 16% unfolded population, a folding rate of 1.8 s(-1), and an unfolding rate of 0.33 s(-1) at pH 6.1. TrAvrPto stability increases with increasing pH, with only 2% unfolded population observed at pH 7.0. The R(1) relaxation of TrAvrPto, which is sensitive to both the global anisotropy of folded TrAvrPto and slow exchange between folded and unfolded conformations, provided independent verification of the global kinetic rate constants. Given the acidic apoplast in which the pathogen resides and the more basic host cytoplasm, these results offer an intriguing mechanism by which the pH dependence of stability and slow folding kinetics of AvrPto would allow efficient translocation of the unfolded form through the TTSS and refolding into its functional folded form once inside the host.

  14. A Tat ménage à trois--The role of Bacillus subtilis TatAc in twin-arginine protein translocation.

    PubMed

    Goosens, Vivianne J; De-San-Eustaquio-Campillo, Alba; Carballido-López, Rut; van Dijl, Jan Maarten

    2015-10-01

    The twin-arginine translocation system (Tat) is a protein transport system that moves fully folded and cofactor-containing proteins across membranes of bacteria, archaea and thylakoids. The minimal Tat pathway is composed of two subunits, TatA and TatC. In some organisms TatA has been duplicated and evolved to form a third specialized subunit, TatB. The Bacillus subtilis genome encodes two TatC subunits (TatCd and TatCy) and three TatA subunits (TatAd, TatAy and TatAc). These subunits combine to form two parallel minimal pathways, TatAy-TatCy and TatAd-TatCd. The purpose and role of the third TatA component, TatAc, has remained ambiguous. In this study we examined the translocation of two natively expressed TatAy-TatCy-dependent substrates, EfeB and QcrA, in various Tat-deficient genetic backgrounds. More specifically, we examined the ability of different mutated TatAy subunits to complement for the absence of wild-type TatAy. We further detailed a graded growth phenotype associated with the functional translocation of EfeB. We found that in various instances where specific amino acid substitutions were made in TatAy, a definite TatAc-associated growth phenotype occurred in genetic backgrounds lacking TatAc. Altogether, our findings show that TatAy and TatAc interact and that this TatAy-TatAc interaction, although not essential, supports the translocation of the Tat substrate EfeB when TatAy function is compromised. This implies that the third TatA-like protein in B. subtilis could represent an intermediate evolutionary step in TatA-TatB specialization. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. 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.

  16. 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.

  17. Making new out of old: recycling and modification of an ancient protein translocation system during eukaryotic evolution. Mechanistic comparison and phylogenetic analysis of ERAD, SELMA and the peroxisomal importomer.

    PubMed

    Bolte, Kathrin; Gruenheit, Nicole; Felsner, Gregor; Sommer, Maik S; Maier, Uwe-G; Hempel, Franziska

    2011-05-01

    At first glance the three eukaryotic protein translocation machineries--the ER-associated degradation (ERAD) transport apparatus of the endoplasmic reticulum, the peroxisomal importomer and SELMA, the pre-protein translocator of complex plastids--appear quite different. However, mechanistic comparisons and phylogenetic analyses presented here suggest that all three translocation machineries share a common ancestral origin, which highlights the recycling of pre-existing components as an effective evolutionary driving force. Editor's suggested further reading in BioEssays ERAD ubiquitin ligases Abstract. Copyright © 2011 WILEY Periodicals, Inc.

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

    PubMed Central

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

    2006-01-01

    APS (Adaptor protein with PH and SH2 domains) initiates a PI 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. Since actin rearrangement is important for insulin-induced 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 co-localized 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 GFP-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 remodelling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue. PMID:16803868

  19. 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.

  20. The 70-Kilodalton Heat Shock Cognate Can Act as a Molecular Chaperone during the Membrane Translocation of a Plant Secretory Protein Precursor.

    PubMed Central

    Miernyk, JA; Duck, NB; Shatters, RG; Folk, WR

    1992-01-01

    When a model secretory precursor was synthesized in vitro and analyzed by rate-zonal sedimentation, it appeared to be associated with other proteins present in a wheat germ extract. At least one of the associated proteins is a member of the 70-kD family of stress proteins. It was possible to immunoprecipitate the secretory precursor with anti-heat shock cognate 70 (Hsc70) antibodies in the absence but not in the presence of ATP, suggesting that the association was specific. ATP-sensitive association is one diagnostic characteristic of molecular chaperone-type proteins. Increasing incubation temperature decreased the amount of precursor associated with Hsc70. A method was developed for the removal of Hsc70 from a wheat germ in vitro translation mixture by immunoprecipitation. Cotranslational translocation and processing of the secretory precursor by maize endosperm microsomes were inefficient in the Hsc70-depleted system but were greatly stimulated by addition of purified preparations of various heat shock 70 proteins (Hsp70s). Cytosolic Hsc70 from maize endosperm was capable of autophosphorylation in vitro. Phosphorylated Hsc70 was much less efficient in promoting membrane translocation of the secretory precursor. These results suggest that chaperone function in vivo could be regulated by phosphorylation. PMID:12297663

  1. RNA interference targeting rye secalins alters flour protein composition in a wheat variety carrying a 1Bl.1RS translocation

    USDA-ARS?s Scientific Manuscript database

    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 ...

  2. Kinetic analysis of the translocator protein positron emission tomography ligand [(18)F]GE-180 in the human brain.

    PubMed

    Feeney, Claire; Scott, Gregory; Raffel, Joel; Roberts, S; Coello, Christopher; Jolly, Amy; Searle, Graham; Goldstone, A P; Brooks, David J; Nicholas, Richard S; Trigg, William; Gunn, Roger N; Sharp, David J

    2016-11-01

    PET can image neuroinflammation by targeting the translocator protein (TSPO), which is upregulated in activated microglia. The high nonspecific binding of the first-generation TSPO radioligand [(11)C]PK-11195 limits accurate quantification. [(18)F]GE-180, a novel TSPO ligand, displays superior binding to [(11)C]PK-11195 in vitro. Our objectives were to: (1) evaluate tracer characteristics of [(18)F]GE-180 in the brains of healthy human subjects; and (2) investigate whether the TSPO Ala147Thr polymorphism influences outcome measures. Ten volunteers (five high-affinity binders, HABs, and five mixed-affinity binders, MABs) underwent a dynamic PET scan with arterial sampling after injection of [(18)F]GE-180. Kinetic modelling of time-activity curves with one-tissue and two-tissue compartment models and Logan graphical analysis was applied to the data. The primary outcome measure was the total volume of distribution (V T) across various regions of interest (ROIs). Secondary outcome measures were the standardized uptake values (SUV), the distribution volume and SUV ratios estimated using a pseudoreference region. The two-tissue compartment model was the best model. The average regional delivery rate constant (K 1) was 0.01 mL cm(-3) min(-1) indicating low extraction across the blood-brain barrier (1 %). The estimated median V T across all ROIs was also low, ranging from 0.16 mL cm(-3) in the striatum to 0.38 mL cm(-3) in the thalamus. There were no significant differences in V T between HABs and MABs across all ROIs. A reversible two-tissue compartment model fitted the data well and determined that the tracer has a low first-pass extraction (approximately 1 %) and low V T estimates in healthy individuals. There was no observable dependency on the rs6971 polymorphism as compared to other second-generation TSPO PET tracers. Investigation of [(18)F]GE-180 in populations with neuroinflammatory disease is needed to determine its suitability for quantitative assessment

  3. 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

  4. 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

  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 Ciliary Protein Nephrocystin-4 Translocates the Canonical Wnt Regulator Jade-1 to the Nucleus to Negatively Regulate β-Catenin Signaling*

    PubMed Central

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

    2012-01-01

    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. PMID:22654112

  7. 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.

  8. 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

  9. The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3.

    PubMed Central

    Fredericks, W J; Galili, N; Mukhopadhyay, S; Rovera, G; Bennicelli, J; Barr, F G; Rauscher, F J

    1995-01-01

    Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family. To determine the role of the fusion protein in transcriptional regulation and oncogenesis, we identified the PAX3-FKHR fusion protein and characterized its function(s) as a transcription factor relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor cell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKHR sera demonstrated expression of a 97-kDa PAX3-FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified that a single polypeptide contains epitopes derived from each protein. The PAX3-FKHR protein was localized to the nucleus in Rh30 cells, as was wild-type PAX3, in t(2;13)-negative A673 cells. In gel shift assays using a canonical PAX binding site (e5 sequence), we found that DNA binding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains. However, the PAX3-FKHR fusion protein was a much more potent transcriptional activator than PAX3 as determined by transient cotransfection assays using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes. PMID:7862145

  10. 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.

  11. 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.

  12. GTP promotes the formation of early-import intermediates but is not required during the translocation step of protein import into chloroplasts

    SciTech Connect

    Young, M.E.; Keegstra, K.; Froehlich, J.E.

    1999-09-01

    Protein import into chloroplasts is an energy-requiring process mediated by a pertinacious import apparatus. Although previous work has shown that low levels of ATP or GTP can support precursor binding, the role of GTP during the import process remains unclear. Specifically, it is unknown whether GTP plays a separate role from ATP during the early stages of protein import and whether GTP has any role in the later stages of transport. The authors investigated the role of GTP during the various stages of protein import into chloroplasts by using purified GTP analogs and an in vitro import assay. GTP, GDP, the nonhydrolyzable analog GMP-PNP, and the slowly hydrolyzable analogs guanosine 5{prime}-O-(2-thiodiphosphate) and guanosine 5{prime}-O-(3-thiotriphosphate) were used in this study. Chromatographically purified 5{prime}-guanylyl-imido-diphosphate and guanosine 5{prime}-O-(3-thiotriphosphate) were found to inhibit the formation of early-import intermediates, even in the presence of ATP. The authors also observed that GTP does not play a role during the translocation of precursors from the intermediate state. They conclude that GTP hydrolysis influences events leading to the formation of early-import intermediates, but not subsequent steps such as precursor translocation.

  13. Safety, Tolerability, and Pharmacokinetic Profile of the Novel Translocator Protein 18 kDa Antagonist ONO-2952 in Healthy Volunteers.

    PubMed

    Suto, Fumitaka; Wood, Andrew T; Kobayashi, Michiyoshi; Komaba, Junji; Duffy, Kevin; Bruce, Mark

    2015-09-01

    To investigate safety, tolerability, and pharmacokinetic properties of single and multiple doses of novel translocator protein 18 kDa antagonist ONO-2952 in healthy subjects. Double-blind, placebo-controlled single (SAD) and multiple (MAD) dose escalation studies were conducted. Healthy men and women aged 18 to 55 years inclusive and without history of psychiatric disorders were eligible. Forty-eight volunteers received single doses of ONO-2952 (3, 10, 30, 100, 200, or 400 mg) or placebo under fasted conditions (SAD study), and 36 received ONO-2952 (30, 60, or 100 mg/d) or placebo for 21 consecutive days under fed conditions (MAD study). ONO-2952 10 and 200 mg were administered under fasted and fed conditions in the SAD study to investigate the effect of food on the absorption of ONO-2952. Safety assessments included adverse events, vital signs, 12-lead ECGs, and clinical laboratory evaluations. Plasma and urine pharmacokinetic profiles of ONO-2952 were determined. Across both studies, mean age ranged from 29.8 to 39.8 years, most participants were white, and the proportion of female volunteers was 52%. No treatment or dose-related trends in adverse events were observed. The most frequent adverse events were headache and presyncope (n = 2 each [SAD study]) and constipation and headache (n = 3 each [MAD study]). All headache and constipation episodes were possibly related to the study drug. Plasma ONO-2952 concentrations peaked 2.5 to 3.5 hours (SAD study) and 3.0 to 4.0 hours (MAD study) postdose. ONO-2952 systemic exposure increased less than dose proportionally under fasted conditions. Fed conditions significantly increased exposure compared with fasted conditions: geometric mean ratios of Cmax (90% CIs) were 229% (176-299 [10 mg]) and 778% (623-971 [200 mg]), and AUClast were 159% (131-192 [10 mg]) and 382% (288-506 [200 mg]). In the MAD study, the systemic exposure of ONO-2952 increased in a slightly greater than dose-proportional manner. Geometric mean

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane

    NASA Astrophysics Data System (ADS)

    Rajapaksha, Suneth P.; Pal, Nibedita; Zheng, Desheng; Lu, H. Peter

    2015-11-01

    We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ˜15 Å diameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a

  2. Translocator protein (18 kDa) polymorphism (rs6971) explains in-vivo brain binding affinity of the PET radioligand [(18)F]-FEPPA.

    PubMed

    Mizrahi, Romina; Rusjan, Pablo M; Kennedy, James; Pollock, Bruce; Mulsant, Benoit; Suridjan, Ivonne; De Luca, Vincenzo; Wilson, Alan A; Houle, Sylvain

    2012-06-01

    [(18)F]-FEPPA binds to the 18-kDa translocator protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of the PET signal with new generation TSPO PET radioligands are confounded by large interindividual variability in binding affinity. This presents as a trimodal distribution, reflecting high-affinity binders (HABs), low-affinity binder (LAB), and mixed-affinity binders (MABs). Here, we show that one polymorphism (rs6971) located in exon 4 of the TSPO gene, which results in a nonconservative amino-acid substitution from alanine to threonine (Ala147Thr) in the TSPO protein, predicts [(18)F]-FEPPA total distribution volume in human brains. In addition, [(18)F]-FEPPA exhibits clearly different features in the shape of the time activity curves between genetic groups. Testing for the rs6971 polymorphism may allow quantitative interpretation of TSPO PET studies with new generation of TSPO PET radioligands.

  3. Translocator protein (18 kDa) polymorphism (rs6971) explains in-vivo brain binding affinity of the PET radioligand [18F]-FEPPA

    PubMed Central

    Mizrahi, Romina; Rusjan, Pablo M; Kennedy, James; Pollock, Bruce; Mulsant, Benoit; Suridjan, Ivonne; De Luca, Vincenzo; Wilson, Alan A; Houle, Sylvain

    2012-01-01

    [18F]-FEPPA binds to the 18-kDa translocator protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of the PET signal with new generation TSPO PET radioligands are confounded by large interindividual variability in binding affinity. This presents as a trimodal distribution, reflecting high-affinity binders (HABs), low-affinity binder (LAB), and mixed-affinity binders (MABs). Here, we show that one polymorphism (rs6971) located in exon 4 of the TSPO gene, which results in a nonconservative amino-acid substitution from alanine to threonine (Ala147Thr) in the TSPO protein, predicts [18F]-FEPPA total distribution volume in human brains. In addition, [18F]-FEPPA exhibits clearly different features in the shape of the time activity curves between genetic groups. Testing for the rs6971 polymorphism may allow quantitative interpretation of TSPO PET studies with new generation of TSPO PET radioligands. PMID:22472607

  4. 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.

  5. 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

  6. 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

  7. Promoter swapping between the genes for a novel zinc finger protein and beta-catenin in pleiomorphic adenomas with t(3;8)(p21;q12) translocations.

    PubMed

    Kas, K; Voz, M L; Röijer, E; Aström, A K; Meyen, E; Stenman, G; Van de Ven, W J

    1997-02-01

    Pleiomorphic adenoma of the salivary glands is a benign epithelial tumour occurring primarily in the major and minor salivary glands. It is by far the most common type of salivary gland tumour. Microscopically, pleiomorphic adenomas show a marked histological diversity with epithelial, myoepithelial and mesenchymal components in a variety of patterns. In addition to a cytogenetic subgroup with normal karyotypes, pleiomorphic adenomas are characterized by recurrent chromosome rearrangements, particularly reciprocal translocations, with breakpoints at 8q12, 3p21, and 12q13-15, in that order of frequency. The most common abnormality is a reciprocal t(3;8)(p21;q12). We here demonstrate that the t(3;8)(p21;q12) results in promoter swapping between PLAG1, a novel, developmentally regulated zinc finger gene at 8q12, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein interface functioning in the WG/WNT signalling pathway and specification of cell fate during embryogenesis. Fusions occur in the 5'-non-coding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. Due to the t(3;8)(p21;q12), PLAG1 is activated and expression levels of CTNNB1 are reduced. Activation of PLAG1 was also observed in an adenoma with a variant translocation t(8;15)(q12;q14). Our results indicate that PLAG1 activation due to promoter swapping is a crucial event in salivary gland tumourigenesis.

  8. 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.

  9. 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.

  10. 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.

  11. Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array.

    PubMed

    Ravid-Hermesh, Orit; Zurgil, Naomi; Shafran, Yana; Sobolev, Maria; Galmidi, Moti; Badihi, Yoav; Israel, Liron Limor; Lellouche, Jean Paul; Lellouche, Emmanuel; Michaeli, Shulamit; Deutsch, Mordechai

    2014-11-01

    Cell populations represent intrinsically heterogeneous systems with a high level of spatiotemporal complexity. Monitoring and understanding cell-to-cell diversity is essential for the research and application of intra- and interpopulation variations. Optical analysis of live cells is challenging since both adherent and nonadherent cells change their spatial location. However, most currently available single-cell techniques do not facilitate treatment and monitoring of the same live cells over time throughout multistep experiments. An imaging-dish-based live cell array (ID-LCA) has been developed and produced for cell handling, culturing, and imaging of numerous live cells. The dish is composed of an array of pico scale cavities-pico wells (PWs) embossed on its glass bottom. Cells are seeded, cultured, treated, and spatiotemporally measured on the ID-LCA, while each cell or small group of cells are locally constrained in the PWs. Finally, predefined cells can be retrieved for further evaluation. Various types of ID-LCAs were used in this proof-of-principle work, to demonstrate on-ID-LCA transfection of fluorescently tagged chimeric proteins, as well as the detection and kinetic analysis of their induced translocation. High variability was evident within cell populations with regard to protein expression levels as well as the extent and dynamics of protein redistribution. The association of these parameters with cell morphology and functional parameters was examined. Both the new methodology and the device facilitate research of the translocation process at individual cell resolution within large populations and thus, can potentially be used in high-throughput fashion.

  12. 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.

  13. Dendritic oligoguanidines as intracellular translocators.

    PubMed

    Chung, Hyun-Ho; Harms, Guido; Seong, Churl Min; Choi, Byung Hyune; Min, Changhee; Taulane, Joseph P; Goodman, Murray

    2004-01-01

    A series of polyguanidylated dendritic structures that can be used as molecular translocators have been designed and synthesized based on nonpeptide units. The dendritic oligoguanidines conjugated with fluorescein or with a green fluorescent protein (GFP) mutant as cargos were isolated and characterized. Quantification and time-course analyses of the cellular uptake of the conjugates using HeLa S3 and human cervical carcinoma cells reveal that the polyguanidylated dendrimers have comparable translocation efficiency to the Tat(49-57) peptide. Furthermore, the deconvolution microscopy image analysis shows that they are located inside the cells. These results clearly show that nonlinear, branched dendritic oligoguanidines are capable of translocation through the cell membrane. This work also demonstrates the potential of these nonpeptidic dendritic oligoguanidines as carriers for intracellular delivery of small molecule drugs, bioactive peptides, and proteins. Copyright 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004

  14. Reversible cAMP-induced translocation of cytoskeleton-associated 300- to 350-kDa proteins from nucleus to cytoplasm

    SciTech Connect

    Nakayama, Tokiko; Nishizawa, Kimiko; Sato, Chicako )

    1988-08-01

    The authors previously reported that treatment of SV-3Y1 cells in an exponential growth state with db-cAMP plus theophylline induced reversible disappearance of nuclear dots stained by monoclonal anti-microtubule-associated protein (MAP)-1 antibody. In the present study, the authors examined the relation between the intracellular localization and phosphorylation of 300- to 350-kDa proteins that are intracellular antigens for our anti-Map-1 and -2 antibodies. Treatment with db-cAMP plus theophylline was found to result in a reversible decrease in immunofluorescent staining of the nucleus with polyclonal MAP-1 or -2 antibody, and a reversible increase in that of the cytoplasm. Simultaneous treatment with colchicine, colcemid, putrescine, or {alpha}-naphthyl phosphate in the presence of db-cAMP plus theophylline almost prevented this effect of db-cAMP plus theophylline. They examined the cytoplasmic and nuclear fractions by immunoperoxidase staining, immunoprecipitation, and {sup 125}I-protein A with anti-MAP-1 and -2 antibodies. The present research indicated that treatment with db-cAMP plus theophylline resulted in the reversible translocation of 300- to 350-kDa proteins from the nucleus to the cytoplasm accompanied by the dephosphorylation of these proteins.

  15. Evolutionary well-conserved region in the signal peptide of parathyroid hormone-related protein is critical for its dual localization through the regulation of ER translocation

    PubMed Central

    Amaya, Yoshihiro; Nakai, Toshiki; Miura, Satoshi

    2016-01-01

    Parathyroid hormone-related protein (PTHrP) has two different targeting signals: an N-terminal signal peptide for the endoplasmic reticulum (ER) targeting and an internal nuclear localization signal. The protein not only functions as a secretory protein, but is also found in the nucleus and/or nucleolus under certain conditions. PTHrP signal peptide is less hydrophobic than most signal peptides mainly due to its evolutionarily well-conserved region (QQWS). The substitution of four tandem leucine residues for this conserved region resulted in a significant inhibition of the signal peptide cleavage. At the same time, proportion of nuclear and/or nucleolar localization decreased, probably due to tethering of the protein to the ER membrane by the uncleaved mutant signal peptide. Almost complete cleavage of the signal peptide accompanied by a lack of nuclear/nucleolar localization was achieved by combining the hydrophobic h-region and an optimized sequence of the cleavage site. In addition, mutational modifications of the distribution of charged residues in and around the signal peptide affect its cleavage and/or nuclear/nucleolar localization of the protein. These results indicate that the well-conserved region in the signal peptide plays an essential role in the dual localization of PTHrP through ER targeting and/or the membrane translocation. PMID:26538570

  16. Evolutionary well-conserved region in the signal peptide of parathyroid hormone-related protein is critical for its dual localization through the regulation of ER translocation.

    PubMed

    Amaya, Yoshihiro; Nakai, Toshiki; Miura, Satoshi

    2016-04-01

    Parathyroid hormone-related protein (PTHrP) has two different targeting signals: an N-terminal signal peptide for the endoplasmic reticulum (ER) targeting and an internal nuclear localization signal. The protein not only functions as a secretory protein, but is also found in the nucleus and/or nucleolus under certain conditions. PTHrP signal peptide is less hydrophobic than most signal peptides mainly due to its evolutionarily well-conserved region (QQWS). The substitution of four tandem leucine residues for this conserved region resulted in a significant inhibition of the signal peptide cleavage. At the same time, proportion of nuclear and/or nucleolar localization decreased, probably due to tethering of the protein to the ER membrane by the uncleaved mutant signal peptide. Almost complete cleavage of the signal peptide accompanied by a lack of nuclear/nucleolar localization was achieved by combining the hydrophobic h-region and an optimized sequence of the cleavage site. In addition, mutational modifications of the distribution of charged residues in and around the signal peptide affect its cleavage and/or nuclear/nucleolar localization of the protein. These results indicate that the well-conserved region in the signal peptide plays an essential role in the dual localization of PTHrP through ER targeting and/or the membrane translocation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  17. Residence Time, a New parameter to Predict Neurosteroidogenic Efficacy of Translocator Protein (TSPO) Ligands: the Case Study of N,N-Dialkyl-2-arylindol-3-ylglyoxylamides.

    PubMed

    Costa, Barbara; Taliani, Sabrina; Da Pozzo, Eleonora; Barresi, Elisabetta; Robello, Marco; Cavallini, Chiara; Cosconati, Sandro; Da Settimo, Federico; Novellino, Ettore; Martini, Claudia

    2017-08-22

    Targeting the biosynthetic pathway of neuroactive steroids with specific 18 kDa translocator protein (TSPO) ligands may be a viable therapeutic approach for a variety of neurodegenerative and neuropsychiatric diseases. However, the lack of correlation between binding affinity and in vitro steroidogenic efficacy has limited the identification of lead compounds by traditional affinity-based drug discovery strategies. Our recent research indicates that the key factor for robust steroidogenic TSPO ligand efficacy is not the binding affinity per se, but rather the time the compound spends in the target, namely its residence time (RT). The assessment of this kinetic parameter during the in vitro characterization of compounds appears mandatory in order to obtain structure-efficacy relationships suitable for the future development of novel molecules with promising pharmacological properties. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. 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.

  19. 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

  20. Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor.

    PubMed

    Pais, June E; Schilke, Brenda; Craig, Elizabeth A

    2011-12-01

    The heat-shock protein 70 (Hsp70)-based import motor, associated with the translocon on the matrix side of the mitochondrial inner membrane, drives translocation of proteins via cycles of binding and release. Stimulation of Hsp70's ATPase activity by the translocon-associated J-protein Pam18 is critical for this process. Pam18 forms a heterodimer with the structurally related protein Pam16, via their J-type domains. This interaction has been proposed to perform a critical regulatory function, inhibiting the ATPase stimulatory activity of Pam18. Using biochemical and genetic assays, we tested this hypothesis by assessing the in vivo function of Pam18 variants having altered abilities to stimulate Hsp70's ATPase activity. The observed pattern of genetic interactions was opposite from that predicted if the heterodimer serves an inhibitory function; instead the pattern was consistent with that of mutations known to cause reduction in the stability of the heterodimer. Analysis of a previously uncharacterized region of Pam16 revealed its requirement for formation of an active Pam18:Pam16 complex able to stimulate Hsp70's ATPase activity. Together, our data are consistent with the idea that Pam18 and Pam16 form a stable heterodimer and that the critical role of the Pam18:Pam16 interaction is the physical tethering of Pam18 to the translocon via its interaction with Pam16.

  1. Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor

    PubMed Central

    Pais, June E.; Schilke, Brenda; Craig, Elizabeth A.

    2011-01-01

    The heat-shock protein 70 (Hsp70)–based import motor, associated with the translocon on the matrix side of the mitochondrial inner membrane, drives translocation of proteins via cycles of binding and release. Stimulation of Hsp70's ATPase activity by the translocon-associated J-protein Pam18 is critical for this process. Pam18 forms a heterodimer with the structurally related protein Pam16, via their J-type domains. This interaction has been proposed to perform a critical regulatory function, inhibiting the ATPase stimulatory activity of Pam18. Using biochemical and genetic assays, we tested this hypothesis by assessing the in vivo function of Pam18 variants having altered abilities to stimulate Hsp70's ATPase activity. The observed pattern of genetic interactions was opposite from that predicted if the heterodimer serves an inhibitory function; instead the pattern was consistent with that of mutations known to cause reduction in the stability of the heterodimer. Analysis of a previously uncharacterized region of Pam16 revealed its requirement for formation of an active Pam18:Pam16 complex able to stimulate Hsp70's ATPase activity. Together, our data are consistent with the idea that Pam18 and Pam16 form a stable heterodimer and that the critical role of the Pam18:Pam16 interaction is the physical tethering of Pam18 to the translocon via its interaction with Pam16. PMID:22031295

  2. 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.

  3. 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

  4. 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

  5. Hepatic Diacylglycerol-Associated Protein Kinase Cε Translocation Links Hepatic Steatosis to Hepatic Insulin Resistance in Humans.

    PubMed

    Ter Horst, Kasper W; Gilijamse, Pim W; Versteeg, Ruth I; Ackermans, Mariette T; Nederveen, Aart J; la Fleur, Susanne E; Romijn, Johannes A; Nieuwdorp, Max; Zhang, Dongyan; Samuel, Varman T; Vatner, Daniel F; Petersen, Kitt F; Shulman, Gerald I; Serlie, Mireille J

    2017-06-06

    Hepatic lipid accumulation has been implicated in the development of insulin resistance, but translational evidence in humans is limited. We investigated the relationship between liver fat and tissue-specific insulin sensitivity in 133 obese subjects. Although the presence of hepatic steatosis in obese subjects was associated with hepatic, adipose tissue, and peripheral insulin resistance, we found that intrahepatic triglycerides were not strictly sufficient or essential for hepatic insulin resistance. Thus, to examine the molecular mechanisms that link hepatic steatosis to hepatic insulin resistance, we comprehensively analyzed liver biopsies from a subset of 29 subjects. Here, hepatic cytosolic diacylglycerol content, but not hepatic ceramide content, was increased in subjects with hepatic insulin resistance. Moreover, cytosolic diacylglycerols were strongly associated with hepatic PKCε activation, as reflected by PKCε translocation to the plasma membrane. These results demonstrate the relevance of hepatic diacylglycerol-induced PKCε activation in the pathogenesis of NAFLD-associated hepatic insulin resistance in humans. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. The Cancer-associated K351N Mutation Affects the Ubiquitination and the Translocation to Mitochondria of p53 Protein*

    PubMed Central

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

    2011-01-01

    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. PMID:21953469

  7. 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.

  8. Dissecting structures and functions of SecA-only protein-conducting channels: ATPase, pore structure, ion channel activity, protein translocation, and interaction with SecYEG/SecDF•YajC.

    PubMed

    Hsieh, Ying-Hsin; Huang, Ying-Ju; Zhang, Hao; Liu, Qian; Lu, Yang; Yang, Hsiuchin; Houghton, John; Jiang, Chun; Sui, Sen-Fang; Tai, Phang C

    2017-01-01

    SecA is an essential protein in the major bacterial Sec-dependent translocation pathways. E. coli SecA has 901 aminoacyl residues which form multi-functional domains that interact with various ligands to impart function. In this study, we constructed and purified tethered C-terminal deletion fragments of SecA to determine the requirements for N-terminal domains interacting with lipids to provide ATPase activity, pore structure, ion channel activity, protein translocation and interactions with SecYEG-SecDF•YajC. We found that the N-terminal fragment SecAN493 (SecA1-493) has low, intrinsic ATPase activity. Larger fragments have greater activity, becoming highest around N619-N632. Lipids greatly stimulated the ATPase activities of the fragments N608-N798, reaching maximal activities around N619. Three helices in amino-acyl residues SecA619-831, which includes the "Helical Scaffold" Domain (SecA619-668) are critical for pore formation, ion channel activity, and for function with SecYEG-SecDF•YajC. In the presence of liposomes, N-terminal domain fragments of SecA form pore-ring structures at fragment-size N640, ion channel activity around N798, and protein translocation capability around N831. SecA domain fragments ranging in size between N643-N669 are critical for functional interactions with SecYEG-SecDF•YajC. In the presence of liposomes, inactive C-terminal fragments complement smaller non-functional N-terminal fragments to form SecA-only pore structures with ion channel activity and protein translocation ability. Thus, SecA domain fragment interactions with liposomes defined critical structures and functional aspects of SecA-only channels. These data provide the mechanistic basis for SecA to form primitive, low-efficiency, SecA-only protein-conducting channels, as well as the minimal parameters for SecA to interact functionally with SecYEG-SecDF•YajC to form high-efficiency channels.

  9. RNA polymerase stalls in a post-translocated register and can hyper-translocate

    PubMed Central

    Nedialkov, Yuri A.; Nudler, Evgeny; Burton, Zachary F.

    2012-01-01

    Exonuclease (Exo) III was used to probe translocation states of RNA polymerase (RNAP) ternary elongation complexes (TECs). Escherichia coli RNAP stalls primarily in a post-translocation register that makes relatively slow excursions to a hyper-translocated state or to a pre-translocated state. Tagetitoxin (TGT) strongly inhibits hyper-translocation and inhibits backtracking, so, as indicated by Exo III mapping, TGT appears to stabilize both the pre- and probably a partially post-translocation state of RNAP. Because the pre-translocated to post-translocated transition is slow at many template positions, these studies appear inconsistent with a model in which RNAP makes frequent and rapid (i.e., millisecond phase) oscillations between pre- and post-translocation states. Nine nucleotides (9-nt) and 10-nt TECs, and TECs with longer nascent RNAs, have distinct translocation properties consistent with a 9–10 nt RNA/DNA hybrid. RNAP mutant proteins in the bridge helix and trigger loop are identified that inhibit or stimulate forward and backward translocation. PMID:23132506

  10. 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

  11. The MN1-TEL fusion protein, encoded by the translocation (12;22)(p13;q11) in myeloid leukemia, is a transcription factor with transforming activity.

    PubMed

    Buijs, A; van Rompaey, L; Molijn, A C; Davis, J N; Vertegaal, A C; Potter, M D; Adams, C; van Baal, S; Zwarthoff, E C; Roussel, M F; Grosveld, G C

    2000-12-01

    The Tel gene (or ETV6) is the target of the translocation (12;22)(p13;q11) in myeloid leukemia. TEL is a member of the ETS family of transcription factors and contains the pointed protein interaction (PNT) domain and an ETS DNA binding domain (DBD). By contrast to other chimeric proteins that contain TEL's PNT domain, such as TEL-platelet-derived growth factor beta receptor in t(5;12)(q33;p13), MN1-TEL contains the DBD of TEL. The N-terminal MN1 moiety is rich in proline residues and contains two polyglutamine stretches, suggesting that MN1-TEL may act as a deregulated transcription factor. We now show that MN1-TEL type I, unlike TEL and MN1, transforms NIH 3T3 cells. The transforming potential depends on both N-terminal MN1 sequences and a functional TEL DBD. Furthermore, we demonstrate that MN1 has transcription activity and that MN1-TEL acts as a chimeric transcription factor on the Moloney sarcoma virus long terminal repeat and a synthetic promoter containing TEL binding sites. The transactivating capacity of MN1-TEL depended on both the DBD of TEL and sequences in MN1. MN1-TEL contributes to leukemogenesis by a mechanism distinct from that of other chimeric proteins containing TEL.

  12. Interaction of the stress protein p8 with Jab1 is required for Jab1-dependent p27 nuclear-to-cytoplasm translocation

    SciTech Connect

    Malicet, Cedric; Hoffmeister, Albrecht; Moreno, Silvia; Closa, Daniel; Dagorn, Jean-Charles; Vasseur, Sophie; Iovanna, Juan L. . E-mail: iovanna@marseille.inserm.fr

    2006-01-06

    p8 is an 80 amino-acid polypeptide identified because of its remarkable over-expression in the stressed pancreas. This protein, apparently devoid of enzymatic activity, is a powerful regulator of several intracellular pathways, suggesting that it has to interact with several molecular partners to modulate their activity. We used two-hybrid screening of a pre-transformed human testes cDNA library to identify some of these partners. One of them was the multifunctional protein Jab1, its interaction with p8 being confirmed by His{sub 6}-pull down and co-immunoprecipitation assays. In addition, we could show that the two proteins co-localized in the cell. Our functional data demonstrate that Jab1 requires direct interaction with p8 to induce the translocation of p27 from nucleus to cytoplasm and its subsequent degradation. Experiments showing that the knock-down of p8 expression results in a strong inhibition of Jab1 activity confirmed that the mechanism by which Jab1 promotes cell growth by decreasing p27 level is p8-dependent.

  13. 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

  14. 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

  15. 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.

  16. Hypoxia induces oncogene yes-associated protein 1 nuclear translocation to promote pancreatic ductal adenocarcinoma invasion via epithelial-mesenchymal transition.

    PubMed

    Wei, Honglong; Xu, Zongzhen; Liu, Feng; Wang, Fuhai; Wang, Xin; Sun, Xueying; Li, Jie

    2017-05-01

    Pancreatic ductal adenocarcinoma is one of the most lethal cancers. The Hippo pathway is involved in tumorigenesis and remodeling of tumor microenvironments. Hypoxia exists in the microenvironment of solid tumors, including pancreatic ductal adenocarcinoma and plays a vital role in tumor progression and metastasis. However, it remains unclear how hypoxia interacts with the Hippo pathway to regulate these events. In this study, expressions of yes-associated protein 1 and hypoxia-inducible factor-1α were found to be elevated in pancreatic ductal adenocarcinoma samples compared with those in matched adjacent non-tumor samples. Moreover, hypoxia-inducible factor-1α expression was positively correlated with yes-associated protein 1 level in pancreatic ductal adenocarcinoma tissues. The higher expression of nuclear yes-associated protein 1 was associated with poor histological grade and prognosis for pancreatic ductal adenocarcinoma patients. In vitro, yes-associated protein 1 was highly expressed in pancreatic ductal adenocarcinoma cells. Depletion of yes-associated protein 1 inhibited the invasion of pancreatic ductal adenocarcinoma cells via downregulation of Vimentin, matrix metalloproteinase-2, and matrix metalloproteinase-13, and upregulation of E-cadherin. In addition, hypoxia promoted the invasion of pancreatic ductal adenocarcinoma cells via regulating the targeted genes. Hypoxia also deactivated the Hippo pathway and induced yes-associated protein 1 nuclear translocation. Furthermore, depletion of yes-associated protein 1 or hypoxia-inducible factor-1α suppressed the invasion of pancreatic ductal adenocarcinoma cells under hypoxia. Mechanism studies showed that nuclear yes-associated protein 1 interacted with hypoxia-inducible factor-1α and activated Snail transcription to participate in epithelial-mesenchymal transition-mediated and matrix metalloproteinase-mediated remodeling of tumor microenvironments. Collectively, yes-associated protein 1 is an

  17. 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