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Sample records for abundant cytoplasmic proteins

  1. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells

    PubMed Central

    Tichon, Ailone; Gil, Noa; Lubelsky, Yoav; Havkin Solomon, Tal; Lemze, Doron; Itzkovitz, Shalev; Stern-Ginossar, Noam; Ulitsky, Igor

    2016-01-01

    Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionarily conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD—an abundant and highly conserved cytoplasmic lncRNA. Here we show that most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two mammalian Pumilio homologues. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA-binding proteins, an activity which positions them at key junctions of cellular signalling pathways. PMID:27406171

  2. Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule- associated protein (cytoplasmic dynein)

    PubMed Central

    1988-01-01

    Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP- 1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule- activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3- nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties. PMID:2972729

  3. Late embryogenesis abundant proteins

    PubMed Central

    Olvera-Carrillo, Yadira; Reyes, José Luis

    2011-01-01

    Late Embryogenesis Abundant (LEA) proteins accumulate at the onset of seed desiccation and in response to water deficit in vegetative plant tissues. The typical LEA proteins are highly hydrophilic and intrinsically unstructured. They have been classified in different families, each one showing distinctive conserved motifs. In this manuscript we present and discuss some of the recent findings regarding their role in plant adaptation to water deficit, as well as those concerning to their possible function, and how it can be related to their intrinsic structural flexibility. PMID:21447997

  4. Cytoplasmic protein methylation is essential for neural crest migration

    PubMed Central

    Vermillion, Katie L.; Lidberg, Kevin A.

    2014-01-01

    As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration. PMID:24379414

  5. Staphylococcal Major Autolysin (Atl) Is Involved in Excretion of Cytoplasmic Proteins*

    PubMed Central

    Pasztor, Linda; Ziebandt, Anne-Kathrin; Nega, Mulugeta; Schlag, Martin; Haase, Sabine; Franz-Wachtel, Mirita; Madlung, Johannes; Nordheim, Alfred; Heinrichs, David E.; Götz, Friedrich

    2010-01-01

    Many microorganisms excrete typical cytoplasmic proteins into the culture supernatant. As none of the classical secretion systems appears to be involved, this type of secretion was referred to as “nonclassical protein secretion.” Here, we demonstrate that in Staphylococcus aureus the major autolysin plays a crucial role in release of cytoplasmic proteins. Comparative secretome analysis revealed that in the wild type S. aureus strain, 22 typical cytoplasmic proteins were excreted into the culture supernatant, although in the atl mutant they were significantly decreased. The presence or absence of prophages had little influence on the secretome pattern. In the atl mutant, secondary peptidoglycan hydrolases were increased in the secretome; the corresponding genes were transcriptionally up-regulated suggesting a compensatory mechanism for the atl mutation. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic indicator enzyme, we showed that all clinical isolates tested excreted this protein. In the wall teichoic acid-deficient tagO mutant with its increased autolysis activity, GAPDH was excreted in even higher amounts than in the WT, confirming the importance of autolysis in excretion of cytoplasmic proteins. To answer the question of how discriminatory the excretion of cytoplasmic proteins is, we performed a two-dimensional PAGE of cytoplasmic proteins isolated from WT. Surprisingly, the most abundant proteins in the cytoplasm were not found in the secretome of the WT, suggesting that there exists a selection mechanism in the excretion of cytoplasmic proteins. As the major autolysin binds at the septum site, we assume that the proteins are preferentially released at and during septum formation. PMID:20847047

  6. Nuclear Proteins Hijacked by Mammalian Cytoplasmic Plus Strand RNA Viruses

    PubMed Central

    Lloyd, Richard E.

    2015-01-01

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. PMID:25818028

  7. PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae.

    PubMed Central

    Anderson, J T; Paddy, M R; Swanson, M S

    1993-01-01

    Proteins that directly associate with nuclear polyadenylated RNAs, or heterogeneous nuclear RNA-binding proteins (hnRNPs), and those that associate with cytoplasmic mRNAs, or mRNA-binding proteins (mRNPs), play important roles in regulating gene expression at the posttranscriptional level. Previous work with a variety of eukaryotic cells has demonstrated that hnRNPs are localized predominantly within the nucleus whereas mRNPs are cytoplasmic. While studying proteins associated with polyadenylated RNAs in Saccharomyces cerevisiae, we discovered an abundant polyuridylate-binding protein, PUB1, which appears to be both an hnRNP and an mRNP. PUB1 and PAB1, the polyadenylate tail-binding protein, are the two major proteins cross-linked by UV light to polyadenylated RNAs in vivo. The deduced primary structure of PUB1 indicates that it is a member of the ribonucleoprotein consensus sequence family of RNA-binding proteins and is structurally related to the human hnRNP M proteins. Even though the PUB1 protein is a major cellular polyadenylated RNA-binding protein, it is nonessential for cell growth. Indirect cellular immunofluorescence combined with digital image processing allowed a detailed comparison of the intracellular distributions of PUB1 and PAB1. While PAB1 is predominantly, and relatively uniformly, distributed within the cytoplasm, PUB1 is localized in a nonuniform pattern throughout both the nucleus and the cytoplasm. The cytoplasmic distribution of PUB1 is considerably more discontinuous than that of PAB1. Furthermore, sucrose gradient sedimentation analysis demonstrates that PAB1 cofractionates with polyribosomes whereas PUB1 does not. These results suggest that PUB1 is both an hnRNP and an mRNP and that it may be stably bound to a translationally inactive subpopulation of mRNAs within the cytoplasm. Images PMID:8413212

  8. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

    SciTech Connect

    Lloyd, Richard E.

    2015-05-15

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

  9. Multiple Poliovirus Proteins Repress Cytoplasmic RNA Granules

    PubMed Central

    Dougherty, Jonathan D.; Tsai, Wei-Chih; Lloyd, Richard E.

    2015-01-01

    We have previously shown that poliovirus (PV) infection induces stress granule (SG) formation early in infection and then inhibits the formation of SG and disperses processing bodies (PBs) by the mid-phase of infection. Loss of SG was linked to cleavage of G3BP1 by viral 3C proteinase (3Cpro), however dispersal of PBs was not strongly linked to cleavage of specific factors by viral proteinases, suggesting other viral proteins may play roles in inhibition of SG or PB formation. Here we have screened all viral proteins for roles in inducing or inhibiting the formation of RNA granules by creating fusions with mCherry and expressing them individually in cells. Expression of viral proteins separately revealed that the capsid region P1, 2Apro, 3A, 3Cpro, the protease precursor 3CD and 3D polymerase all affect RNA granules to varying extents, whereas 2BC does not. 2Apro, which cleaves eIF4GI, induced SGs as expected, and entered novel foci containing the SG nucleating protein G3BP1. Of the two forms of G3BP, only G3BP1 is cleaved by a virus proteinase, 3Cpro, whereas G3BP2 is not cleaved by 3Cpro or 2Apro. Surprisingly, 3CD, which contains proteinase activity, differentially repressed PBs but not SGs. Further, both 2Apro and 3Cpro expression dispersed PBs, however molecular targets were different since PB dispersal due to 2Apro and heat shock protein (Hsp)90 inhibition but not 3Cpro, could be rescued by application of oxidative stress to cells. The data indicate that PV repression of SGs and PBs is multifactorial, though protease function is dominant. PMID:26610553

  10. Proteins contribute insignificantly to the intrinsic buffering capacity of yeast cytoplasm

    SciTech Connect

    Poznanski, Jaroslaw; Szczesny, Pawel; Ruszczynska, Katarzyna; Zielenkiewicz, Piotr; Paczek, Leszek

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We predicted buffering capacity of yeast proteome from protein abundance data. Black-Right-Pointing-Pointer We measured total buffering capacity of yeast cytoplasm. Black-Right-Pointing-Pointer We showed that proteins contribute insignificantly to buffering capacity. -- Abstract: Intracellular pH is maintained by a combination of the passive buffering of cytoplasmic dissociable compounds and several active systems. Over the years, a large portion of and possibly most of the cell's intrinsic (i.e., passive non-bicarbonate) buffering effect was attributed to proteins, both in higher organisms and in yeast. This attribution was not surprising, given that the concentration of proteins with multiple protonable/deprotonable groups in the cell exceeds the concentration of free protons by a few orders of magnitude. Using data from both high-throughput experiments and in vitro laboratory experiments, we tested this concept. We assessed the buffering capacity of the yeast proteome using protein abundance data and compared it to our own titration of yeast cytoplasm. We showed that the protein contribution is less than 1% of the total intracellular buffering capacity. As confirmed with NMR measurements, inorganic phosphates play a crucial role in the process. These findings also shed a new light on the role of proteomes in maintaining intracellular pH. The contribution of proteins to the intrinsic buffering capacity is negligible, and proteins might act only as a recipient of signals for changes in pH.

  11. Quality Control of a Cytoplasmic Protein Complex

    PubMed Central

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H.

    2015-01-01

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2. PMID:25564609

  12. Biotoxicity assays for fruiting body lectins and other cytoplasmic proteins.

    PubMed

    Künzler, Markus; Bleuler-Martinez, Silvia; Butschi, Alex; Garbani, Mattia; Lüthy, Peter; Hengartner, Michael O; Aebi, Markus

    2010-01-01

    Recent studies suggest that a specific class of fungal lectins, commonly referred to as fruiting body lectins, play a role as effector molecules in the defense of fungi against predators and parasites. Hallmarks of these fungal lectins are their specific expression in reproductive structures, fruiting bodies, and/or sclerotia and their synthesis on free ribosomes in the cytoplasm. Fruiting body lectins are released upon damage of the fungal cell and bind to specific carbohydrate structures of predators and parasites, which leads to deterrence, inhibition of growth, and development or even killing of these organisms. Here, we describe assays to assess the toxicity of such lectins and other cytoplasmic proteins toward three different model organisms: the insect Aedes aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on heterologous expression of the examined proteins in the cytoplasm of Escherichia coli and feeding of these recombinant bacteria to omnivorous and bacterivorous organisms. PMID:20816208

  13. Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis.

    PubMed Central

    Lam, E; Green, P J; Wong, M; Chua, N H

    1989-01-01

    We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organellar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes. Images PMID:2583082

  14. RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation

    PubMed Central

    Reyes, Juan M; Chitwood, James L; Ross, Pablo J

    2014-01-01

    Molecular changes occurring during mammalian oocyte maturation are partly regulated by cytoplasmic polyadenylation (CP) and affect oocyte quality, yet the extent of CP activity during oocyte maturation remains unknown. Single bovine oocyte RNA sequencing (RNA-Seq) was performed to examine changes in transcript abundance during in vitro oocyte maturation in cattle. Polyadenylated RNA from individual germinal-vesicle and metaphase-II oocytes was amplified and processed for Illumina sequencing, producing approximately 30 million reads per replicate for each sample type. A total of 10,494 genes were found to be expressed, of which 2,455 were differentially expressed (adjusted P<0.05 and fold change >2) between stages, with 503 and 1,952 genes respectively increasing and decreasing in abundance. Differentially expressed genes with complete 3’-untranslated-region sequence (279 increasing and 918 decreasing in polyadenylated transcript abundance) were examined for the presence, position, and distribution of motifs mediating CP, revealing enrichment (85%) and lack there of (18%) in up- and down-regulated genes, respectively. Examination of total and polyadenylated RNA abundance by quantitative PCR validated these RNA-Seq findings. The observed increases in polyadenylated transcript abundance within the RNA-Seq data are likely due to CP, providing novel insight into targeted transcripts and resultant differential gene expression profiles that contribute to oocyte maturation. PMID:25560149

  15. RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation.

    PubMed

    Reyes, Juan M; Chitwood, James L; Ross, Pablo J

    2015-02-01

    Molecular changes occurring during mammalian oocyte maturation are partly regulated by cytoplasmic polyadenylation (CP) and affect oocyte quality, yet the extent of CP activity during oocyte maturation remains unknown. Single bovine oocyte RNA sequencing (RNA-Seq) was performed to examine changes in transcript abundance during in vitro oocyte maturation in cattle. Polyadenylated RNA from individual germinal-vesicle and metaphase-II oocytes was amplified and processed for Illumina sequencing, producing approximately 30 million reads per replicate for each sample type. A total of 10,494 genes were found to be expressed, of which 2,455 were differentially expressed (adjusted P < 0.05 and fold change >2) between stages, with 503 and 1,952 genes respectively increasing and decreasing in abundance. Differentially expressed genes with complete 3'-untranslated-region sequence (279 increasing and 918 decreasing in polyadenylated transcript abundance) were examined for the presence, position, and distribution of motifs mediating CP, revealing enrichment (85%) and lack thereof (18%) in up- and down-regulated genes, respectively. Examination of total and polyadenylated RNA abundance by quantitative PCR validated these RNA-Seq findings. The observed increases in polyadenylated transcript abundance within the RNA-Seq data are likely due to CP, providing novel insight into targeted transcripts and resultant differential gene expression profiles that contribute to oocyte maturation.

  16. Female breast carcinomas: nuclear and cytoplasmic proteins versus steroid receptors.

    PubMed

    Bryś, M; Romanowicz-Makowska, H; Nawrocka, A; Krajewska, W M

    2000-01-01

    Nuclear and cytoplasmic proteins of human female breast cancer were analysed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Oestrogen receptor and progesterone receptor expression was determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. The electropherograms were developed by silver nitrate staining and quantitative analysis was carried out by video densitometer using the software Gel-Pro Analyzer. Nuclear and cytoplasmic proteins of breast carcinomas and normal tissue differed both qualitatively and quantitatively. Nuclear polypeptides of 108, 53 and 48 kD as well as the 36 kD cytoplasmic polypeptide were specific for tumour samples, while the 51 kD nuclear polypeptide was detected only in normal tissue. Quantitative differences in band density were noted in the 32 kD nuclear polypeptide. This polypeptide was expressed in greatest concentration in infiltrating ductal carcinomas which also indicated the greatest oestrogen receptor gene expression. This relationship appeared to be statistically significant (p < 0.005). No correlations were evident between the 32 kD protein expression and the progesterone receptor gene expression in any of the tissue types examined, nor between the 32 kD protein and the patient's age or tumour grade. PMID:10756981

  17. Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress

    PubMed Central

    Baqader, Noor O.; Radulovic, Marko; Crawford, Mark; Stoeber, Kai; Godovac-Zimmermann, Jasminka

    2014-01-01

    We have used a subcellular spatial razor approach based on LC–MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function. PMID:25133973

  18. Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

    PubMed Central

    Pietrocola, Federico; Mariño, Guillermo; Lissa, Delphine; Vacchelli, Erika; Malik, Shoaib Ahmad; Niso-Santano, Mireia; Zamzami, Naoufal; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Kroemer, Guido

    2012-01-01

    Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components. PMID:23070521

  19. Predicting the Dynamics of Protein Abundance

    PubMed Central

    Mehdi, Ahmed M.; Patrick, Ralph; Bailey, Timothy L.; Bodén, Mikael

    2014-01-01

    Protein synthesis is finely regulated across all organisms, from bacteria to humans, and its integrity underpins many important processes. Emerging evidence suggests that the dynamic range of protein abundance is greater than that observed at the transcript level. Technological breakthroughs now mean that sequencing-based measurement of mRNA levels is routine, but protocols for measuring protein abundance remain both complex and expensive. This paper introduces a Bayesian network that integrates transcriptomic and proteomic data to predict protein abundance and to model the effects of its determinants. We aim to use this model to follow a molecular response over time, from condition-specific data, in order to understand adaptation during processes such as the cell cycle. With microarray data now available for many conditions, the general utility of a protein abundance predictor is broad. Whereas most quantitative proteomics studies have focused on higher organisms, we developed a predictive model of protein abundance for both Saccharomyces cerevisiae and Schizosaccharomyces pombe to explore the latitude at the protein level. Our predictor primarily relies on mRNA level, mRNA–protein interaction, mRNA folding energy and half-life, and tRNA adaptation. The combination of key features, allowing for the low certainty and uneven coverage of experimental observations, gives comparatively minor but robust prediction accuracy. The model substantially improved the analysis of protein regulation during the cell cycle: predicted protein abundance identified twice as many cell-cycle-associated proteins as experimental mRNA levels. Predicted protein abundance was more dynamic than observed mRNA expression, agreeing with experimental protein abundance from a human cell line. We illustrate how the same model can be used to predict the folding energy of mRNA when protein abundance is available, lending credence to the emerging view that mRNA folding affects translation

  20. In Silico Classification of Proteins from Acidic and Neutral Cytoplasms

    PubMed Central

    Fang, Yaping; Middaugh, C. Russell; Fang, Jianwen

    2012-01-01

    Protein acidostability is a common problem in biopharmaceutical and other industries. However, it remains a great challenge to engineer proteins for enhanced acidostability because our knowledge of protein acidostabilization is still very limited. In this paper, we present a comparative study of proteins from bacteria with acidic (AP) and neutral cytoplasms (NP) using an integrated statistical and machine learning approach. We construct a set of 393 non-redundant AP-NP ortholog pairs and calculate a total of 889 sequence based features for these proteins. The pairwise alignments of these ortholog pairs are used to build a residue substitution propensity matrix between APs and NPs. We use Gini importance provided by the Random Forest algorithm to rank the relative importance of these features. A scoring function using the 10 most significant features is developed and optimized using a hill climbing algorithm. The accuracy of the score function is 86.01% in predicting AP-NP ortholog pairs and is 76.65% in predicting non-ortholog AP-NP pairs, suggesting that there are significant differences between APs and NPs which can be used to predict relative acidostability of proteins. The overall trends uncovered in the study can be used as general guidelines for designing acidostable proteins. To best of our knowledge, this work represents the first systematic comparative study of the acidostable proteins and their non-acidostable orthologs. PMID:23049817

  1. On protein abundance distributions in complex mixtures

    PubMed Central

    2013-01-01

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

  2. Diffusion, Crowding & Protein Stability in a Dynamic Molecular Model of the Bacterial Cytoplasm

    PubMed Central

    McGuffee, Sean R.; Elcock, Adrian H.

    2010-01-01

    A longstanding question in molecular biology is the extent to which the behavior of macromolecules observed in vitro accurately reflects their behavior in vivo. A number of sophisticated experimental techniques now allow the behavior of individual types of macromolecule to be studied directly in vivo; none, however, allow a wide range of molecule types to be observed simultaneously. In order to tackle this issue we have adopted a computational perspective, and, having selected the model prokaryote Escherichia coli as a test system, have assembled an atomically detailed model of its cytoplasmic environment that includes 50 of the most abundant types of macromolecules at experimentally measured concentrations. Brownian dynamics (BD) simulations of the cytoplasm model have been calibrated to reproduce the translational diffusion coefficients of Green Fluorescent Protein (GFP) observed in vivo, and “snapshots” of the simulation trajectories have been used to compute the cytoplasm's effects on the thermodynamics of protein folding, association and aggregation events. The simulation model successfully describes the relative thermodynamic stabilities of proteins measured in E. coli, and shows that effects additional to the commonly cited “crowding” effect must be included in attempts to understand macromolecular behavior in vivo. PMID:20221255

  3. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  4. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein

    PubMed Central

    Much, Christian; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O’Carroll, Dónal

    2016-01-01

    Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse. PMID:27254021

  5. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

    PubMed

    Much, Christian; Auchynnikava, Tania; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O'Carroll, Dónal

    2016-06-01

    Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse. PMID:27254021

  6. Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA.

    PubMed

    Woerner, Andreas C; Frottin, Frédéric; Hornburg, Daniel; Feng, Li R; Meissner, Felix; Patra, Maria; Tatzelt, Jörg; Mann, Matthias; Winklhofer, Konstanze F; Hartl, F Ulrich; Hipp, Mark S

    2016-01-01

    Amyloid-like protein aggregation is associated with neurodegeneration and other pathologies. The nature of the toxic aggregate species and their mechanism of action remain elusive. Here, we analyzed the compartment specificity of aggregate toxicity using artificial β-sheet proteins, as well as fragments of mutant huntingtin and TAR DNA binding protein-43 (TDP-43). Aggregation in the cytoplasm interfered with nucleocytoplasmic protein and RNA transport. In contrast, the same proteins did not inhibit transport when forming inclusions in the nucleus at or around the nucleolus. Protein aggregation in the cytoplasm, but not the nucleus, caused the sequestration and mislocalization of proteins containing disordered and low-complexity sequences, including multiple factors of the nuclear import and export machinery. Thus, impairment of nucleocytoplasmic transport may contribute to the cellular pathology of various aggregate deposition diseases. PMID:26634439

  7. Cytoplasmic N-Terminal Protein Acetylation Is Required for Efficient Photosynthesis in ArabidopsisW⃞

    PubMed Central

    Pesaresi, Paolo; Gardner, Nora A.; Masiero, Simona; Dietzmann, Angela; Eichacker, Lutz; Wickner, Reed; Salamini, Francesco; Leister, Dario

    2003-01-01

    The Arabidopsis atmak3-1 mutant was identified on the basis of a decreased effective quantum yield of photosystem II. In atmak3-1, the synthesis of the plastome-encoded photosystem II core proteins D1 and CP47 is affected, resulting in a decrease in the abundance of thylakoid multiprotein complexes. DNA array–based mRNA analysis indicated that extraplastid functions also are altered. The mutation responsible was localized to AtMAK3, which encodes a homolog of the yeast protein Mak3p. In yeast, Mak3p, together with Mak10p and Mak31p, forms the N-terminal acetyltransferase complex C (NatC). The cytoplasmic AtMAK3 protein can functionally replace Mak3p, Mak10p, and Mak31p in acetylating N termini of endogenous proteins and the L-A virus Gag protein. This result, together with the finding that knockout of the Arabidopsis MAK10 homolog does not result in obvious physiological effects, indicates that AtMAK3 function does not require NatC complex formation, as it does in yeast. We suggest that N-acetylation of certain chloroplast precursor protein(s) is necessary for the efficient accumulation of the mature protein(s) in chloroplasts. PMID:12897255

  8. Further analysis of cytoplasmic polyadenylation in Xenopus embryos and identification of embryonic cytoplasmic polyadenylation element-binding proteins.

    PubMed

    Simon, R; Richter, J D

    1994-12-01

    Early development in Xenopus laevis is programmed in part by maternally inherited mRNAs that are synthesized and stored in the growing oocyte. During oocyte maturation, several of these messages are translationally activated by poly(A) elongation, which in turn is regulated by two cis elements in the 3' untranslated region, the hexanucleotide AAUAAA and a cytoplasmic polyadenylation element (CPE) consisting of UUUUUAU or similar sequence. In the early embryo, a different set of maternal mRNAs is translationally activated. We have shown previously that one of these, C12, requires a CPE consisting of at least 12 uridine residues, in addition to the hexanucleotide, for its cytoplasmic polyadenylation and subsequent translation (R. Simon, J.-P. Tassan, and J.D. Richter, Genes Dev. 6:2580-2591, 1992). To assess whether this embryonic CPE functions in other maternal mRNAs, we have chosen Cl1 RNA, which is known to be polyadenylated during early embryogenesis (J. Paris, B. Osborne, A. Couturier, R. LeGuellec, and M. Philippe, Gene 72:169-176, 1988). Wild-type as well as mutated versions of Cl1 RNA were injected into fertilized eggs and were analyzed for cytoplasmic polyadenylation at times up to the gastrula stage. This RNA also required a poly(U) CPE for cytoplasmic polyadenylation in embryos, but in this case the CPE consisted of 18 uridine residues. In addition, the timing and extent of cytoplasmic poly(A) elongation during early embryogenesis were dependent upon the distance between the CPE and the hexanucleotide. Further, as was the case with Cl2 RNA, Cl1 RNA contains a large masking element that prevents premature cytoplasmic polyadenylation during oocyte maturation. To examine the factors that may be involved in the cytoplasmic polyadenylation of both C12 and C11 RNAs, we performed UV cross-linking experiments in egg extracts. Two proteins with sizes of ~36 and ~45 kDa interacted specifically with the CPEs of both RNAs, although they bound preferentially to the C12

  9. Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.

    PubMed

    Okumus, Burak; Landgraf, Dirk; Lai, Ghee Chuan; Bakhsi, Somenath; Arias-Castro, Juan Carlos; Yildiz, Sadik; Huh, Dann; Fernandez-Lopez, Raul; Peterson, Celeste N; Toprak, Erdal; El Karoui, Meriem; Paulsson, Johan

    2016-01-01

    Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting. PMID:27189321

  10. Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells

    PubMed Central

    Okumus, Burak; Landgraf, Dirk; Lai, Ghee Chuan; Bakhsi, Somenath; Arias-Castro, Juan Carlos; Yildiz, Sadik; Huh, Dann; Fernandez-Lopez, Raul; Peterson, Celeste N.; Toprak, Erdal; El Karoui, Meriem; Paulsson, Johan

    2016-01-01

    Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3–4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting. PMID:27189321

  11. Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells

    NASA Astrophysics Data System (ADS)

    Okumus, Burak; Landgraf, Dirk; Lai, Ghee Chuan; Bakhsi, Somenath; Arias-Castro, Juan Carlos; Yildiz, Sadik; Huh, Dann; Fernandez-Lopez, Raul; Peterson, Celeste N.; Toprak, Erdal; El Karoui, Meriem; Paulsson, Johan

    2016-05-01

    Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting.

  12. Late Embryogenesis Abundant (LEA) proteins in legumes.

    PubMed

    Battaglia, Marina; Covarrubias, Alejandra A

    2013-01-01

    Plants are exposed to different external conditions that affect growth, development, and productivity. Water deficit is one of these adverse conditions caused by drought, salinity, and extreme temperatures. Plants have developed different responses to prevent, ameliorate or repair the damage inflicted by these stressful environments. One of these responses is the activation of a set of genes encoding a group of hydrophilic proteins that typically accumulate to high levels during seed dehydration, at the last stage of embryogenesis, hence named Late Embryogenesis Abundant (LEA) proteins. LEA proteins also accumulate in response to water limitation in vegetative tissues, and have been classified in seven groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. These proteins are widely distributed in the plant kingdom, from ferns to angiosperms, suggesting a relevant role in the plant response to this unfavorable environmental condition. In this review, we analyzed the LEA proteins from those legumes whose complete genomes have been sequenced such as Phaseolus vulgaris, Glycine max, Medicago truncatula, Lotus japonicus, Cajanus cajan, and Cicer arietinum. Considering their distinctive motifs, LEA proteins from the different groups were identified, and their sequence analysis allowed the recognition of novel legume specific motifs. Moreover, we compile their transcript accumulation patterns based on publicly available data. In spite of the limited information on these proteins in legumes, the analysis and data compiled here confirm the high correlation between their accumulation and water deficit, reinforcing their functional relevance under this detrimental conditions. PMID:23805145

  13. The HEX 110 Hexamerin Is a Cytoplasmic and Nucleolar Protein in the Ovaries of Apis mellifera

    PubMed Central

    Martins, Juliana Ramos; Bitondi, Márcia Maria Gentile

    2016-01-01

    Hexamerins are insect storage proteins abundantly secreted by the larval fat body into the haemolymph. The canonical role of hexamerins consists of serving as an amino acid reserve for development toward the adult stage. However, in Apis mellifera, immunofluorescence assays coupled to confocal laser-scanning microscopy, and high-throughput sequencing, have recently shown the presence of hexamerins in other organs than the fat body. These findings have led us to study these proteins with the expectation of uncovering additional functions in insect development. We show here that a honeybee hexamerin, HEX 110, localizes in the cytoplasm and nucleus of ovarian cells. In the nucleus of somatic and germline cells, HEX 110 colocalized with a nucleolar protein, fibrillarin, suggesting a structural or even regulatory function in the nucleolus. RNase A provoked the loss of HEX 110 signals in the ovarioles, indicating that the subcellular localization depends on RNA. This was reinforced by incubating ovaries with pyronin Y, a RNA-specific dye. Together, the colocalization with fibrillarin and pyronin Y, and the sensitivity to RNase, highlight unprecedented roles for HEX110 in the nucleolus, the nuclear structure harbouring the gene cluster involved in ribosomal RNA production. However, the similar patterns of HEX 110 foci distribution in the active and inactive ovaries of queens and workers preclude its association with the functional status of these organs. PMID:26954256

  14. Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

    PubMed Central

    Ke, Na; Landgraf, Dirk; Paulsson, Johan

    2016-01-01

    ABSTRACT The use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryote Escherichia coli. We show that functional protein fusions of the HaloTag can be detected both in vivo and in vitro when expressed within the cytoplasmic or periplasmic compartments of E. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications. IMPORTANCE Visualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryote Escherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells. PMID:26787765

  15. Beyond the cytoplasm of Escherichia coli: localizing recombinant proteins where you want them.

    PubMed

    Boock, Jason T; Waraho-Zhmayev, Dujduan; Mizrachi, Dario; DeLisa, Matthew P

    2015-01-01

    Recombinant protein expression in Escherichia coli represents a cornerstone of the biotechnology enterprise. While cytoplasmic expression in this host has received the most attention, achieving substantial yields of correctly folded proteins in this compartment can sometimes be met with difficulties. These issues can often be overcome by targeting protein expression to extracytoplasmic compartments (e.g., membrane, periplasm) or to the culture medium. This chapter discusses various strategies for exporting proteins out of the cytoplasm as well as tools for monitoring and optimizing these different export mechanisms. PMID:25447860

  16. A new nomenclature for the cytoplasmic ribosomal proteins of Saccharomyces cerevisiae.

    PubMed Central

    Mager, W H; Planta, R J; Ballesta, J G; Lee, J C; Mizuta, K; Suzuki, K; Warner, J R; Woolford, J

    1997-01-01

    The availability of the complete sequence of the Saccharomyces cerevisiae genome has allowed a comprehensive analysis of the genes encoding cytoplasmic ribosomal proteins in this organism. On the basis of this complete inventory a new nomenclature for the yeast ribosomal proteins is presented. PMID:9396790

  17. Cytoplasmic Sequestration of Rel Proteins by IκBα Requires CRM1-Dependent Nuclear Export

    PubMed Central

    Tam, Winnie F.; Lee, Linda H.; Davis, Laura; Sen, Ranjan

    2000-01-01

    Rel and IκB protein families form a complex cellular regulatory network. A major regulatory function of IκB proteins is to retain Rel proteins in the cell cytoplasm. In addition, IκB proteins have also been postulated to serve nuclear functions. These include the maintenance of inducible NF-κB-dependent gene transcription, as well as termination of inducible transcription. We show that IκBα shuttles between the nucleus and the cytoplasm, utilizing the nuclear export receptor CRM1. A CRM1-binding export sequence was identified in the N-terminal domain of IκBα but not in that of IκBβ or IκBɛ. By reconstituting major aspects of NF-κB–IκB sequestration in yeast, we demonstrate that cytoplasmic retention of p65 (also called RelA) by IκBα requires Crm1p-dependent nuclear export. In mammalian cells, inhibition of CRM1 by leptomycin B resulted in nuclear localization of cotransfected p65 and IκBα in COS cells and enhanced nuclear relocation of endogenous p65 in T cells. These observations suggest that the main function of IκBα is that of a nuclear export chaperone rather than a cytoplasmic tether. We propose that the nucleus is the major site of p65-IκBα association, from where these complexes must be exported in order to create the cytoplasmic pool. PMID:10688673

  18. Identification of Missing Proteins Defined by Chromosome-Centric Proteome Project in the Cytoplasmic Detergent-Insoluble Proteins.

    PubMed

    Chen, Yang; Li, Yaxing; Zhong, Jiayong; Zhang, Jing; Chen, Zhipeng; Yang, Lijuan; Cao, Xin; He, Qing-Yu; Zhang, Gong; Wang, Tong

    2015-09-01

    Finding protein evidence (PE) for protein coding genes is a primary task of the Phase I Chromosome-Centric Human Proteome Project (C-HPP). Currently, there are 2948 PE level 2-4 coding genes per neXtProt, which are deemed missing proteins in the human proteome. As most samples prepared and analyzed in the C-HPP framework were focusing on detergent soluble proteins, we posit that as a natural composition the cytoplasmic detergent-insoluble proteins (DIPs) represent a source of finding missing proteins. We optimized a workflow and separated cytoplasmic DIPs from three human lung and three human hepatoma cell lines via differential speed centrifugation. We verified that the detergent-soluble proteins (DSPs) could be sufficiently depleted and the cytoplasmic DIP isolation was partially reproducible with Spearman r > 0.70 according to two independent SILAC MS experiments. Through label-free MS, we identified 4524 and 4156 DIPs from lung and liver cells, respectively. Among them, a total of 23 missing proteins (22 PE2 and 1 PE4) were identified by MS, and 18 of them had translation evidence; in addition, six PE5 proteins were identified by MS, three with translation evidence. We showed that cytoplasmic DIPs were not an enrichment of transmembrane proteins and were chromosome-, cell type-, and tissue-specific. Furthermore, we demonstrated that DIPs were distinct from DSPs in terms of structural and physical-chemical features. In conclusion, we have found 23 missing proteins and 6 PE5 proteins from the cytoplasmic insoluble proteome that is biologically and physical-chemically different from the soluble proteome, suggesting that cytoplasmic DIPs carry comprehensive and valuable information for finding PE of missing proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001694.

  19. Protein misfolding specifies recruitment to cytoplasmic inclusion bodies

    PubMed Central

    Bersuker, Kirill; Brandeis, Michael

    2016-01-01

    Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington’s disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs. PMID:27114501

  20. Protein misfolding specifies recruitment to cytoplasmic inclusion bodies.

    PubMed

    Bersuker, Kirill; Brandeis, Michael; Kopito, Ron R

    2016-04-25

    Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.

  1. Transcriptional abundance is not the single force driving the evolution of bacterial proteins

    PubMed Central

    2013-01-01

    Background Despite rapid progress in understanding the mechanisms that shape the evolution of proteins, the relative importance of various factors remain to be elucidated. In this study, we have assessed the effects of 16 different biological features on the evolutionary rates (ERs) of protein-coding sequences in bacterial genomes. Results Our analysis of 18 bacterial species revealed new correlations between ERs and constraining factors. Previous studies have suggested that transcriptional abundance overwhelmingly constrains the evolution of yeast protein sequences. This transcriptional abundance leads to selection against misfolding or misinteractions. In this study we found that there was no single factor in determining the evolution of bacterial proteins. Not only transcriptional abundance (codon adaptation index and expression level), but also protein-protein associations (PPAs), essentiality (ESS), subcellular localization of cytoplasmic membrane (SLM), transmembrane helices (TMH) and hydropathicity score (HS) independently and significantly affected the ERs of bacterial proteins. In some species, PPA and ESS demonstrate higher correlations with ER than transcriptional abundance. Conclusions Different forces drive the evolution of protein sequences in yeast and bacteria. In bacteria, the constraints are involved in avoiding a build-up of toxic molecules caused by misfolding/misinteraction (transcriptional abundance), while retaining important functions (ESS, PPA) and maintaining the cell membrane (SLM, TMH and HS). Each of these independently contributes to the variation in protein evolution. PMID:23914835

  2. Cytoplasmic Ig-Domain Proteins: Cytoskeletal Regulators with a Role in Human Disease

    PubMed Central

    Otey, Carol A.; Dixon, Richard; Stack, Christianna; Goicoechea, Silvia M.

    2009-01-01

    Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain proteins are more than structural elements of the cell, and may have evolved to fill different needs in different cellular compartments. PMID:19466753

  3. Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

    PubMed Central

    Pézolet, M; Pigeon, M; Ménard, D; Caillé, J P

    1988-01-01

    The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation. Images FIGURE 2 PMID:3349128

  4. Phage Display Based Cloning of Proteins Interacting with the Cytoplasmic Tail of Membrane Immunoglobulins

    PubMed Central

    Geisberger, Roland; Prlic, Martin; Achatz-Straussberger, Gertrude; Oberndorfer, Iris; Luger, Elke; Lamers, Marinus; Crameri, Reto; Appenzeller, Ulrich; Wienands, Jürgen; Breitenbach, Michael; Ferreira, Fatima; Achatz, Gernot

    2002-01-01

    The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Igα/Igβ heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation. PMID:12885153

  5. Cytoplasmic polyadenylation element binding protein-dependent protein synthesis is regulated by calcium/calmodulin-dependent protein kinase II.

    PubMed

    Atkins, Coleen M; Nozaki, Naohito; Shigeri, Yasushi; Soderling, Thomas R

    2004-06-01

    Phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) regulates protein synthesis in hippocampal dendrites. CPEB binds the 3' untranslated region (UTR) of cytoplasmic mRNAs and, when phosphorylated, initiates mRNA polyadenylation and translation. We report that, of the protein kinases activated in the hippocampus during synaptic plasticity, calcium/calmodulin-dependent protein kinase II (CaMKII) robustly phosphorylated the regulatory site (threonine 171) in CPEB in vitro. In postsynaptic density fractions or hippocampal neurons, CPEB phosphorylation increased when CaMKII was activated. These increases in CPEB phosphorylation were attenuated by a specific peptide inhibitor of CaMKII and by the general CaM-kinase inhibitor KN-93. Inhibitors of protein phosphatase 1 increased basal CPEB phosphorylation in neurons; this was also attenuated by a CaM-kinase inhibitor. To determine whether CaM-kinase activity regulates CPEB-dependent mRNA translation, hippocampal neurons were transfected with luciferase fused to a 3' UTR containing CPE-binding elements. Depolarization of neurons stimulated synthesis of luciferase; this was abrogated by inhibitors of protein synthesis, mRNA polyadenylation, and CaMKII. These results demonstrate that CPEB phosphorylation and translation are regulated by CaMKII activity and provide a possible mechanism for how dendritic protein synthesis in the hippocampus may be stimulated during synaptic plasticity.

  6. In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus.

    PubMed Central

    Kang, K I; Devin, J; Cadepond, F; Jibard, N; Guiochon-Mantel, A; Baulieu, E E; Catelli, M G

    1994-01-01

    In target tissue extracts, heat shock protein hsp90 has been found associated to all unliganded steroid receptors. Modulation of important functions of these receptors, including prevention of DNA binding and optimization of transcriptional activity, has been attributed to hsp90. However no unequivocal in vivo demonstration of interaction between receptors and hsp90 has been presented. We targeted chicken hsp90, a mainly cytoplasmic protein, with the nucleoplasmin nuclear localization signal (90NLS). After transfection into COS-7 cells, 90NLS was found in the nucleus with specific immunofluorescence and confocal microscopy techniques. A human glucocorticosteroid receptor mutant devoid of NLS sequence was also expressed in COS-7 cells and found exclusively cytoplasmic. Coexpression of 90NLS and of the cytoplasmic human glucocorticosteroid receptor mutant led to complete nuclear localization of the receptor, indicating its piggyback transport by 90NLS and thus physical and functional interaction between the two proteins in the absence of hormone. The same nuclear localization was obtained after cotransfection of 90NLS and a cytoplasmic rabbit progesterone receptor mutant. Finally, coexpression of wild-type rabbit progesterone receptor (nuclear) and wildtype hsp90 (cytoplasmic) into COS-7 cells provoked partial relocalization of hsp90 into the nucleus. These experiments lay the groundwork on which to study hsp90 as a chaperone, regulating activities of steroid receptors and possibly participating in their nuclear-cytoplasmic shuttling. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8278390

  7. Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor.

    PubMed Central

    Matsumoto, M; Hsieh, T Y; Zhu, N; VanArsdale, T; Hwang, S B; Jeng, K S; Gorbalenya, A E; Lo, S Y; Ou, J H; Ware, C F; Lai, M M

    1997-01-01

    Hepatitis C virus (HCV) core protein is a multifunctional protein. We examined whether it can interact with cellular proteins, thus contributing to viral pathogenesis. Using the HCV core protein as a bait to screen a human liver cDNA library in a yeast two-hybrid screening system, we have isolated several positive clones encoding cellular proteins that interact with the HCV core protein. Interestingly, more than half of these clones encode the cytoplasmic domain of lymphotoxin-beta receptor (LT betaR), which is a member of the tumor necrosis factor receptor family. Their binding was confirmed by in vitro glutathione S-transferase fusion protein binding assay and protein-protein blotting assay to be direct and specific. The binding sites were mapped within a 58-amino-acid region of the cytoplasmic tail of LT betaR. The binding site in the HCV core protein was localized within amino acid residues 36 to 91 from the N terminus, corresponding to the hydrophilic region of the protein. In mammalian cells, the core protein was found to be associated with the membrane-bound LT betaR. Since the LT betaR is involved in germinal center formation and developmental regulation of peripheral lymphoid organs, lymph node development, and apoptotic signaling, the binding of HCV core protein to LT betaR suggests the possibility that this viral protein has an immunomodulating function and may explain the mechanism of viral persistence and pathogenesis of HCV. PMID:8995654

  8. Detection of cytoplasmic proteins from Helicobacter pylori in Colony Lift Immunoassay.

    PubMed

    Rojas-Rengifo, Diana F; Jaramillo, Carlos A; Haas, Rainer; Jiménez-Soto, Luisa F

    2015-12-01

    Use of the Colony Lift Immunoassay has been described for several Gram negative bacteria of medical interest. In all cases detection was limited to the use of antibodies against outer membrane proteins. Here we describe the adaptation of this method for detection of the cytoplasmic CagA toxin from Helicobacter pylori.

  9. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

    PubMed Central

    Ansseau, Eugénie; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q.; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  10. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.

    PubMed

    Ansseau, Eugénie; Eidahl, Jocelyn O; Lancelot, Céline; Tassin, Alexandra; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  11. The human immunodeficiency virus type 1 Rev protein shuttles between the cytoplasm and nuclear compartments.

    PubMed Central

    Kalland, K H; Szilvay, A M; Brokstad, K A; Saetrevik, W; Haukenes, G

    1994-01-01

    A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (HIV). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation. Images PMID:7935458

  12. Two Novel Heat-Soluble Protein Families Abundantly Expressed in an Anhydrobiotic Tardigrade

    PubMed Central

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D.; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals. PMID:22937162

  13. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

    PubMed

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

  14. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

    PubMed

    Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

    2012-01-01

    Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals. PMID:22937162

  15. Transport of the outer dynein arm complex to cilia requires a cytoplasmic protein Lrrc6.

    PubMed

    Inaba, Yasuko; Shinohara, Kyosuke; Botilde, Yanick; Nabeshima, Ryo; Takaoka, Katsuyoshi; Ajima, Rieko; Lamri, Lynda; Takeda, Hiroyuki; Saga, Yumiko; Nakamura, Tetsuya; Hamada, Hiroshi

    2016-07-01

    Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism.

  16. ERAD of proteins containing aberrant transmembrane domains requires ubiquitylation of cytoplasmic lysine residues

    PubMed Central

    Briant, Kit; Koay, Yee-Hui; Otsuka, Yuka; Swanton, Eileithyia

    2015-01-01

    ABSTRACT Clearance of misfolded proteins from the endoplasmic reticulum (ER) is mediated by the ubiquitin-proteasome system in a process known as ER-associated degradation (ERAD). The mechanisms through which proteins containing aberrant transmembrane domains are degraded by ERAD are poorly understood. To address this question, we generated model ERAD substrates based on CD8 with either a non-native transmembrane domain but a folded ER luminal domain (CD8TMD*), or the native transmembrane domain but a misfolded luminal domain (CD8LUM*). Although both chimeras were degraded by ERAD, we found that the location of the folding defect determined the initial site of ubiquitylation. Ubiquitylation of cytoplasmic lysine residues was required for the extraction of CD8TMD* from the ER membrane during ERAD, whereas CD8LUM* continued to be degraded in the absence of cytoplasmic lysine residues. Cytoplasmic lysine residues were also required for degradation of an additional ERAD substrate containing an unassembled transmembrane domain and when a non-native transmembrane domain was introduced into CD8LUM*. Our results suggest that proteins with defective transmembrane domains are removed from the ER through a specific ERAD mechanism that depends upon ubiquitylation of cytoplasmic lysine residues. PMID:26446255

  17. The nuclear protein Sam68 is recruited to the cytoplasmic stress granules during enterovirus 71 infection.

    PubMed

    Zhang, Hua; Chen, Ning; Li, Pengfei; Pan, Ziye; Ding, Yun; Zou, Dehua; Li, Liyang; Xiao, Lijie; Shen, Binglei; Liu, Shuxia; Cao, Hongwei; Cui, Yudong

    2016-07-01

    Our previous study found that the nuclear protein, 68-kDa Src-associated in mitosis protein (Sam68), is translocated to the cytoplasm and forms punctate pattern during enterovirus 71 (EV71) infection [Virus Research, 180 (2014), 1-11]. However, the exact function of this punctate pattern in cytoplasm during EV71 infection remains unknown. In this study, we firstly have examined this punctate pattern of Sam68 re-localization in the cytoplasm, and observed the obvious recruitments of Sam68 to the EV71-induced stress granules (SGs). Sam68, belongs to the KH domain family of RNA binding proteins (RBPs), was then confirmed that its KH domain was essential for this recruitment. Nevertheless, Knockdown of Sam68 expression using ShRNA had no effects on SGs assembly, indicating that Sam68 is not a constitutive component of the SGs during EV71 infection. Lastly, we investigated the importance of microtubulin transport to SGs aggregation, and revealed that microtubule depolymerization inhibited SGs formation, suggesting that EV71-induced SGs move throughout the cytoplasm in a microtubule-dependent manner. Taken together, these results illuminated that EV71 infections can induce SGs formation, and Sam68, as a SGs component, migrates alone with SGs dependent on intact microtubule upon the viral infections. These findings may provide novel underlying mechanism for delineating the role of SGs during EV71 infection. PMID:27057671

  18. Transport of the outer dynein arm complex to cilia requires a cytoplasmic protein Lrrc6.

    PubMed

    Inaba, Yasuko; Shinohara, Kyosuke; Botilde, Yanick; Nabeshima, Ryo; Takaoka, Katsuyoshi; Ajima, Rieko; Lamri, Lynda; Takeda, Hiroyuki; Saga, Yumiko; Nakamura, Tetsuya; Hamada, Hiroshi

    2016-07-01

    Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism. PMID:27353389

  19. Depletion of Abundant Plasma Proteins and Limitations of Plasma Proteomics

    PubMed Central

    Tu, Chengjian; Rudnick, Paul A.; Martinez, Misti Y.; Cheek, Kristin L.; Stein, Stephen E.; Slebos, Robbert J. C.; Liebler, Daniel C.

    2010-01-01

    Immunoaffinity depletion with antibodies to the top 7 or top 14 high abundance plasma proteins is used to enhance detection of lower abundance proteins in both shotgun and targeted proteomic analyses. We evaluated the effects of top 7/top 14 immunodepletion on the shotgun proteomic analysis of human plasma. Our goal was to evaluate the impact of immunodepletion on detection of proteins across detectable ranges of abundance. The depletion columns afforded highly repeatable and efficient plasma protein fractionation. Relatively few nontargeted proteins were captured by the depletion columns. Analyses of unfractionated and immunodepleted plasma by peptide isoelectric focusing (IEF), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrated enrichment of nontargeted plasma proteins by an average of 4-fold, as assessed by MS/MS spectral counting. Either top 7 or top 14 immunodepletion resulted in a 25% increase in identified proteins compared to unfractionated plasma. Although 23 low abundance (<10 ng mL−1) plasma proteins were detected, they accounted for only 5–6% of total protein identifications in immunodepleted plasma. In both unfractionated and immunodepleted plasma, the 50 most abundant plasma proteins accounted for 90% of cumulative spectral counts and precursor ion intensities, leaving little capacity to sample lower abundance proteins. Untargeted proteomic analyses using current LC-MS/MS platforms—even with immunodepletion—cannot be expected to efficiently discover low abundance, disease-specific biomarkers in plasma. PMID:20677825

  20. Phage shock proteins B and C prevent lethal cytoplasmic membrane permeability in Yersinia enterocolitica.

    PubMed

    Horstman, N Kaye; Darwin, Andrew J

    2012-08-01

    The bacterial phage shock protein (Psp) stress response system is activated by events affecting the cytoplasmic membrane. In response, Psp protein levels increase, including PspA, which has been implicated as the master effector of stress tolerance. Yersinia enterocolitica and related bacteria with a defective Psp system are highly sensitive to the mislocalization of pore-forming secretin proteins. However, why secretins are toxic to psp null strains, whereas some other Psp inducers are not, has not been explained. Furthermore, previous work has led to the confounding and disputable suggestion that PspA is not involved in mitigating secretin toxicity. Here we have established a correlation between the amount of secretin toxicity in a psp null strain and the extent of cytoplasmic membrane permeability to large molecules. This leads to a morphological change resembling cells undergoing plasmolysis. Furthermore, using novel strains with dis-regulated Psp proteins has allowed us to obtain unequivocal evidence that PspA is not required for secretin-stress tolerance. Together, our data suggest that the mechanism by which secretin multimers kill psp null cells is by causing a profound defect in the cytoplasmic membrane permeability barrier. This allows lethal molecular exchange with the environment, which the PspB and PspC proteins can prevent. PMID:22646656

  1. Protein kinases are associated with multiple, distinct cytoplasmic granules in quiescent yeast cells.

    PubMed

    Shah, Khyati H; Nostramo, Regina; Zhang, Bo; Varia, Sapna N; Klett, Bethany M; Herman, Paul K

    2014-12-01

    The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival.

  2. Protein Kinases Are Associated with Multiple, Distinct Cytoplasmic Granules in Quiescent Yeast Cells

    PubMed Central

    Shah, Khyati H.; Nostramo, Regina; Zhang, Bo; Varia, Sapna N.; Klett, Bethany M.; Herman, Paul K.

    2014-01-01

    The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival. PMID:25342717

  3. Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels.

    PubMed

    Schraivogel, Daniel; Schindler, Susann G; Danner, Johannes; Kremmer, Elisabeth; Pfaff, Janina; Hannus, Stefan; Depping, Reinhard; Meister, Gunter

    2015-09-01

    MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago - TNRC6 levels.

  4. Critical Role of the Fusion Protein Cytoplasmic Tail Sequence in Parainfluenza Virus Assembly

    PubMed Central

    Stone, Raychel; Takimoto, Toru

    2013-01-01

    Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F (rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites. PMID:23593451

  5. Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.

    PubMed Central

    Guénette, S Y; Chen, J; Jondro, P D; Tanzi, R E

    1996-01-01

    We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855266

  6. Excretion of cytoplasmic proteins in Staphylococcus is most likely not due to cell lysis.

    PubMed

    Ebner, Patrick; Rinker, Janina; Götz, Friedrich

    2016-02-01

    The excretion of cytoplasmic proteins (ECP) is a long-known phenomenon in bacteria and eukaryotes. So far, it was not possible to associate either a signal peptide-dependent or a signal peptide-independent pathway to ECP. Nevertheless 25% of the proteins found in Staphylococcus aureus supernatants were cytoplasmic proteins. Because the excreted proteins do not possess a common motive, the most widespread opinion is that ECP is due to cell lysis. This explanation seems to be too easy since several indications imply that there exists a yet unknown mechanism for ECP. Certainly, the up-regulation of autolysins as well as decreased peptidoglycan cross-linking increased ECP. However, in recent years, several evidences arose that cell lysis is not the only reason for ECP. It seems that ECP is a part of the normal cell cycle of S. aureus as it turned out that ECP with several model proteins occurs mainly during cell growth. It has common features as proteins secreted via the Sec translocon and finally the excretion site is the cross wall of dividing cells.

  7. A testis cytoplasmic RNA-binding protein that has the properties of a translational repressor.

    PubMed Central

    Lee, K; Fajardo, M A; Braun, R E

    1996-01-01

    Translation of the mouse protamine 1 (Prm-1) mRNA is repressed for several days during male germ cell differentiation. With the hope of cloning genes that regulate the translational repression of Prm-1, we screened male germ cell cDNA expression libraries with the 3' untranslated region of the Prm-1 RNA. From this screen we obtained two independent clones that encode Prbp, a Prm-1 RNA-binding protein. Prbp contains two copies of a double-stranded-RNA-binding domain. In vitro, the protein binds to a portion of the Prm-1 3' untranslated region previously shown to be sufficient for translational repression in transgenic mice, as well as to poly(I). poly(C). Prbp protein is present in multiple forms in cytoplasmic extracts prepared from wild-type mouse testes and is absent from testes of germ cell-deficient mouse mutants, suggesting that Prbp is restricted to the germ cells of the testis. Immunocytochemical localization confirmed that Prbp is present in the cytoplasmic compartment of late-stage meiotic cells and haploid round spermatids. Recombinant Prbp protein inhibits the translation of multiple mRNAs in a wheat germ lysate, suggesting that Prbp acts to repress translation in round spermatids. While this protein lacks complete specificity for Prm-1-containing RNAs in vitro, the properties of Prbp are consistent with it acting as a general repressor of translation. PMID:8649414

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

    PubMed

    Keller, Rob C A

    2013-01-01

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

  9. A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption.

    PubMed Central

    Kiino, D R; Licudine, R; Wilt, K; Yang, D H; Rothman-Denes, L B

    1993-01-01

    At least four genes are required for irreversible adsorption of bacteriophage N4. nfrA and nfrB have been characterized previously and encode an outer membrane protein and inner membrane protein, respectively. The nfrC gene product is characterized in detail in this study. We have mapped the nfrD locus to min 52 on the Escherichia coli linkage map. Maxicell analysis of nfrC and a null allele (nfrC2) cloned into a high-copy-number plasmid shows its gene product to be 42 kDa in size. We determined the nfrC nucleotide sequence which predicts a gene product of 42 kDa. Western blots (immunoblots) of Escherichia coli proteins after cellular fractionation show NfrC to be a cytoplasmic protein which is required for irreversible bacteriophage N4 adsorption, an event occurring at the cell surface. Images PMID:8226648

  10. Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus.

    PubMed

    Horie, Ryo; Yoneda, Misako; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

    2016-10-01

    Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21-24 and 110-139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60-75 and 72-75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm. PMID:27501340

  11. Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus.

    PubMed

    Horie, Ryo; Yoneda, Misako; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

    2016-10-01

    Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21-24 and 110-139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60-75 and 72-75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm.

  12. Identification of domains mediating transcriptional activation and cytoplasmic export in the caudal homeobox protein Cdx-3.

    PubMed

    Trinh, K Y; Jin, T; Drucker, D J

    1999-02-26

    The caudal genes have important functions in embryonic development and cell differentiation. The caudal-related protein Cdx-2/3 (the protein designated Cdx-2 in the mouse and Cdx-3 in the hamster) is expressed in the gastrointestinal epithelium and in islet and enteroendocrine cells, where it activates proglucagon gene transcription. We show here that Cdx-3 sequences amino-terminal to the homeodomain (amino acids 1-180) function as a heterologous transcriptional activation domain when fused to the LexA DNA binding domain. A Cdx-3-Pit-1 fusion protein containing only the first 83 amino acids of Cdx-3 linked to the POU domain of Pit-1 markedly stimulated the transcriptional activity of a Pit-1-responsive promoter. Analysis of the transcriptional properties of Cdx-3 mutants in fibroblasts and islet cells revealed distinct amino-terminal subdomains that function in a cell-specific manner. Point mutations within the amino-terminal A domain were associated with reduced transcriptional activity. Furthermore, internal deletions and selected point mutations within domain A, but not the B or C domains, resulted in accumulation of mutant Cdx-3 in the cytoplasm. Unexpectedly, mutation of an Asp-Lys-Asp motif within domain A identified a putative cytoplasmic membrane-associated export signal that mediates Cdx-3 compartmentalization. These experiments delineate unique activities for specific amino-terminal sequences that are functionally important for Cdx-3 biological activity.

  13. Holophytochrome-Interacting Proteins in Physcomitrella: Putative Actors in Phytochrome Cytoplasmic Signaling

    PubMed Central

    Ermert, Anna Lena; Mailliet, Katharina; Hughes, Jon

    2016-01-01

    Phytochromes are the principle photoreceptors in light-regulated plant development, primarily acting via translocation of the light-activated photoreceptor into the nucleus and subsequent gene regulation. However, several independent lines of evidence indicate unambiguously that an additional cytoplasmic signaling mechanism must exist. Directional responses in filament tip cells of the moss Physcomitrella patens are steered by phy4 which has been shown to interact physically with the blue light receptor phototropin at the plasma membrane. This complex might perceive and transduce vectorial information leading to cytoskeleton reorganization and finally a directional growth response. We developed yeast two-hybrid procedures using photochemically functional, full-length phy4 as bait in Physcomitrella cDNA library screens and growth assays under different light conditions, revealing Pfr-dependent interactions possibly associated with phytochrome cytoplasmic signaling. Candidate proteins were then expressed in planta with fluorescent protein tags to determine their intracellular localization in darkness and red light. Of 14 candidates, 12 were confirmed to interact with phy4 in planta using bimolecular fluorescence complementation. We also used database information to study their expression patterns relative to those of phy4. We discuss the likely functional characteristics of these holophytochrome-interacting proteins (HIP’s) and their possible roles in signaling. PMID:27242820

  14. Urea may regulate urea transporter protein abundance during osmotic diuresis.

    PubMed

    Kim, Dongun; Klein, Janet D; Racine, Sandy; Murrell, Brian P; Sands, Jeff M

    2005-01-01

    Rats with diabetes mellitus have an increase in UT-A1 urea transporter protein abundance and absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased due to the marked glucosuria. Urea-specific signaling pathways have been identified in mIMCD3 cells and renal medulla, suggesting the possibility that changes in the percentage or concentration of urea could be a factor that regulates UT-A1 abundance. In this study, we tested the hypothesis that an increase in a urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus, whereas an increase in urine urea would not. In both inner medullary base and tip, UT-A1 protein abundance increased during NaCl- or glucose-induced osmotic diuresis but not during urea-induced osmotic diuresis. Next, rats undergoing NaCl or glucose diuresis were given supplemental urea to increase the percentage of urine urea to control values. UT-A1 abundance did not increase in these urea-supplemented rats compared with control rats. Additionally, both UT-A2 and UT-B protein abundances in the outer medulla increased during urea-induced osmotic diuresis but not in NaCl or glucose diuresis. We conclude that during osmotic diuresis, UT-A1 abundance increases when the percentage of urea in total urinary solute is low and UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high. These findings suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability.

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

    PubMed Central

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

    1989-01-01

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

  16. Tumour suppressor death-associated protein kinase targets cytoplasmic HIF-1α for Th17 suppression

    PubMed Central

    Chou, Ting-Fang; Chuang, Ya-Ting; Hsieh, Wan-Chen; Chang, Pei-Yun; Liu, Hsin-Yu; Mo, Shu-Ting; Hsu, Tzu-Sheng; Miaw, Shi-Chuen; Chen, Ruey-Hwa; Kimchi, Adi; Lai, Ming-Zong

    2016-01-01

    Death-associated protein kinase (DAPK) is a tumour suppressor. Here we show that DAPK also inhibits T helper 17 (Th17) and prevents Th17-mediated pathology in a mouse model of autoimmunity. We demonstrate that DAPK specifically downregulates hypoxia-inducible factor 1α (HIF-1α). In contrast to the predominant nuclear localization of HIF-1α in many cell types, HIF-1α is located in both the cytoplasm and nucleus in T cells, allowing for a cytosolic DAPK–HIF-1α interaction. DAPK also binds prolyl hydroxylase domain protein 2 (PHD2) and increases HIF-1α-PHD2 association. DAPK thereby promotes the proline hydroxylation and proteasome degradation of HIF-1α. Consequently, DAPK deficiency leads to excess HIF-1α accumulation, enhanced IL-17 expression and exacerbated experimental autoimmune encephalomyelitis. Additional knockout of HIF-1α restores the normal differentiation of Dapk−/− Th17 cells and prevents experimental autoimmune encephalomyelitis development. Our results reveal a mechanism involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that raising DAPK levels could be used for treatment of Th17-associated inflammatory diseases. PMID:27312851

  17. Possible relationship of brain cytoplasmic tetrodotoxin-sensitive protein to voltage-gated sodium channel shown by monoclonal antibody.

    PubMed

    Pinchuk, G V; Malysheva, M K; Pinchuk, L N; Gerasymenko, O V; Zhukareva, V A

    1990-02-01

    Biochemical events leading to the formation of mature membrane-associated sodium channel proteins are not completely understood. We have recently purified a protein from the cytoplasm of brain cells, which is able to become incorporated into liposomes and induce neurotoxin-dependent sodium permeability. Here we report data on a monoclonal antibody derived against this protein. This antibody crossreacts with cell membrane preparations. The antibody binding to viable neuroblastoma cells is inhibited by veratrine, indicating that membrane molecules antigenically related to the cytoplasmic protein may also be related to the voltage-gated sodium channel.

  18. Novel hexamerization motif is discovered in a conserved cytoplasmic protein from Salmonella typhimurium.

    SciTech Connect

    Petrova, T.; Cuff, M.; Wu, R.; Kim, Y.; Holzle, D.; Joachimiak, A.; Biosciences Division; Inst. of Mathematical Problems of Biology

    2007-01-01

    The cytoplasmic protein Stm3548 of unknown function obtained from a strain of Salmonella typhimurium was determined by X-ray crystallography at a resolution of 2.25 A. The asymmetric unit contains a hexamer of structurally identical monomers. The monomer is a globular domain with a long beta-hairpin protrusion that distinguishes this structure. This beta-hairpin occupies a central position in the hexamer, and its residues participate in the majority of interactions between subunits of the hexamer. We suggest that the structure of Stm3548 presents a new hexamerization motif. Because the residues participating in interdomain interactions are highly conserved among close members of protein family DUF1355 and buried solvent accessible area for the hexamer is significant, the hexamer is most likely conserved as well. A light scattering experiment confirmed the presence of hexamer in solution.

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

    SciTech Connect

    Srinivasan, K.; Bhargava, M.M.

    1986-05-01

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

  20. Novel alternatively spliced endoplasmic reticulum retention signal in the cytoplasmic loop of Proteolipid Protein-1.

    PubMed

    Southwood, Cherie; Olson, Kevin; Wu, Chia-Yen; Gow, Alexander

    2007-02-15

    Increased awareness about the importance of protein folding and trafficking to the etiology of gain-of-function diseases has driven extensive efforts to understand the cell and molecular biology underlying the life cycle of normal secretory pathway proteins and the detrimental effects of abnormal proteins. In this regard, the quality-control machinery in the endoplasmic reticulum (ER) has emerged as a major mechanism by which cells ensure that secreted and transmembrane proteins either adopt stable secondary, tertiary, and quaternary structures or are retained in the ER and degraded. Here we examine cellular and molecular aspects of ER retention in transfected fibroblasts expressing missense mutations in the Proteolipid Protein-1 (PLP1) gene that cause mild or severe forms of neurodegenerative disease in humans. Mild mutations cause protein retention in the ER that is partially dependent on the presence of a cytoplasmically exposed heptapeptide, KGRGSRG. In contrast, retention associated with severe mutations occurs independently of this peptide. Accordingly, the function of this novel heptapeptide has a significant impact on pathogenesis and provides new insight into the functions of the two splice isoforms encoded by the PLP1 gene, PLP1 and DM-20. PMID:17171701

  1. SoyProLow: A protein database enriched in low abundant soybean proteins

    PubMed Central

    Tavakolan, Mona; Alkharouf, Nadim W; Matthews, Benjamin F; Natarajan, Savithiry S

    2014-01-01

    Soybeans are an important legume crop that contain 2 major storage proteins, β-conglycinin and glycinin, which account about 70- 80% of total seed proteins. These abundant proteins hinder the isolation and characterization of several low abundant proteins in soybean seeds. Several protein extraction methodologies were developed in our laboratory to decrease these abundant storage proteins in seed extracts and to also decrease the amount of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), which is normally very abundant in leaf extracts. One of the extraction methodologies used 40% isopropanol and was more effective in depleting soybean storage proteins and enhancing low abundant seed proteins than similar methods using 10-80% isopropanol. Extractions performed with 40% isopropanol decreased the amount of storage proteins and revealed 107 low abundant proteins when using the combined approaches of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Mass Spectrometry (MS). The separation of proteins was achieved by iso-electric focusing (IEF) and 2D-PAGE. The proteins were analyzed with MS techniques to provide amino acid sequence. The proteins were identified by comparing their amino acid sequences with those in different databases including NCBI-non redundant, UniprotKB and MSDB databases. In this investigation, previously published results on low abundant soybean seed proteins were used to create an online database (SoyProLow) to provide a data repository that can be used as a reference to identify and characterize low abundance proteins. This database is freely accessible to individuals using similar techniques and can be for the subsequent genetic manipulation to produce value added soybean traits. An intuitive user interface based on dynamic HTML enables users to browse the network and the profiles of the low abundant proteins. Availability http://bioinformatics.towson.edu/Soybean_low_abundance_proteins_2D_Gel_DB/Gel1.aspx PMID:25352730

  2. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization.

    PubMed

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability.

  3. Selection of intracellular single-domain antibodies targeting the HIV-1 Vpr protein by cytoplasmic yeast two-hybrid system.

    PubMed

    Matz, Julie; Hérate, Cécile; Bouchet, Jérôme; Dusetti, Nelson; Gayet, Odile; Baty, Daniel; Benichou, Serge; Chames, Patrick

    2014-01-01

    The targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system.

  4. Selection of Intracellular Single-Domain Antibodies Targeting the HIV-1 Vpr Protein by Cytoplasmic Yeast Two-Hybrid System

    PubMed Central

    Matz, Julie; Hérate, Cécile; Bouchet, Jérôme; Dusetti, Nelson; Gayet, Odile; Baty, Daniel; Benichou, Serge; Chames, Patrick

    2014-01-01

    The targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system. PMID:25436999

  5. Nuclear and Cytoplasmic Soluble Proteins Extraction from a Small Quantity of Drosophila’s Whole Larvae and Tissues

    PubMed Central

    Lo Piccolo, Luca; Bonaccorso, Rosa; Onorati, Maria Cristina

    2015-01-01

    The identification and study of protein’s function in several model organisms is carried out using both nuclear and cytoplasmic extracts. For a long time, Drosophila’s embryos have represented the main source for protein extractions, although in the last year, the importance of collecting proteins extracts also from larval tissues has also been understood. Here we report a very simple protocol, improved by a previously developed method, to produce in a single extraction both highly stable nuclear and cytoplasmic protein extracts from a small quantity of whole Drosophila’s larvae or tissues, suitable for biochemical analyses like co-immunoprecipitation. PMID:26039237

  6. ANALYSIS OF THE FUNCTION OF CYTOPLASMIC FIBERS FORMED BY THE RUBELLA VIRUS NONSTRUCTURAL REPLICASE PROTEINS

    PubMed Central

    Matthews, Jason D.; Tzeng, Wen-Pin; Frey, Teryl K.

    2010-01-01

    The P150 and P90 replicase proteins of rubella virus (RUBV), a plus-strand RNA Togavirus, produce a unique cytoplasmic fiber network resembling microtubules. Pharmacological and mutagenic approaches were used to determine if these fibers functioned in virus replication. The pharmacological approach revealed that microtubules were required for fiber formation, but neither was necessary for virus replication. Through the mutagenic approach it was found that α-helices near both termini of P150 were necessary for fiber assembly and infectivity, but fiber formation and viability could not be correlated because most of these mutations were lethal. The N-terminal α-helix of P150 affected both proteolytic processing of P150 and P90 from the P200 precursor and targeting of P200, possibly through directing conformational folding of P200. Finally, we made the unexpected discovery that RUBV genomes can spread from cell-to-cell without virus particles, a process that we hypothesize utilizes RUBV-induced cytoplasmic projections containing fibers and replication complexes. PMID:20696450

  7. Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein

    PubMed Central

    Marina, Alberto; Waldburger, Carey D; Hendrickson, Wayne A

    2005-01-01

    The large majority of histidine kinases (HKs) are multifunctional enzymes having autokinase, phosphotransfer and phosphatase activities, and most of these are transmembrane sensor proteins. Sensor HKs possess conserved cytoplasmic phosphorylation and ATP-binding kinase domains. The different enzymatic activities require participation by one or both of these domains, implying the need for different conformational states. The catalytic domains are linked to the membrane through a coiled-coil segment that sometimes includes other domains. We describe here the first crystal structure of the complete cytoplasmic region of a sensor HK, one from the thermophile Thermotoga maritima in complex with ADPβN at 1.9 Å resolution. The structure reveals previously unidentified functions for several conserved residues and reveals the relative disposition of domains in a state seemingly poised for phosphotransfer. The structure thereby inspires hypotheses for the mechanisms of autophosphorylation, phosphotransfer and response-regulator dephosphorylation, and for signal transduction through the coiled-coil segment. Mutational tests support the functional relevance of interdomain contacts. PMID:16319927

  8. Structure of the Entire Cytoplasmic Portion of a Sensor Histidine-kinase Protein

    SciTech Connect

    Marina,A.; Waldburger, C.; Hendrickson, W.

    2005-01-01

    The large majority of histidine kinases (HKs) are multifunctional enzymes having autokinase, phosphotransfer and phosphatase activities, and most of these are transmembrane sensor proteins. Sensor HKs possess conserved cytoplasmic phosphorylation and ATP-binding kinase domains. The different enzymatic activities require participation by one or both of these domains, implying the need for different conformational states. The catalytic domains are linked to the membrane through a coiled-coil segment that sometimes includes other domains. We describe here the first crystal structure of the complete cytoplasmic region of a sensor HK, one from the thermophile Thermotoga maritima in complex with ADP{beta}N at 1.9 Angstrom resolution. The structure reveals previously unidentified functions for several conserved residues and reveals the relative disposition of domains in a state seemingly poised for phosphotransfer. The structure thereby inspires hypotheses for the mechanisms of autophosphorylation, phosphotransfer and response-regulator dephosphorylation, and for signal transduction through the coiled-coil segment. Mutational tests support the functional relevance of interdomain contacts.

  9. Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain

    PubMed Central

    1996-01-01

    The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when

  10. Cytoplasmic localization of wild-type p53 in glioblastomas correlates with expression of vimentin and glial fibrillary acidic protein.

    PubMed Central

    Sembritzki, Olivier; Hagel, Christian; Lamszus, Katrin; Deppert, Wolfgang; Bohn, Wolfgang

    2002-01-01

    Cytoplasmic accumulation of wild-type p53 in tumor cells indicates that the tumor suppressor is inactive with regard to growth suppressive functions. Whether this occurs randomly during tumor development or characterizes a certain tumor cell subset is not known. Here we assayed primary glioblastomas for expression and subcellular localization of p53 and determined a correlation with expression of intermediate filament proteins characterizing glial cell development. Sixty-nine percent of the tumors were p53 positive in immunohistochemistry. A significant number of tumors (23%) accumulated wild-type p53 in the cytoplasm, which correlated with the presence of vimentin and glial fibrillary acidic protein, except for 1 case. Tumors with exclusive nuclear p53 contained none or only one of these intermediate filament proteins. In an alternative approach, tumors positive for glial fibrillary acidic protein were screened for expression of p53 and vimentin. Thirty-eight percent of these tumors showed cytoplasmic p53, and all of those also expressed vimentin. Tumors with only nuclear p53 were vimentin negative, except for 1 case. No mutation was detected in p53 exons 5 to 8 in tumors with cytoplasmic p53, suggesting that they express wild-type p53. The data indicate that a cytoplasmic accumulation of wild-type p53 in human primary glioblastomas correlates with a certain intermediate filament protein expression, suggesting that it identifies a certain subset of tumors. PMID:12084347

  11. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.

    PubMed Central

    Ozawa, M; Baribault, H; Kemler, R

    1989-01-01

    Uvomorulin belongs to the group of Ca2+-dependent cell adhesion molecules, which are integral membrane proteins with several structural features in common. In particular, the cytoplasmic part of these proteins is highly conserved in different species, suggesting a common biological function. To test this assumption we transfected a uvomorulin full-length cDNA into uvomorulin-negative mouse NIH 3T3 and L cells. Immunoprecipitations with anti-uvomorulin antibodies detected, in addition to uvomorulin, three independent proteins of 102, 88 and 80 kd which are of host origin and which form complexes with uvomorulin. Using cDNA constructs coding for uvomorulin with cytoplasmic or extracellular deletions it is shown that the 102, 88 and 80 kd proteins complex with the cytoplasmic domain of uvomorulin. Peptide pattern analysis revealed that these three proteins are identical in different mouse cells. When uvomorulin cDNA was introduced into cell lines from other species, such as human HeLa and avian fibroblasts, the expressed uvomorulin was also associated with endogenous 102, 88 and 80 kd proteins and, moreover, each of these proteins showed structural similarities to the respective mouse molecule. A panel of antibodies specific for known cytoplasmic proteins of mol. wts similar to those of the three proteins did not react with any of the described components. This suggests that the 102, 88 and 80 kd proteins constitute a new group of proteins for which we propose the nomenclature of catenin alpha, beta and gamma respectively. The characterization of these proteins provides a first molecular basis for a possible cytoplasmic anchorage of uvomorulin to the cytoskeleton. Images PMID:2788574

  12. Relocalization of nuclear ALY proteins to the cytoplasm by the tomato bushy stunt virus P19 pathogenicity protein.

    PubMed

    Uhrig, Joachim F; Canto, Tomas; Marshall, David; MacFarlane, Stuart A

    2004-08-01

    The P19 protein of tomato bushy stunt virus (TBSV) is a multifunctional pathogenicity determinant involved in suppression of posttranscriptional gene silencing, virus movement, and symptom induction. Here, we report that P19 interacts with the conserved RNA-binding domain of an as yet uncharacterized family of plant ALY proteins that, in animals, are involved in export of RNAs from the nucleus and transcriptional coactivation. We show that the four ALY proteins encoded by the Arabidopsis genome and two ALY proteins from Nicotiana benthamiana are localized to the nucleus. Moreover, and in contrast to animal ALY, all but one of the proteins are also in the nucleolus, with distinct subnuclear localizations. Infection of plants by TBSV or expression of P19 from Agrobacterium results in relocation of three of the six ALY proteins from the nucleus to the cytoplasm demonstrating specific targeting of the ALY proteins by P19. The differential effects on subcellular localization indicate that, in plants, the various ALY proteins may have different functions. Interaction with and relocalization of ALY is prevented by mutation of P19 at residues previously shown to be important for P19 function in plants. Down-regulation of expression of two N. benthamiana ALY genes by virus-induced gene silencing did not interfere with posttranscriptional gene silencing. Targeting of ALY proteins during TBSV infection may therefore be related to functions of P19 in addition to its silencing suppression activity. PMID:15299117

  13. Expression of aquaporin1, a water channel protein, in cytoplasm is negatively correlated with prognosis of breast cancer patients

    PubMed Central

    Shao, Ying; Liu, Xiaoli; Yang, Limin; Huang, Yong; Fu, Li; Gu, Feng; Ma, Yongjie

    2016-01-01

    Aquaporin1 (AQP1) belongs to a highly conserved family of aquaporin proteins which facilitate water flux across cell membranes. Although emerging evidences indicated the cytoplasm was important for AQP1 localization, the function of AQP1 corresponding to its cytoplasmic distribution has rarely been explored until present. In our clinical study, we reported for the first time that AQP1 was localized dominantly in the cytoplasm of cancer cells of invasive breast cancer patients and cytoplasmic AQP1 was an independent prognostic factor. High expression of AQP1 indicated a shorter survival, especially in luminal subtype. Moreover, in line with our findings in clinic, cytoplasmic expression of AQP1 was further validated in both primary cultured breast cancer cells and AQP1 over-expressing cell lines, in which the functional importance of cytoplasmic AQP1 was confirmed in vitro. In conclusion, our study provided the first evidence that cytoplasmic expression of AQP1 promoted breast cancer progression and it could be a potential prognostic biomarker for breast cancer. PMID:26812884

  14. An Abundant, Highly Conserved Tonoplast Protein in Seeds 1

    PubMed Central

    Johnson, Kenneth D.; Herman, Eliot M.; Chrispeels, Maarten J.

    1989-01-01

    We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with Mr 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:16667102

  15. The Cytoplasmic Adaptor Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization

    SciTech Connect

    Bergamin, E.; Hallock, P; Burden, S; Hubbard, S

    2010-01-01

    Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.

  16. MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion.

    PubMed

    Oswald, Felix; Varadarajan, Aravindan; Lill, Holger; Peterman, Erwin J G; Bollen, Yves J M

    2016-03-01

    The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs. PMID:26958890

  17. Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane

    SciTech Connect

    Kranz, R.G.; Gennis, R.B.

    1982-04-01

    Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100. Two bands in IEF stained for heme with pI values of 4.7 and 5.3. One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylenediamine (TMPD) oxidase activity. The pI 4.7 band was present in cells harvested in both mid-log and stationary phases. Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component. The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study. The component which was present in cells grown to stationary phase contained hemes b, a/sub 1/, and d. The other two fractions contained only b heme. One of these corresponded to the component with pI 4.7 in IEF and had catalase activity. Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex. With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme. Three of these corresponded to the components identified by IEF and agarose electrophoresis. We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins. In particular, this work represents a substantial initial step toward a structural elucidation of the E. coli aerobic respiratory chain.

  18. Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

    PubMed Central

    Guan, J L; Ruusala, A; Cao, H; Rose, J K

    1988-01-01

    Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane. Images PMID:2841589

  19. High-molecular-mass, iron-repressed cytoplasmic proteins in fluorescent Pseudomonas: potential peptide-synthetases for pyoverdine biosynthesis.

    PubMed

    Georges, C; Meyer, J M

    1995-10-01

    High molecular-mass cytoplasmic proteins were detected in iron-starved, pyoverdine-producing Pseudomonas aeruginosa, P. chlororaphis, P. fluorescens, P. putida, P. aptata and P. tolaasii. They appeared to be specifically located in the cytoplasm and thus were termed 'IRCPs', for iron-repressed cytoplasmic proteins. A strain-dependent gel electrophoresis pattern with multiple bands of M(r) values ranging from 180 to 600 kDa was usually observed for these proteins. Strains synthesizing pyoverdines differing in their peptide part presented different IRCP gel electrophoresis profiles, whereas strains synthesizing identical pyoverdines had identical IRCP gel electrophoresis profiles. Some mutants affected in pyoverdine biosynthesis presented a perturbed IRCP pattern, and no IRCPs were detected in non-fluorescent Pseudomonas strains either unable to synthesize siderophores or synthesizing non-peptidic siderophores. The data strongly suggest that the IRCPs could be related to peptide synthetases involved in the biosynthesis of the peptidic part of pyoverdine-type siderophores. PMID:7590169

  20. Optimized RNA gel-shift and UV cross-linking assays for characterization of cytoplasmic RNA-protein interactions.

    PubMed

    Thomson, A M; Rogers, J T; Walker, C E; Staton, J M; Leedman, P J

    1999-11-01

    Considerable interest has recently focused on defining the mechanisms involved in the regulation of gene expression at the level of mRNA stability and translational efficiency. However, the assays used to directly investigate interactions between RNA and cytoplasmic proteins have been difficult to establish, and methods are not widely available. Here, we describe a robust method for RNA electrophoretic mobility shift and UV cross-linking assays that allows rapid detection of cytoplasmic RNA-protein interactions. For added convenience to new investigators, these assays use mini-gels with an electrophoresis time of 15-20 min, enabling a high throughput of samples. The method works successfully with many different probes and cytoplasmic extracts from a variety of cell lines. Furthermore, we provide a system to optimize characterization of the RNA-protein complex and troubleshoot most assay difficulties.

  1. The CMS-associated 16 kDa protein encoded by orfH522 in the PET1 cytoplasm is also present in other male-sterile cytoplasms of sunflower.

    PubMed

    Horn, R; Hustedt, J E; Horstmeyer, A; Hahnen, J; Zetsche, K; Friedt, W

    1996-02-01

    In sunflower plants carrying the PET1 cytoplasm male sterility (CMS) is associated with a new open reading frame (orfH522) in the 3'-flanking region of the atpA gene and an additional 16 kDa protein. Twenty-seven male-sterile cytoplasms of different origin were studied for the expression of the 16 kDa protein. In addition to the PET1 cytoplasm nine other male-sterile cytoplasms express the CMS-associated protein. These CMS sources originate from different interspecific crosses, from spontaneously occurring male-sterile plants in wild sunflower and from induced mutagenesis. Polyclonal antisera were raised against fusion proteins which contain 421 bp of the 3'-coding region of orfH522 to verify by immunological methods the identity of the other CMS cytoplasms. The anti-ORFH522 antiserum showed a positive reaction in the immunoblot with all CMS cytoplasms which expressing the 16 kDa protein. Investigations of the mitochondrial DNA demonstrated that all ten CMS cytoplasms which express the 16 kDa protein have the same organization at the atpA locus. OrfH522 as probes gave the same transcript pattern for the investigated CMS cytoplasms, just as for PET1. The MAX1 cytoplasm has an orfH522-related sequence but does not synthesize the 16 kDa protein. Using the sodium carbonate treatment the 16 kDa protein proved to be membrane-bound. Computer analyses predict that the hydrophobic N-terminal region of ORFH522 may form a transmembrane helix functioning as membrane anchor. PMID:8605303

  2. An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

    PubMed

    Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

    2016-01-01

    A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

  3. Downregulation of human immunodeficiency virus type 1 Gag expression by a gp41 cytoplasmic domain fusion protein

    SciTech Connect

    Chan, W.-E.; Chen, Steve S.-L. . E-mail: schen@ibms.sinica.edu.tw

    2006-05-10

    The cytoplasmic domain of human immunodeficiency virus type 1 (HIV-1) envelope (Env) transmembrane protein gp41 interacts with the viral matrix MA protein, which facilitates incorporation of the trimeric Env complex into the virus. It is thus feasible to design an anti-HIV strategy targeting this interaction. We herein describe that Gag expression can be downregulated by a cytoplasmic domain fusion protein of the Env transmembrane protein, {beta}-galactosidase ({beta}-gal)/706-856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli {beta}-gal. This mediator depleted intracellular Gag molecules in a dose-dependent manner. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag and {beta}-gal/706-856 had stable interactions and formed aggregated complexes in perinuclear, intracellular sites. Pulse-chase and cycloheximide chase analyses demonstrated that this mediator enhanced unmyristylated Gag degradation. The results demonstrate a novel mode of HIV-1 Gag downregulation by directing Gag to an intracellular site via the interaction of Gag with a gp41 cytoplasmic domain fusion protein.

  4. Identification of adducin-binding residues on the cytoplasmic domain of erythrocyte membrane protein, band 3.

    PubMed

    Franco, Taina; Chu, Haiyan; Low, Philip S

    2016-10-01

    Two major complexes form structural bridges that connect the erythrocyte membrane to its underlying spectrin-based cytoskeleton. Although the band 3-ankyrin bridge may account for most of the membrane-to-cytoskeleton interactions, the linkage between the cytoplasmic domain of band 3 (cdb3) and adducin has also been shown to be critical to membrane integrity. In the present paper, we demonstrate that adducin, a major component of the spectrin-actin junctional complex, binds primarily to residues 246-264 of cdb3, and mutation of two exposed glutamic acid residues within this sequence completely abrogates both α- and β-adducin binding. Because these residues are located next to the ankyrin-binding site on cdb3, it seems unlikely that band 3 can bind ankyrin and adducin concurrently, reducing the chances of an association between the ankyrin and junctional complexes that would significantly compromise erythrocyte membrane integrity. We also demonstrate that adducin binds the kidney isoform of cdb3, a spliceoform that lacks the first 65 amino acids of erythrocyte cdb3, including the central strand of a large β-pleated sheet. Because kidney cdb3 is not known to bind any of the common peripheral protein partners of erythrocyte cdb3, including ankyrin, protein 4.1, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and phosphofructokinase, retention of this affinity for adducin was unexpected.

  5. Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems.

    PubMed

    Siwaszek, Aleksandra; Ukleja, Marta; Dziembowski, Andrzej

    2014-01-01

    The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5'-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3'>5'direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals.

  6. Characterization, occurrence, and molecular cloning of a 39-kilodalton Brucella abortus cytoplasmic protein immunodominant in cattle.

    PubMed Central

    Denoel, P A; Vo, T K; Tibor, A; Weynants, V E; Trunde, J M; Dubray, G; Limet, J N; Letesson, J J

    1997-01-01

    Monoclonal antibodies and polyclonal antisera recognizing a 39-kDa protein (P39) of brucellin, a cytoplasmic extract from Brucella melitensis rough strain B115, were produced. The P39 was purified by anion-exchange chromatography. Eleven of fourteen Brucella-infected cows whose infections had been detected by the delayed-type hypersensitivity (DTH) test with brucellergen also developed a DTH reaction when purified P39 was used as the trigger. The T-cell proliferative responses to P39 of peripheral blood lymphocytes from Brucella-infected cows were also positive. None of the animals infected with other bacterial species that are presumed to induce immunological cross-reactions with Brucella spp. reacted to P39, either in DTH tests or in lymphocyte proliferation assays. A lambda gt11 genomic library of Brucella abortus was screened with a monoclonal antibody specific for P39, and the gene coding for this protein was subsequently isolated. The nucleotide sequence of the P39 gene was determined, and the deduced amino acid sequence is in accordance with the sequence of an internal peptide isolated from P39. PMID:9009303

  7. Cytoplasmic dynein and its regulatory proteins in Golgi pathology in nervous system disorders.

    PubMed

    Jaarsma, Dick; Hoogenraad, Casper C

    2015-01-01

    The Golgi apparatus is a dynamic organelle involved in processing and sorting of lipids and proteins. In neurons, the Golgi apparatus is important for the development of axons and dendrites and maintenance of their highly complex polarized morphology. The motor protein complex cytoplasmic dynein has an important role in Golgi apparatus positioning and function. Together, with dynactin and other regulatory factors it drives microtubule minus-end directed motility of Golgi membranes. Inhibition of dynein results in fragmentation and dispersion of the Golgi ribbon in the neuronal cell body, resembling the Golgi abnormalities observed in some neurodegenerative disorders, in particular motor neuron diseases. Mutations in dynein and its regulatory factors, including the dynactin subunit p150Glued, BICD2 and Lis-1, are associated with several human nervous system disorders, including cortical malformation and motor neuropathy. Here we review the role of dynein and its regulatory factors in Golgi function and positioning, and the potential role of dynein malfunction in causing Golgi apparatus abnormalities in nervous system disorders. PMID:26578860

  8. Membrane protein assembly: two cytoplasmic phosphorylated serine sites of Vpu from HIV-1 affect oligomerization

    PubMed Central

    Chen, Chin-Pei; Lin, Meng-Han; Chan, Ya-Ting; Chen, Li-Chyong; Ma, Che; Fischer, Wolfgang B.

    2016-01-01

    Viral protein U (Vpu) encoded by human immunodeficiency virus type 1 (HIV-1) is a short integral membrane protein which is known to self-assemble within the lipid membrane and associate with host factors during the HIV-1 infectivity cycle. In this study, full-length Vpu (M group) from clone NL4-3 was over-expressed in human cells and purified in an oligomeric state. Various single and double mutations were constructed on its phosphorylation sites to mimic different degrees of phosphorylation. Size exclusion chromatography of wild-type Vpu and mutants indicated that the smallest assembly unit of Vpu was a dimer and over time Vpu formed higher oligomers. The rate of oligomerization increased when (i) the degree of phosphorylation at serines 52 and 56 was decreased and (ii) when the ionic strength was increased indicating that the cytoplasmic domain of Vpu affects oligomerization. Coarse-grained molecular dynamic simulations with models of wild-type and mutant Vpu in a hydrated lipid bilayer supported the experimental data in demonstrating that, in addition to a previously known role in downregulation of host factors, the phosphorylation sites of Vpu also modulate oligomerization. PMID:27353136

  9. GNL3L Is a Nucleo-Cytoplasmic Shuttling Protein: Role in Cell Cycle Regulation.

    PubMed

    Thoompumkal, Indu Jose; Subba Rao, Malireddi Rama Krishna; Kumaraswamy, Anbarasu; Krishnan, Rehna; Mahalingam, Sundarasamy

    2015-01-01

    GNL3L is an evolutionarily conserved high molecular weight GTP binding nucleolar protein belonging to HSR1-MMR1 subfamily of GTPases. The present investigation reveals that GNL3L is a nucleo-cytoplasmic shuttling protein and its export from the nucleus is sensitive to Leptomycin B. Deletion mutagenesis reveals that the C-terminal domain (amino acids 501-582) is necessary and sufficient for the export of GNL3L from the nucleus and the exchange of hydrophobic residues (M567, L570 and 572) within the C-terminal domain impairs this process. Results from the protein-protein interaction analysis indicate that GNL3L interaction with CRM1 is critical for its export from the nucleus. Ectopic expression of GNL3L leads to lesser accumulation of cells in the 'G2/M' phase of cell cycle whereas depletion of endogenous GNL3L results in 'G2/M' arrest. Interestingly, cell cycle analysis followed by BrdU labeling assay indicates that significantly increased DNA synthesis occurs in cells expressing nuclear export defective mutant (GNL3L∆NES) compared to the wild type or nuclear import defective GNL3L. Furthermore, increased hyperphosphorylation of Rb at Serine 780 and the upregulation of E2F1, cyclins A2 and E1 upon ectopic expression of GNL3L∆NES results in faster 'S' phase progression. Collectively, the present study provides evidence that GNL3L is exported from the nucleus in CRM1 dependent manner and the nuclear localization of GNL3L is important to promote 'S' phase progression during cell proliferation.

  10. New Proteins Involved in Sulfur Trafficking in the Cytoplasm of Allochromatium vinosum*

    PubMed Central

    Stockdreher, Yvonne; Sturm, Marga; Josten, Michaele; Sahl, Hans-Georg; Dobler, Nadine; Zigann, Renate; Dahl, Christiane

    2014-01-01

    The formation of periplasmic sulfur globules is an intermediate step during the oxidation of reduced sulfur compounds in various sulfur-oxidizing microorganisms. The mechanism of how this sulfur is activated and crosses the cytoplasmic membrane for further oxidation to sulfite by the dissimilatory reductase DsrAB is incompletely understood, but it has been well documented that the pathway involves sulfur trafficking mediated by sulfur-carrying proteins. So far sulfur transfer from DsrEFH to DsrC has been established. Persulfurated DsrC very probably serves as a direct substrate for DsrAB. Here, we introduce further important players in oxidative sulfur metabolism; the proteins Rhd_2599, TusA, and DsrE2 are strictly conserved in the Chromatiaceae, Chlorobiaceae, and Acidithiobacillaceae families of sulfur-oxidizing bacteria and are linked to genes encoding complexes involved in sulfur oxidation (Dsr or Hdr) in the latter two. Here we show via relative quantitative real-time PCR and microarray analysis an increase of mRNA levels under sulfur-oxidizing conditions for rhd_2599, tusA, and dsrE2 in Allochromatium vinosum. Transcriptomic patterns for the three genes match those of major genes for the sulfur-oxidizing machinery rather than those involved in biosynthesis of sulfur-containing biomolecules. TusA appears to be one of the major proteins in A. vinosum. A rhd_2599-tusA-dsrE2-deficient mutant strain, although not viable in liquid culture, was clearly sulfur oxidation negative upon growth on solid media containing sulfide. Rhd_2599, TusA, and DsrE2 bind sulfur atoms via conserved cysteine residues, and experimental evidence is provided for the transfer of sulfur between these proteins as well as to DsrEFH and DsrC. PMID:24648525

  11. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    SciTech Connect

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  12. Abundant protein phosphorylation potentially regulates Arabidopsis anther development

    PubMed Central

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-01-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana. However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4–7 and 8–12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  13. Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

    PubMed

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-09-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  14. Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition.

    PubMed

    Mishima, Masaki; Maesaki, Ryoko; Kasa, Miyuki; Watanabe, Takashi; Fukata, Masaki; Kaibuchi, Kozo; Hakoshima, Toshio

    2007-06-19

    Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end. PMID:17563362

  15. The adenovirus E4 11 k protein binds and relocalizes the cytoplasmic P-body component Ddx6 to aggresomes

    SciTech Connect

    Greer, Amy E.; Hearing, Patrick; Ketner, Gary

    2011-08-15

    The adenovirus E4 11 k protein, product of E4 ORF3, is required in infection for processes including normal accumulation of viral late mRNAs. 11 k restructures both the nucleus and cytoplasm of infected cells by relocalizing specific host cell target proteins, most strikingly components of nuclear PML oncogenic domains. It is likely that in many cases relocalization inactivates target proteins to produce 11 k's effects, although the mechanism and targets for stimulation of late mRNA accumulation is unknown. We have identified a new set of proteins relocalized by 11 k: at least five protein components of cytoplasmic mRNA processing bodies (p-bodies) are found in 11 k-induced cytoplasmic aggresomes, sites where proteins are inactivated or destroyed. One of these p-body proteins, RNA helicase Ddx6, binds 11 k, suggesting a mechanism for relocalization. Because p-bodies are sites for mRNA degradation, their modification by 11 k may provide an explanation for the role of 11 k in viral late mRNA accumulation.

  16. Topology of Protein Interaction Network Shapes Protein Abundances and Strengths of Their Functional and Nonspecific Interactions

    SciTech Connect

    Maslov, S.; Heo, M.; Shakhnovich, E.

    2011-03-08

    How do living cells achieve sufficient abundances of functional protein complexes while minimizing promiscuous nonfunctional interactions? Here we study this problem using a first-principle model of the cell whose phenotypic traits are directly determined from its genome through biophysical properties of protein structures and binding interactions in a crowded cellular environment. The model cell includes three independent prototypical pathways, whose topologies of protein-protein interaction (PPI) subnetworks are different, but whose contributions to the cell fitness are equal. Model cells evolve through genotypic mutations and phenotypic protein copy number variations. We found a strong relationship between evolved physical-chemical properties of protein interactions and their abundances due to a 'frustration' effect: Strengthening of functional interactions brings about hydrophobic interfaces, which make proteins prone to promiscuous binding. The balancing act is achieved by lowering concentrations of hub proteins while raising solubilities and abundances of functional monomers. On the basis of these principles we generated and analyzed a possible realization of the proteome-wide PPI network in yeast. In this simulation we found that high-throughput affinity capture-mass spectroscopy experiments can detect functional interactions with high fidelity only for high-abundance proteins while missing most interactions for low-abundance proteins.

  17. Human Dectin-1 isoform E is a cytoplasmic protein and interacts with RanBPM.

    PubMed

    Xie, Jianhui; Sun, Maoyun; Guo, Liang; Liu, Weicheng; Jiang, Jianhai; Chen, Xiaoning; Zhou, Lei; Gu, Jianxin

    2006-09-01

    Human Dectin-1, a type II transmembrane receptor, is alternatively spliced, generating eight isoforms. Of these isoforms, the isoform E (hDectin-1E) is structurally unique, containing a complete C-type lectin-like domain as well as an ITAM-like sequence. So far, little is known about its function. In the present study, we demonstrated that hDectin-1E was not secreted and it mainly resided in the cytoplasm. Using yeast two-hybrid screening, we identified a Ran-binding protein, RanBPM, as an interacting partner of hDectin-1E. GST pull-down assays showed that RanBPM interacted directly with hDectin-1E and the region containing SPRY domain was sufficient for the interaction. The binding of hDectin-1E and RanBPM was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Taken together, our data provide a clue to the understanding of the function about hDectin-1E. PMID:16870151

  18. Detection of Antibodies to Brucella Cytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis

    PubMed Central

    Baldi, Pablo C.; Araj, George F.; Racaro, Graciela C.; Wallach, Jorge C.; Fossati, Carlos A.

    1999-01-01

    The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of Brucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis. PMID:10473531

  19. Identification of cytoplasmic proteins interacting with unliganded estrogen receptor α and β in human breast cancer cells.

    PubMed

    Stellato, Claudia; Nassa, Giovanni; Tarallo, Roberta; Giurato, Giorgio; Ravo, Maria; Rizzo, Francesca; Marchese, Giovanna; Alexandrova, Elena; Cordella, Angela; Baumann, Marc; Nyman, Tuula A; Weisz, Alessandro; Ambrosino, Concetta

    2015-06-01

    Estrogen receptor subtypes (ERα and ERβ) are transcription factors sharing a similar structure but exerting opposite roles in breast cancer cells. Besides the well-characterized genomic actions of nuclear ERs upon ligand binding, specific actions of ligand-free ERs in the cytoplasm also affect cellular functions. The identification of cytoplasmic interaction partners of unliganded ERα and ERβ may help characterize the molecular basis of the extra-nuclear mechanism of action of these receptors, revealing novel mechanisms to explain their role in breast cancer response or resistance to endocrine therapy. To this aim, cytoplasmic extracts from human breast cancer MCF-7 cells stably expressing tandem affinity purification-tagged ERα and ERβ and maintained in estrogen-free medium were subject to affinity-purification and MS analysis, leading to the identification of 84 and 142 proteins associated with unliganded ERα and ERβ, respectively. Functional analyses of ER subtype-specific interactomes revealed significant differences in the molecular pathways targeted by each receptor in the cytoplasm. This work, reporting the first identification of the unliganded ERα and ERβ cytoplasmic interactomes in breast cancer cells, provides novel experimental evidence on the nongenomic effects of ERs in the absence of hormonal stimulus. All MS data have been deposited in the ProteomeXchange with identifier PXD001202 (http://proteomecentral.proteomexchange.org/dataset/PXD001202). PMID:25604459

  20. Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

    PubMed Central

    Autieri, M V; Fresa, K L; Coffman, F D; Katz, M E; Cohen, S

    1990-01-01

    We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein. PMID:1725128

  1. Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

    PubMed

    Autieri, M V; Fresa, K L; Coffman, F D; Katz, M E; Cohen, S

    1990-12-01

    We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein. PMID:1725128

  2. Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

    PubMed Central

    Li, Shumin; Chakraborty, Nilay; Borcar, Apurva; Menze, Michael A.; Toner, Mehmet; Hand, Steven C.

    2012-01-01

    Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporter was used for intracellular loading of this disaccharide. Cells were rapidly and uniformly desiccated to low water content (<0.12 g H2O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, compared with 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state. PMID:23185012

  3. Cortical Polarity of the RING Protein PAR-2 Is Maintained by Exchange Rate Kinetics at the Cortical-Cytoplasmic Boundary.

    PubMed

    Arata, Yukinobu; Hiroshima, Michio; Pack, Chan-Gi; Ramanujam, Ravikrishna; Motegi, Fumio; Nakazato, Kenichi; Shindo, Yuki; Wiseman, Paul W; Sawa, Hitoshi; Kobayashi, Tetsuya J; Brandão, Hugo B; Shibata, Tatsuo; Sako, Yasushi

    2016-08-23

    Cell polarity arises through the spatial segregation of polarity regulators. PAR proteins are polarity regulators that localize asymmetrically to two opposing cortical domains. However, it is unclear how the spatially segregated PAR proteins interact to maintain their mutually exclusive partitioning. Here, single-molecule detection analysis in Caenorhabditis elegans embryos reveals that cortical PAR-2 diffuses only short distances, and, as a result, most PAR-2 molecules associate and dissociate from the cortex without crossing into the opposing domain. Our results show that cortical PAR-2 asymmetry is maintained by the local exchange reactions that occur at the cortical-cytoplasmic boundary. Additionally, we demonstrate that local exchange reactions are sufficient to maintain cortical asymmetry in a parameter-free mathematical model. These findings suggest that anterior and posterior PAR proteins primarily interact through the cytoplasmic pool and not via cortical diffusion. PMID:27524610

  4. The Extracellular Matrix of Staphylococcus aureus Biofilms Comprises Cytoplasmic Proteins That Associate with the Cell Surface in Response to Decreasing pH

    PubMed Central

    Foulston, Lucy; Elsholz, Alexander K. W.; DeFrancesco, Alicia S.

    2014-01-01

    ABSTRACT Biofilm formation by Staphylococcus aureus involves the formation of an extracellular matrix, but the composition of this matrix has been uncertain. Here we report that the matrix is largely composed of cytoplasmic proteins that reversibly associate with the cell surface in a manner that depends on pH. We propose a model for biofilm formation in which cytoplasmic proteins are released from cells in stationary phase. These proteins associate with the cell surface in response to decreasing pH during biofilm formation. Rather than utilizing a dedicated matrix protein, S. aureus appears to recycle cytoplasmic proteins that moonlight as components of the extracellular matrix. PMID:25182325

  5. Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A*

    PubMed Central

    Svozil, Julia; Hirsch-Hoffmann, Matthias; Dudler, Robert; Gruissem, Wilhelm; Baerenfaller, Katja

    2014-01-01

    As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures

  6. Functional significance of a protein conformation change at the cytoplasmic end of helix F during the bacteriorhodopsin photocycle.

    PubMed

    Brown, L S; Váró, G; Needleman, R; Lanyi, J K

    1995-11-01

    The second half of the photocycle of the light-driven proton pump bacteriorhodopsin includes proton transfers between D96 and the retinal Schiff base (the M to N reaction) and between the cytoplasmic surface and D96 (decay of the N intermediate). The inhibitory effects of decreased water activity and increased hydrostatic pressure have suggested that a conformational change resulting in greater hydration of the cytoplasmic region is required for proton transfer from D96 to the Schiff base, and have raised the possibility that the reversal of this process might be required for the subsequent reprotonation of D96 from the cytoplasmic surface. Tilt of the cytoplasmic end of helix F has been suggested by electron diffraction of the M intermediate. Introduction of bulky groups, such as various maleimide labels, to engineered cysteines at the cytoplasmic ends of helices A, B, C, E, and G produce only minor perturbation of the decays of M and N, but major changes in these reactions when the label is linked to helix F. In these samples the reprotonation of the Schiff base is accelerated and the reprotonation of D96 is strongly retarded. Cross-linking with benzophenone introduced at this location, but not at the others, causes the opposite change: the reprotonation of the Schiff base is greatly slowed while the reprotonation of D96 is accelerated. We conclude that, consistent with the structure from diffraction, the proton transfers in the second half of the photocycle are facilitated by motion of the cytoplasmic end of helix F, first away from the center of the protein and then back.

  7. Evidence that fungal MEP proteins mediate diffusion of the uncharged species NH(3) across the cytoplasmic membrane.

    PubMed

    Soupene, E; Ramirez, R M; Kustu, S

    2001-09-01

    Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH(4)(+) + NH(3)). Transport and accumulation of the ammonium analog [(14)C]methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH(4)(+). In bacteria, however, ATP-dependent conversion of methylammonium to gamma-N-methylglutamine by glutamine synthetase precludes its use in assessing concentrative transport across the cytoplasmic membrane. We have confirmed that methylammonium is not metabolized in the yeast S. cerevisiae and have shown that it is little metabolized in the filamentous fungus Neurospora crassa. However, its accumulation depends on the energy-dependent acidification of vacuoles. A Deltavph1 mutant of S. cerevisiae and a Deltavma1 mutant, which lack vacuolar H(+)-ATPase activity, had large (fivefold or greater) defects in the accumulation of methylammonium, with little accompanying defect in the initial rate of transport. A vma-1 mutant of N. crassa largely metabolized methylammonium to methylglutamine. Thus, in fungi as in bacteria, subsequent energy-dependent utilization of methylammonium precludes its use in assessing active transport across the cytoplasmic membrane. The requirement for a proton gradient to sequester the charged species CH(3)NH(3)(+) in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH(3)NH(2). By inference, their natural substrate is NH(3), a gas. We postulate that MEP proteins facilitate diffusion of NH(3) across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO(2).

  8. Phosphorylation of synaptosomal cytoplasmic proteins: Inhibition of calcium-activated, phospholipid-dependent protein kinase (protein kinase c) by bay k 8644.

    PubMed

    Robinson, P J; Lovenberg, W

    1988-01-01

    The phosphorylation of specific substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was examined in striatal synaptosomal cytoplasm. The phosphoprotein substrata were termed group C phosphoprotems and were divided into two subgroups: group C(1) phosphoproteins (P83, P45A, P21 and P18) were found in both cytoplasm and synaptosomal membranes and, although stimulated by phosphatidylserine, only required exogamous calcium for their labeling; group C(2) phosphoproteins (P120, P96, P21.5, P18.5 and P16) were found predominantly in the cytoplasm and were absolutely dependent upon exogenous calcium and phosphatidylserme for their labeling. Several criteria were used to identify these proteins as specific protein kinase C substrates: (a) their phosphorylation was stimulated to a greater extent by Ca(2+) /phosphatidylserine/diolein than by Ca(2+) alone or Cal(2+) /calmodulin (group C(1)) or was completely dependent upon Ca(2+) /phosphatdylserine/diolein (group C(2)); (b) supermaximal concentrations of the cAMP-dependent protein kinase inhibitor were without effect; (c) their phosphorylation was stimulated by oleic acid, which selectively activates protein kinase C in the absence of Ca(2+); (d) NaCl, which inhibited cAMP- and Ca(2+)/calmodulindependent phosphorylation, slightly increased phosphorylation of group C(1) and slightly decreased phosphorylation of group C(2) phosphoproteins. Maximal phosphorylation of P96 and other group C phosphoproteins occurred within 60 s and was followed by a slow decay rate while substrata of calmodulin-dependent protein kinase were maximally labeled within 20-30 s and rapidly dephosphorylated. The phosphorylation of all group C phosphoproteins was inhibited by the calcium channel agomst BAY K 8644, however, group C(2) phosphoproteins were considerably more sensitive. The IC(50) for inhibition of P96 labeling was 19 ?M. but for P83 was 190 ?M. Group B phosphoproteins were also slightly inhibited, and the

  9. Practical Immunoaffinity-Enrichment LC-MS for Measuring Protein Kinetics of Low-Abundance Proteins

    PubMed Central

    Lassman, Michael E.; McAvoy, Thomas; Lee, Anita Y.H.; Chappell, Derek; Wong, Oitak; Zhou, Haihong; Reyes-Soffer, Gissette; Ginsberg, Henry N.; Millar, John S.; Rader, Daniel J.; Gutstein, David E.; Laterza, Omar

    2016-01-01

    BACKGROUND For a more complete understanding of pharmacodynamic, metabolic, and pathophysiologic effects, protein kinetics, such as production rate and fractional catabolic rate, can offer substantially more information than protein concentration alone. Kinetic experiments with stable isotope tracers typically require laborious sample preparation and are most often used for studying abundant proteins. Here we describe a practical methodology for measuring isotope enrichment into low-abundance proteins that uses an automated procedure and immunoaffinity enrichment (IA) with LC-MS. Low-abundance plasma proteins cholesteryl ester transfer protein (CETP) and proprotein convertase subtilisin/kexin type 9 (PCSK9) were studied as examples. METHODS Human participants (n = 39) were infused with [2H3]leucine, and blood samples were collected at multiple time points. Sample preparation and analysis were automated and multiplexed to increase throughput. Proteins were concentrated from plasma by use of IA and digested with trypsin to yield proteotypic peptides that were analyzed by microflow chromatography-mass spectrometry to measure isotope enrichment. RESULTS The IA procedure was optimized to provide the greatest signal intensity. Use of a gel-free method increased throughput while increasing the signal. The intra- and interassay CVs were <15% at all isotope enrichment levels studied. More than 1400 samples were analyzed in <3 weeks without the need for instrument stoppages or user interventions. CONCLUSIONS The use of automated gel-free methods to multiplex the measurement of isotope enrichment was applied to the low-abundance proteins CETP and PCSK9. PMID:24751376

  10. Live cell cytoplasm staining and selective labeling of intracellular proteins by non-toxic cell-permeant thiophene fluorophores.

    PubMed

    Di Maria, F; Palamà, I E; Baroncini, M; Barbieri, A; Bongini, A; Bizzarri, R; Gigli, G; Barbarella, G

    2014-03-14

    A structurally correlated series of cell-permeant thiophene fluorophores, characterized by intense green or red fluorescence inside live mouse embryonic fibroblasts, was developed. The fluorophores displayed rapid internalization, excellent retention inside the cells, and high optical stability in the cytosolic environment and did not alter cell viability and reproducibility. Depending on the molecular structure, they experienced distinct fate inside the cells: from bright and lasting staining of the cytoplasm to selective tagging of a small set of globular proteins.

  11. Cytoplasmic sequestration of FUS/TLS associated with ALS alters histone marks through loss of nuclear protein arginine methyltransferase 1.

    PubMed

    Tibshirani, Michael; Tradewell, Miranda L; Mattina, Katie R; Minotti, Sandra; Yang, Wencheng; Zhou, Hongru; Strong, Michael J; Hayward, Lawrence J; Durham, Heather D

    2015-02-01

    Mutations in the RNA-binding protein FUS/TLS (FUS) have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Although predominantly nuclear, this heterogenous nuclear ribonuclear protein (hnRNP) has multiple functions in RNA processing including intracellular trafficking. In ALS, mutant or wild-type (WT) FUS can form neuronal cytoplasmic inclusions. Asymmetric arginine methylation of FUS by the class 1 arginine methyltransferase, protein arginine methyltransferase 1 (PRMT1), regulates nucleocytoplasmic shuttling of FUS. In motor neurons of primary spinal cord cultures, redistribution of endogenous mouse and that of ectopically expressed WT or mutant human FUS to the cytoplasm led to nuclear depletion of PRMT1, abrogating methylation of its nuclear substrates. Specifically, hypomethylation of arginine 3 of histone 4 resulted in decreased acetylation of lysine 9/14 of histone 3 and transcriptional repression. Distribution of neuronal PRMT1 coincident with FUS also was detected in vivo in the spinal cord of FUS(R495X) transgenic mice. However, nuclear PRMT1 was not stable postmortem obviating meaningful evaluation of ALS autopsy cases. This study provides evidence for loss of PRMT1 function as a consequence of cytoplasmic accumulation of FUS in the pathogenesis of ALS, including changes in the histone code regulating gene transcription.

  12. A Protein Allergen Microarray Detects Specific IgE to Pollen Surface, Cytoplasmic, and Commercial Allergen Extracts

    PubMed Central

    Vigh-Conrad, Katinka A.; Conrad, Donald F.; Preuss, Daphne

    2010-01-01

    Background Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens. Methodology/Principal Findings We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences. Conclusions/Significance These results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time

  13. Differential effects of depleting agents on cytoplasmic and nuclear non-protein sulphydryls: a fluorescence image cytometry study.

    PubMed Central

    Thomas, M.; Nicklee, T.; Hedley, D. W.

    1995-01-01

    The intracellular distribution of glutathione (GSH) was measured by a quantitative image cytometry method, using the sulphydryl-reactive agent mercury orange. This readily forms fluorescent adducts with GSH and other non-protein sulphydryls (NPSH), but reacts much more slowly with protein sulphydryls. Under optimum staining conditions mean integrated mercury orange fluorescence per cell was closely correlated with a standard biochemical assay for GSH. Use of the DNA dye DAPI as a counterstain allowed measurement of nuclear NPSH. The mean nuclear-cytoplasmic ratio was 0.57 +/- 0.05. Isolation of nuclei under aqueous conditions resulted in the loss of approximately 90% of mercury orange fluorescence, compared with nuclear fluorescence from intact cells, suggesting that background labelling of protein sulphydryls or other macromolecules is low. Depletion of GSH with N-ethylmaleimide or diethylmaleate decreased mercury orange fluorescence in the nucleus and cytoplasm to a similar extent. In contrast, mercury orange fluorescence in the nucleus was much more resistant to DL-buthionine-S,R-sulphoximine (BSO) depletion than that in the cytoplasm. This finding is compatible with a distinct pool of GSH in the nucleus that is comparatively resistant to BSO depletion. Alternatively, the retention of fluorescence in the nucleus following GSH depletion by BSO treatment might be due to accumulation of cysteine. These findings have implications for cancer treatment since the level of NPSH in the nucleus might be a more important determinant of resistance to DNA-damaging agents than that in cytoplasm. The image cytometry method described here is quantitative, allows a measure of tumour cell heterogeneity and can be applied to small biopsy samples obtained by fine-needle aspiration. Thus it appears suitable for prospective clinical studies in cancer patients, and for monitoring the effects of GSH-depleting agents used as adjuncts to cancer chemotherapy or radiotherapy. Images Figure

  14. [Antibody adsorption to tetrodotoxin-sensitive cytoplasmic proteins by murine neuroblastoma cells].

    PubMed

    Pinchuk, G V; Pinchuk, L N; Gerasimenko, O V

    1988-01-01

    The study was aimed to examine properties of polyclonal antibodies elicited after immunization by cytoplasmic nerve cell glycoproteins forming sodium channels in liposomes. It was shown that intact murine neuroblastoma cells can absorb these antibodies. Absorbing cell dose-effect curves were found to have a characteristic form able to shift when the cell culture time or serum concentration in the growth medium varied.

  15. Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

    SciTech Connect

    Perez-Ramirez, B.; Martinez-Carrion, M. )

    1989-06-13

    A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with ({sup 3}H)pyridoxine 5-phosphate (({sup 3}H)PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of ({sup 3}H)PNP at 37{degree}C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the {alpha}-subunit, internal surface labeling by PLP was greatest for the highest molecular weight ({delta}) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles.

  16. A Cytoplasmic Protein Ssl3829 Is Important for NDH-1 Hydrophilic Arm Assembly in Synechocystis sp. Strain PCC 6803.

    PubMed

    Wang, Xiaozhuo; Gao, Fudan; Zhang, Jingsong; Zhao, Jiaohong; Ogawa, Teruo; Ma, Weimin

    2016-06-01

    Despite significant progress in clarifying the subunit compositions and functions of the multiple NDH-1 complexes in cyanobacteria, the assembly factors and their roles in assembling these NDH-1 complexes remain elusive. Two mutants sensitive to high light for growth and impaired in NDH-1-dependent cyclic electron transport around photosystem I were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-tagged library. Both mutants were tagged in the ssl3829 gene encoding an unknown protein, which shares significant similarity with Arabidopsis (Arabidopsis thaliana) CHLORORESPIRATORY REDUCTION7. The ssl3829 product was localized in the cytoplasm and associates with an NDH-1 hydrophilic arm assembly intermediate (NAI) of about 300 kD (NAI300) and an NdhI maturation factor, Slr1097. Upon deletion of Ssl3829, the NAI300 complex was no longer visible on gels, thereby impeding the assembly of the NDH-1 hydrophilic arm. The deletion also abolished Slr1097 and consequently reduced the amount of mature NdhI in the cytoplasm, which repressed the dynamic assembly process of the NDH-1 hydrophilic arm because mature NdhI was essential to stabilize all functional NAIs. Therefore, Ssl3829 plays an important role in the assembly of the NDH-1 hydrophilic arm by accumulating the NAI300 complex and Slr1097 protein in the cytoplasm.

  17. Cytoplasmic Poly(A) Binding Protein C4 Serves a Critical Role in Erythroid Differentiation

    PubMed Central

    Kini, Hemant K.; Kong, Jian

    2014-01-01

    The expression of an mRNA is strongly impacted by its 3′ poly(A) tail and associated poly(A)-binding proteins (PABPs). Vertebrates encode six PABP isoforms that vary in abundance, distribution, developmental control, and subcellular localization. Here we demonstrate that the minor PABP isoform PABPC4 is expressed in erythroid cells and impacts the steady-state expression of a subset of erythroid mRNAs. Motif analyses reveal a high-value AU-rich motif in the 3′ untranslated regions (UTRs) of PABPC4-impacted mRNAs. This motif enhances the association of PABPC4 with mRNAs containing critically shortened poly(A) tails. This association may serve to protect a subset of mRNAs from accelerated decay. Finally, we demonstrate that selective depletion of PABPC4 in an erythroblast cell line inhibits terminal erythroid maturation with corresponding alterations in the erythroid gene expression. These observations lead us to conclude that PABPC4 plays an essential role in posttranscriptional control of a major developmental pathway. PMID:24469397

  18. A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

    SciTech Connect

    You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

    2008-08-15

    Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus.

  19. BTBD1 and BTBD2 colocalize to cytoplasmic bodies with the RBCC/tripartite motif protein, TRIM5delta.

    PubMed

    Xu, Lixin; Yang, Lihong; Moitra, Prasun K; Hashimoto, Keiko; Rallabhandi, Prasad; Kaul, Sunil; Meroni, Germana; Jensen, Jane P; Weissman, Allan M; D'Arpa, Peter

    2003-08-01

    We previously identified BTBD1 and BTBD2 as novel topoisomerase I-interacting proteins that share 80% amino acid identity. Here we report the characterization of their subcellular localization. In a number of mouse and human cells, BTBD1 and BTBD2 (BTBD1/2) colocalized to punctate or elongated cytoplasmic bodies (< 5 microm long and several per cell) that were larger and more elongated in cancer cell lines than in fibroblasts and myoblasts. A search for potential colocalizing proteins identified TRIM family members that localize to morphologically similar cytoplasmic bodies, which were then tested for colocalization with BTBD1/2. TRIM5delta, expressed as a GFP fusion, colocalized with BTBD1/2 immunostaining and appeared to serve as a scaffold for the assembly of endogenous BTBD1/2 proteins. TRIM family members contain a RING domain, B-box(es), and coiled-coil regions, which have a characteristic order and spacing (RBCC domain). RING-dependent ubiquitin ligase activity and multimerization via the coiled-coil region may be defining properties of the RBCC/TRIM protein family. We found that TRIM5delta with a deleted coiled-coil region or a mutated RING domain failed to colocalize with BTBD1/2. Additionally, TRIM5delta ubiquitylated itself in a RING finger- and UbcH5B-dependent manner. BTBD1/2 each contain a PHR-similarity region, repeated twice on the putative ubiquitin ligases PAM, highwire and RPM-1, which also contain a RING and B-box. Thus, four protein modules found on each of these putative ubiquitin ligases, a RING, a B-box and two PHR repeats, are present on BTBD1/2 and TRIM5delta that are colocalized to cytoplasmic bodies.

  20. Nuclear and cytoplasmic tau proteins from human nonneuronal cells share common structural and functional features with brain tau.

    PubMed

    Cross, D C; Muñoz, J P; Hernández, P; Maccioni, R B

    2000-05-01

    The heterogeneous family of tau proteins interacts with microtubules, actin filaments, and intermediate filaments. The tau isoforms have been shown to play a major role in neuronal polarity. However, tau-like proteins have been found in several other types of cells. Previous studies have also indicated the presence of a nuclear tau. The relationships between nuclear and cytoplasmic tau as well as the functional aspects of the nuclear tau are unknown. In this study, we demonstrate by reverse transcriptase polymerase chain reaction using specific primers that a transcript with features of neuronal tau is present in human fibroblast and Huh-7 hepatoma cell lines. Additionally, we present the first isolation and characterization of cytosolic and nuclear tau-like proteins from nonneuronal cells. Nonneuronal cytosolic tau components were isolated using the perchloric acid precipitation approach, while nuclear tau was isolated after selective extractions using high-ionic strength buffers. The cytoplasmic tau of nonneuronal cells is composed of at least three isoforms, whereas two main isoforms were detected in nuclear tau. Interestingly, the cytoplasmic and nuclear tau components exhibited the capacity to promote tubulin polymerization in vitro. Immunofluorescence studies using monoclonal anti-tau antibodies indicated a discrete distribution of tau protein in both the interphase and mitotic nucleus. In the latter, tau colocalized with the chromosomal scaffold. These studies, together with previous evidence on tau roles in modulating microtubule growth from centrosomes, and its role in the interaction patterns that stabilize the integrity of the cytoskeletal network, strongly support the idea that tau is a multifunctional protein involved in fundamental cellular processes. PMID:10842324

  1. Human stanniocalcin-1 interacts with nuclear and cytoplasmic proteins and acts as a SUMO E3 ligase.

    PubMed

    dos Santos, Marcos Tadeu; Trindade, Daniel Maragno; Gonçalves, Kaliandra de Almeida; Bressan, Gustavo Costa; Anastassopoulos, Filipe; Yunes, José Andres; Kobarg, Jörg

    2011-01-01

    Human stanniocalcin-1 (STC1) is a glycoprotein that has been implicated in different physiological process, including angiogenesis, apoptosis and carcinogenesis. Here we identified STC1 as a putative molecular marker for the leukemic bone marrow microenvironment and identified new interacting protein partners for STC1. Seven selected interactions retrieved from yeast two-hybrid screens were confirmed by GST-pull down assays in vitro. The N-terminal region was mapped to be the region that mediates the interaction with cytoplasmic, mitochondrial and nuclear proteins. STC1 interacts with SUMO-1 and several proteins that have been shown to be SUMOylated and localized to SUMOylation related nuclear bodies. Although STC1 interacts with SUMO-1 and has a high theoretical prediction score for a SUMOylation site, endogenous co-immunoprecipitation and in vitro SUMOylation assays with the purified recombinant protein could not detect STC1 SUMOylation. However, when we tested STC1 for SUMO E3 ligase activity, we found in an in vitro assay, that it significantly increases the SUMOylation of two other proteins. Confocal microscopic subcellular localization studies using both transfected cells and specific antibodies for endogenous STC1 revealed a cytoplasmic and nuclear deposition, the latter in the form of some specific dot-like substructure resembling SUMOylation related nuclear bodies. Together, these findings suggest a new role for STC1 in SUMOylation pathways, in nuclear bodies.

  2. Cytoplasmic dynein nomenclature

    PubMed Central

    Pfister, K. Kevin; Fisher, Elizabeth M.C.; Gibbons, Ian R.; Hays, Thomas S.; Holzbaur, Erika L.F.; McIntosh, J. Richard; Porter, Mary E.; Schroer, Trina A.; Vaughan, Kevin T.; Witman, George B.; King, Stephen M.; Vallee, Richard B.

    2005-01-01

    A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms. PMID:16260502

  3. Cytoplasmic LSM-1 protein regulates stress responses through the insulin/IGF-1 signaling pathway in Caenorhabditis elegans.

    PubMed

    Cornes, Eric; Porta-De-La-Riva, Montserrat; Aristizábal-Corrales, David; Brokate-Llanos, Ana María; García-Rodríguez, Francisco Javier; Ertl, Iris; Díaz, Mònica; Fontrodona, Laura; Reis, Kadri; Johnsen, Robert; Baillie, David; Muñoz, Manuel J; Sarov, Mihail; Dupuy, Denis; Cerón, Julián

    2015-09-01

    Genes coding for members of the Sm-like (LSm) protein family are conserved through evolution from prokaryotes to humans. These proteins have been described as forming homo- or heterocomplexes implicated in a broad range of RNA-related functions. To date, the nuclear LSm2-8 and the cytoplasmic LSm1-7 heteroheptamers are the best characterized complexes in eukaryotes. Through a comprehensive functional study of the LSm family members, we found that lsm-1 and lsm-3 are not essential for C. elegans viability, but their perturbation, by RNAi or mutations, produces defects in development, reproduction, and motility. We further investigated the function of lsm-1, which encodes the distinctive protein of the cytoplasmic complex. RNA-seq analysis of lsm-1 mutants suggests that they have impaired Insulin/IGF-1 signaling (IIS), which is conserved in metazoans and involved in the response to various types of stress through the action of the FOXO transcription factor DAF-16. Further analysis using a DAF-16::GFP reporter indicated that heat stress-induced translocation of DAF-16 to the nuclei is dependent on lsm-1. Consistent with this, we observed that lsm-1 mutants display heightened sensitivity to thermal stress and starvation, while overexpression of lsm-1 has the opposite effect. We also observed that under stress, cytoplasmic LSm proteins aggregate into granules in an LSM-1-dependent manner. Moreover, we found that lsm-1 and lsm-3 are required for other processes regulated by the IIS pathway, such as aging and pathogen resistance.

  4. Cytoplasmic LSM-1 protein regulates stress responses through the insulin/IGF-1 signaling pathway in Caenorhabditis elegans

    PubMed Central

    Cornes, Eric; Porta-De-La-Riva, Montserrat; Aristizábal-Corrales, David; Brokate-Llanos, Ana María; García-Rodríguez, Francisco Javier; Ertl, Iris; Díaz, Mònica; Fontrodona, Laura; Reis, Kadri; Johnsen, Robert; Baillie, David; Muñoz, Manuel J.; Sarov, Mihail; Dupuy, Denis; Cerón, Julián

    2015-01-01

    Genes coding for members of the Sm-like (LSm) protein family are conserved through evolution from prokaryotes to humans. These proteins have been described as forming homo- or heterocomplexes implicated in a broad range of RNA-related functions. To date, the nuclear LSm2-8 and the cytoplasmic LSm1-7 heteroheptamers are the best characterized complexes in eukaryotes. Through a comprehensive functional study of the LSm family members, we found that lsm-1 and lsm-3 are not essential for C. elegans viability, but their perturbation, by RNAi or mutations, produces defects in development, reproduction, and motility. We further investigated the function of lsm-1, which encodes the distinctive protein of the cytoplasmic complex. RNA-seq analysis of lsm-1 mutants suggests that they have impaired Insulin/IGF-1 signaling (IIS), which is conserved in metazoans and involved in the response to various types of stress through the action of the FOXO transcription factor DAF-16. Further analysis using a DAF-16::GFP reporter indicated that heat stress-induced translocation of DAF-16 to the nuclei is dependent on lsm-1. Consistent with this, we observed that lsm-1 mutants display heightened sensitivity to thermal stress and starvation, while overexpression of lsm-1 has the opposite effect. We also observed that under stress, cytoplasmic LSm proteins aggregate into granules in an LSM-1-dependent manner. Moreover, we found that lsm-1 and lsm-3 are required for other processes regulated by the IIS pathway, such as aging and pathogen resistance. PMID:26150554

  5. Antitermination of amidase expression in Pseudomonas aeruginosa is controlled by a novel cytoplasmic amide-binding protein.

    PubMed Central

    Wilson, S A; Wachira, S J; Drew, R E; Jones, D; Pearl, L H

    1993-01-01

    Amide-inducible expression of the aliphatic amidase system of Pseudomonas aeruginosa can be reconstituted in Escherichia coli with only the amidase structural gene amiE, the negative regulator amiC and the positive regulator amiR, a transcription antitermination factor. Complementation experiments in E. coli suggest that negative control of amidase expression by AmiC is mediated by a protein-protein interaction with AmiR. Purified AmiC binds acetamide with a KD of 3.7 microM in equilibrium dialysis studies, and therefore AmiC appears to be the sensory partner of the AmiC/AmiR pair of regulatory proteins, responding to the presence of amides. Sequence analysis techniques suggest that AmiC is a member of the structural family of periplasmic binding proteins, but has a distinct and novel cytoplasmic role. PMID:8253087

  6. Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor.

    PubMed

    Gould, S J; Kalish, J E; Morrell, J C; Bjorkman, J; Urquhart, A J; Crane, D I

    1996-10-01

    Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.

  7. Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance1[W

    PubMed Central

    Nikolovski, Nino; Shliaha, Pavel V.; Gatto, Laurent; Dupree, Paul; Lilley, Kathryn S.

    2014-01-01

    The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (data-independent acquisition method using ion mobility separation known as LC-IMS-MSE [or HDMSE]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle. PMID:25122472

  8. Cytoplasmic domain of the beta-amyloid protein precursor of Alzheimer's disease: function, regulation of proteolysis, and implications for drug development.

    PubMed

    Kerr, Megan L; Small, David H

    2005-04-15

    The beta-amyloid protein precursor (APP) has been extensively studied for its role in amyloid production and the pathogenesis of Alzheimer's disease (AD). However, little is known about the normal function of APP and its biological interactions. In this Mini-Review, the role of the cytoplasmic domain of APP in APP trafficking and proteolysis is described. These studies suggest that proteins that bind to the cytoplasmic domain may be important targets for drug development in AD. PMID:15672415

  9. Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

    PubMed Central

    Weber, K; Plessmann, U; Ulrich, W

    1989-01-01

    The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor. Images PMID:2583097

  10. Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study.

    PubMed

    Lee, Hye Min; Gupta, Ravi; Kim, Sun Hyung; Wang, Yiming; Rakwal, Randeep; Agrawal, Ganesh Kumar; Kim, Sun Tae

    2015-05-01

    High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates.

  11. Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study.

    PubMed

    Lee, Hye Min; Gupta, Ravi; Kim, Sun Hyung; Wang, Yiming; Rakwal, Randeep; Agrawal, Ganesh Kumar; Kim, Sun Tae

    2015-05-01

    High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates. PMID:25689267

  12. Characterization of Raphanus sativus pentatricopeptide repeat proteins encoded by the fertility restorer locus for Ogura cytoplasmic male sterility.

    PubMed

    Uyttewaal, M; Arnal, N; Quadrado, M; Martin-Canadell, A; Vrielynck, N; Hiard, S; Gherbi, H; Bendahmane, A; Budar, F; Mireau, H

    2008-12-01

    Cytoplasmic male sterility is a maternally inherited trait in higher plants that prevents the production of functional pollen. Ogura cytoplasmic male sterility in radish (Raphanus sativus) is regulated by the orf138 mitochondrial locus. Male fertility can be restored when orf138 accumulation is suppressed by the nuclear Rfo locus, which consists of three genes putatively encoding highly similar pentatricopeptide repeat proteins (PPR-A, -B, and -C). We produced transgenic rapeseed (Brassica napus) plants separately expressing PPR-A and PPR-B and demonstrated that both encoded proteins accumulated preferentially in the anthers of young flower buds. Immunodetection of ORF138 showed that, unlike PPR-B, PPR-A had no effect on the synthesis of the sterility protein. Moreover, immunolocalization experiments indicated that complete elimination of ORF138 from the tapetum of anthers correlated with the restoration of fertility. Thus, the primary role of PPR-B in restoring fertility is to inhibit ORF138 synthesis in the tapetum of young anthers. In situ hybridization experiments confirmed, at the cellular level, that PPR-B has no effect on the accumulation of orf138 mRNA. Lastly, immunoprecipitation experiments demonstrated that PPR-B, but not PPR-A, is associated with the orf138 RNA in vivo, linking restoration activity with the ability to directly or indirectly interact with the orf138 RNA. Together, our data support a role for PPR-B in the translational regulation of orf138 mRNA.

  13. Characterization of Raphanus sativus pentatricopeptide repeat proteins encoded by the fertility restorer locus for Ogura cytoplasmic male sterility.

    PubMed

    Uyttewaal, M; Arnal, N; Quadrado, M; Martin-Canadell, A; Vrielynck, N; Hiard, S; Gherbi, H; Bendahmane, A; Budar, F; Mireau, H

    2008-12-01

    Cytoplasmic male sterility is a maternally inherited trait in higher plants that prevents the production of functional pollen. Ogura cytoplasmic male sterility in radish (Raphanus sativus) is regulated by the orf138 mitochondrial locus. Male fertility can be restored when orf138 accumulation is suppressed by the nuclear Rfo locus, which consists of three genes putatively encoding highly similar pentatricopeptide repeat proteins (PPR-A, -B, and -C). We produced transgenic rapeseed (Brassica napus) plants separately expressing PPR-A and PPR-B and demonstrated that both encoded proteins accumulated preferentially in the anthers of young flower buds. Immunodetection of ORF138 showed that, unlike PPR-B, PPR-A had no effect on the synthesis of the sterility protein. Moreover, immunolocalization experiments indicated that complete elimination of ORF138 from the tapetum of anthers correlated with the restoration of fertility. Thus, the primary role of PPR-B in restoring fertility is to inhibit ORF138 synthesis in the tapetum of young anthers. In situ hybridization experiments confirmed, at the cellular level, that PPR-B has no effect on the accumulation of orf138 mRNA. Lastly, immunoprecipitation experiments demonstrated that PPR-B, but not PPR-A, is associated with the orf138 RNA in vivo, linking restoration activity with the ability to directly or indirectly interact with the orf138 RNA. Together, our data support a role for PPR-B in the translational regulation of orf138 mRNA. PMID:19098270

  14. In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

    PubMed Central

    2011-01-01

    Background Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1) in vitro (derived from LB cell cultures) and in vivo (derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Results Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria

  15. Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM.

    PubMed

    Briegel, Ariane; Ortega, Davi R; Mann, Petra; Kjær, Andreas; Ringgaard, Simon; Jensen, Grant J

    2016-09-13

    Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array. PMID:27573843

  16. Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation*

    PubMed Central

    Fernández-Pevida, Antonio; Rodríguez-Galán, Olga; Díaz-Quintana, Antonio; Kressler, Dieter; de la Cruz, Jesús

    2012-01-01

    Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process. PMID:22995916

  17. lingerer, a Drosophila gene involved in initiation and termination of copulation, encodes a set of novel cytoplasmic proteins.

    PubMed Central

    Kuniyoshi, Hisato; Baba, Kotaro; Ueda, Ryu; Kondo, Shunzo; Awano, Wakae; Juni, Naoto; Yamamoto, Daisuke

    2002-01-01

    In an effort to uncover genetic components underlying the courtship behavior of Drosophila melanogaster, we have characterized a novel gene, lingerer (lig), mutations of which result in abnormal copulation. Males carrying a hypomorphic mutation in lig fail to withdraw their genitalia upon termination of copulation, but display no overt abnormalities in their genitalia. A severe reduction in the dosage of the lig gene causes repeated attempted copulations but no successful copulations. Complete loss of lig function results in lethality during early pupal stages. lig is localized to polytene segment 44A on the second chromosome and encodes three alternatively spliced transcripts that generate two types of 150-kD proteins, Lig-A and Lig-B, differing only at the C terminus. Lig proteins show no similarity to known proteins. However, a set of homologous proteins in mammals suggest that Drosophila Lig belongs to a family of proteins that share five highly conserved domains. Lig is a cytoplasmic protein expressed in the central nervous system (CNS), imaginal discs, and gonads. Lig-A expression is selectively reduced in lig mutants and the ubiquitous supply of this protein at the beginning of metamorphosis restores the copulatory defects of the lig mutant. We propose that lig may act in the nervous system to mediate the control of copulatory organs during courtship. PMID:12524348

  18. Retention of OsNMD3 in the cytoplasm disturbs protein synthesis efficiency and affects plant development in rice

    PubMed Central

    Shi, Yanyun; Liu, Xiangling; Li, Rui; Gao, Yaping; Xu, Zuopeng; Zhang, Baocai; Zhou, Yihua

    2014-01-01

    The ribosome is the basic machinery for translation, and biogenesis of ribosomes involves many coordinated events. However, knowledge about ribosomal dynamics in higher plants is very limited. This study chose a highly conserved trans-factor, the 60S ribosomal subunit nuclear export adaptor NMD3, to characterize the mechanism of ribosome biogenesis in the monocot plant Oryza sativa (rice). O. sativa NMD3 (OsNMD3) shares all the common motifs and shuttles between the nucleus and cytoplasm via CRM1/XPO1. A dominant negative form of OsNMD3 with a truncated nuclear localization sequence (OsNMD3ΔNLS) was retained in the cytoplasm, consequently interfering with the release of OsNMD3 from pre-60S particles and disturbing the assembly of ribosome subunits. Analyses of the transactivation activity and cellulose biosynthesis level revealed low protein synthesis efficiency in the transgenic plants compared with the wild-type plants. Pharmaceutical treatments demonstrated structural alterations in ribosomes in the transgenic plants. Moreover, global expression profiles of the wild-type and transgenic plants were investigated using the Illumina RNA sequencing approach. These expression profiles suggested that overexpression of OsNMD3ΔNLS affected ribosome biogenesis and certain basic pathways, leading to pleiotropic abnormalities in plant growth. Taken together, these results strongly suggest that OsNMD3 is important for ribosome assembly and the maintenance of normal protein synthesis efficiency. PMID:24723395

  19. Specific expression in reproductive tissues and fate of a mitochondrial sterility-associated protein in cytoplasmic male-sterile bean.

    PubMed Central

    Abad, A R; Mehrtens, B J; Mackenzie, S A

    1995-01-01

    In common bean, cytoplasmic male sterility has been associated with a unique sequence found in the mitochondrial genome, designated pvs (for Phaseolus vulgaris sterility sequence). Within the pvs sequence, two open reading frames are encoded, ORF98 and ORF239. We have raised rabbit polyclonal antibodies against Pvs-ORF239 to evaluate the role of this putative male sterility-associated protein. Histological investigation of pollen development revealed that in the male-sterile bean line, callose deposition was abnormal and microspores remained as tetrads as previously reported. Pvs-ORF239 was found to be localized within the reproductive tissues of the male-sterile bean line, in contrast to all other cytoplasmic male sterility systems studied to date. This protein was associated with mitochondria, the callose layer, and developing primary cell walls during microsporogenesis. Expression of pvs-orf239 was not detected in fertile plants containing restorer gene Fr2. These observations, together with previous reports, suggest that nuclear restorer gene Fr2 interferes with expression of the pvs region post-transcriptionally. PMID:7734962

  20. Phosphorylation modulates rapid nucleocytoplasmic shuttling and cytoplasmic accumulation of Neurospora clock protein FRQ on a circadian time scale

    PubMed Central

    Diernfellner, Axel C.R.; Querfurth, Christina; Salazar, Carlos; Höfer, Thomas; Brunner, Michael

    2009-01-01

    The Neurospora clock protein FREQUENCY (FRQ) is an essential regulator of the circadian transcription factor WHITE COLLAR COMPLEX (WCC). In the course of a circadian period, the subcellular distribution of FRQ shifts from mainly nuclear to mainly cytosolic. This shift is crucial for coordinating the negative and positive limbs of the clock. We show that the subcellular redistribution of FRQ on a circadian time scale is governed by rapid, noncircadian cycles of nuclear import and export. The rate of nuclear import of newly synthesized FRQ is progressively reduced in a phosphorylation-dependent manner, leading to an increase in the steady-state level of cytoplasmic FRQ. The long-period frq7 mutant displays reduced kinetics of FRQ7 protein phosphorylation and a prolonged accumulation in the nucleus. We present a mathematical model that describes the cytoplasmic accumulation of wild-type and mutant FRQ on a circadian time scale on the basis of frequency-modulated rapid nucleocytoplasmic shuttling cycles. PMID:19759264

  1. Cytoplasmic localization of SBR (Dm NXF1) protein and its zonal distribution in the ganglia of Drosophila melanogaster larvae.

    PubMed

    Yakimova, Anna O; Pugacheva, Olga M; Golubkova, Elena V; Mamon, Ludmila A

    2016-09-01

    The Drosophila gene Dm nxf1 (nuclear export factor 1) previously known as small bristles (sbr) controls nuclear export of various mRNA transcripts. We found that Dm NXF1 is present not only in nucleoplasm or at the nuclear rim but also in the cytoplasm. On the spatiotemporal level, anti-SBR antibodies labeled some neuroblasts and their lineages in the brains of Drosophila larvae. The number of Dm NXF1-rich lineages increased during larval development, but Dm NXF1 expression was not evident in all lineages. In all larval stages, Dm NXF1 concentrated in the midline cells of the ventral nerve cord, which reflects a specific status of those cells. In neurites, Dm NXF1 was present in the form of cytoplasmic granules, which is similar to the behavior of another RNA-binding protein, dFMR. Interestingly, though, the granule expression pattern of Dm NXF1 and dFMR did not always overlap, as some granules stained exclusively for one or the other protein. It suggests the existence of specific mRNA partners for Dm NXF1 in neurites. PMID:27389771

  2. Regular Patterns for Proteome-Wide Distribution of Protein Abundance across Species

    PubMed Central

    Jiang, Ying; Ying, Wantao; Wu, Songfeng; Zhu, Yunping; Liu, Siqi; Yang, Pengyuan; Qian, Xiaohong; He, Fuchu

    2012-01-01

    A proteome of the bio-entity, including cell, tissue, organ, and organism, consists of proteins of diverse abundance. The principle that determines the abundance of different proteins in a proteome is of fundamental significance for an understanding of the building blocks of the bio-entity. Here, we report three regular patterns in the proteome-wide distribution of protein abundance across species such as human, mouse, fly, worm, yeast, and bacteria: in most cases, protein abundance is positively correlated with the protein's origination time or sequence conservation during evolution; it is negatively correlated with the protein's domain number and positively correlated with domain coverage in protein structure, and the correlations became stronger during the course of evolution; protein abundance can be further stratified by the function of the protein, whereby proteins that act on material conversion and transportation (mass category) are more abundant than those that act on information modulation (information category). Thus, protein abundance is intrinsically related to the protein's inherent characters of evolution, structure, and function. PMID:22427835

  3. Agrobacterium VirE2 proteins can form a complex with T strands in the plant cytoplasm.

    PubMed

    Gelvin, S B

    1998-08-01

    Wild-type VirE2 and VirD2 proteins from Agrobacterium tumefaciens contain nuclear targeting sequences (NLS) that are likely involved in directing transferred T strands to the plant nucleus. An A. tumefaciens virE2 virD2DeltaNLS double mutant was able to form tumors on VirE2-producing transgenic tobacco but not on wild-type tobacco. Because this mutant bacterial strain contains no known T-strand nuclear targeting signal, the data indicate that wild-type VirE2 proteins produced by the plant can interact with the T strands in the plant cytoplasm and direct them to the nucleus. PMID:9696783

  4. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  5. Neutrophil anti-neutrophil cytoplasmic autoantibody proteins: bactericidal increasing protein, lactoferrin, cathepsin, and elastase as serological markers of inflammatory bowel and other diseases

    PubMed Central

    Kyriakidi, Kallirroi S.; Tsianos, Vasileios E.; Karvounis, Evaggelos; Christodoulou, Dimitrios K.; Katsanos, Konstantinos H.; Tsianos, Epameinondas V.

    2016-01-01

    Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract comprising Crohn’s disease and ulcerative colitis. Although the pathogenesis of the disease is not clearly defined yet, environmental, genetic and other factors contribute to the onset of the disease. Apart from the clinical and histopathological findings, several serological biomarkers are also employed to detect IBD. One of the most thoroughly studied biomarker is anti-neutrophil cytoplasmic autoantibody (ANCA). We herein provide an overview of the current knowledge on the use of ANCA and certain ANCA proteins, such as bactericidal increasing protein, lactoferrin, cathepsin G and elastase, as serological markers for IBD and other diseases. PMID:27366026

  6. Aberrant Cytoplasm Localization and Protein Stability of SIRT1 is Regulated by PI3K/IGF-1R Signaling in Human Cancer Cells

    PubMed Central

    Byles, Vanessa; Chmilewski, Laura K.; Wang, Joyce; Zhu, Lijia; Forman, Lora W.; Faller, Douglas V.; Dai, Yan

    2010-01-01

    SIRT1, an NAD-dependent histone/protein deacetylase, has classically been thought of as a nuclear protein. In this study, we demonstrate that SIRT1 is mainly localized in the nucleus of normal cells, but is predominantly localized in the cytoplasm of the cancer / transformed cells we tested. We found this predominant cytoplasmic localization of SIRT1 is regulated by elevated mitotic activity and PI3K/IGF-1R signaling in cancer cells. We show that aberrant cytoplasmic localization of SIRT1 is due to increased protein stability and is regulated by PI3K/IGF-1R signaling. In addition, we determined that SIRT1 is required for PI3K-mediated cancer cell growth. Our study represents the first identification that aberrant cytoplasm localization is one of the specific alternations to SIRT1 that occur in cancer cells, and PI3K/IGF-1R signaling plays an important role in the regulation of cytoplasmic SIRT1 stability. Our findings suggest that the over-expressed cytoplasmic SIRT1 in cancer cells may greatly contribute to its cancer-specific function by working downstream of the PI3K/IGF-1R signaling pathway. PMID:20941378

  7. Characterization of the restricted component of Epstein-Barr virus early antigens as a cytoplasmic filamentous protein.

    PubMed Central

    Luka, J; Miller, G; Jörnvall, H; Pearson, G R

    1986-01-01

    Four monoclonal antibodies produced against the restricted component of the Epstein-Barr virus (EBV) early antigen (EA-R) precipitated a polypeptide with an approximate molecular weight of 85,000. Three of these antibodies prepared against the native 85,000-molecular-weight protein (85K protein) reacted by immunofluorescence with acetone-fixed smears but not methanol-fixed smears of EBV-producing cells activated with tumor-promoting agent and sodium butyrate. The fourth monoclonal antibody which was produced against the denatured 85K protein reacted with both acetone-fixed cells and methanol-fixed cells. Blocking of direct immunofluorescence by the different monoclonal antibodies established that these monoclonal antibodies were directed against three different epitopes expressed on the 85K protein. The cytoplasmic staining pattern produced by each antibody was granular during the first 24 to 28 h after induction, developed into filamentous structures about 36 h after induction, and then began to aggregate after 48 h. Similar structures were observed in human placental cells transfected by EBV DNA and stained with three of the monoclonal antibodies. These results suggest that the EA-R polypeptide is assembled into filaments during the EBV lytic cycle. The significance of this in regards to replication has yet to be determined. Biochemical characterization of this major EA-R component did not reveal any major differences in this protein isolated from different cell lines. Images PMID:2422401

  8. An unrecognized extracellular function for an intracellular adapter protein released from the cytoplasm into the tumor microenvironment.

    PubMed

    Mintz, Paul J; Cardó-Vila, Marina; Ozawa, Michael G; Hajitou, Amin; Rangel, Roberto; Guzman-Rojas, Liliana; Christianson, Dawn R; Arap, Marco A; Giordano, Ricardo J; Souza, Glauco R; Easley, Jeffrey; Salameh, Ahmad; Oliviero, Salvatore; Brentani, Ricardo R; Koivunen, Erkki; Arap, Wadih; Pasqualini, Renata

    2009-02-17

    Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of beta(1) integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein-protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.

  9. Group 3 late embryogenesis abundant proteins from embryos of Artemia franciscana: structural properties and protective abilities during desiccation.

    PubMed

    Boswell, Leaf C; Menze, Michael A; Hand, Steven C

    2014-01-01

    Group 3 late embryogenesis abundant (LEA) proteins are highly hydrophilic, and their expression is associated with desiccation tolerance in both plants and animals. Here we show that two LEA proteins from embryos of Artemia franciscana, AfrLEA2 and AfrLEA3m, are intrinsically disordered in solution but upon desiccation gain secondary structure, as measured by circular dichroism. Trifluoroethanol and sodium dodecyl sulfate are both shown to induce α-helical structure in AfrLEA2 and AfrLEA3m. Bioinformatic predictions of secondary-structure content for both proteins correspond most closely to conformations measured in the dry state. Because some LEA proteins afford protection to desiccation-sensitive proteins during drying and subsequent rehydration, we tested for this capacity in AfrLEA2 and AfrLEA3m. The protective capacities vary, depending on the target enzyme. For the cytoplasmic enzyme lactate dehydrogenase, neither AfrLEA2 nor AfrLEA3m, with or without trehalose present, was able to afford protection better than that provided by bovine serum albumin (BSA) under the same conditions. However, for another cytoplasmic enzyme, phosphofructokinase, both AfrLEA2 and AfrLEA3m in the presence of trehalose were able to afford protection far greater than that provided by BSA with trehalose. Finally, for the mitochondrial enzyme citrate synthase, 400-μg/mL AfrLEA3m without trehalose provided significantly more protection than the same concentration of either AfrLEA2 or BSA. PMID:25244376

  10. Measuring cell viscoelastic properties using a force-spectrometer: influence of protein-cytoplasm interactions

    PubMed Central

    Canetta, Elisabetta; Duperray, Alain; Leyrat, Anne; Verdier, Claude

    2005-01-01

    Cell adhesive and rheological properties play a very important role in cell transmigration through the endothelial barrier, in particular in the case of inflammation (leukocytes) or cancer metastasis (cancer cells). In order to characterize cell viscoelastic properties, we have designed a force spectrometer (AFM) which can stretch cells thereby allowing measurement of their rheological properties. This custom-made force spectrometer allows two different visualizations, one lateral and one from below. It allows investigation of the effects of rheology involved during cell stretching. To test the ability of our system to characterize such viscoelastic properties, ICAM-1 transfected CHO cells were analyzed. Two forms of ICAM-1 were tested; wild type ICAM-1, which can interact with the cytoskeleton, and a mutant form which lacks the cytoplasmic domain, and is unable to associate with the cytoskeleton. Stretching experiments carried out on these cells show the formation of long filaments. Using a previous model of filament elongation, we could determine the viscoelastic properties of a single cell. As expected, different viscoelastic components were found between the wild type and the mutant, which reveal that the presence of interactions between ICAM-1 and the cytoskeleton increases the stiffness of the cell. PMID:16308464

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-01

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

  13. Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells

    PubMed Central

    Miyazaki, Yusuke; Chen, Ling-chun; Chu, Bernard W; Swigut, Tomek; Wandless, Thomas J

    2015-01-01

    Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called ‘destabilizing domains’, whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies. DOI: http://dx.doi.org/10.7554/eLife.07687.001 PMID:26314864

  14. Middle East respiratory syndrome coronavirus ORF4b protein inhibits type I interferon production through both cytoplasmic and nuclear targets

    PubMed Central

    Yang, Yang; Ye, Fei; Zhu, Na; Wang, Wenling; Deng, Yao; Zhao, Zhengdong; Tan, Wenjie

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel and highly pathogenic human coronavirus and has quickly spread to other countries in the Middle East, Europe, North Africa and Asia since 2012. Previous studies have shown that MERS-CoV ORF4b antagonizes the early antiviral alpha/beta interferon (IFN-α/β) response, which may significantly contribute to MERS-CoV pathogenesis; however, the underlying mechanism is poorly understood. Here, we found that ORF4b in the cytoplasm could specifically bind to TANK binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), suppress the molecular interaction between mitochondrial antiviral signaling protein (MAVS) and IKKε, and inhibit IFN regulatory factor 3 (IRF3) phosphorylation and subsequent IFN-β production. Further analysis showed that ORF4b could also inhibit IRF3 and IRF7-induced production of IFN-β, whereas deletion of the nuclear localization signal of ORF4b abrogated its ability to inhibit IRF3 and IRF7-induced production of IFN-β, but not IFN-β production induced by RIG-I, MDA5, MAVS, IKKε, and TBK-1, suggesting that ORF4b could inhibit the induction of IFN-β in both the cytoplasm and nucleus. Collectively, these results indicate that MERS-CoV ORF4b inhibits the induction of type I IFN through a direct interaction with IKKε/TBK1 in the cytoplasm, and also in the nucleus with unknown mechanism. Viruses have evolved multiple strategies to evade or thwart a host’s antiviral responses. A novel human coronavirus (HCoV), Middle East respiratory syndrome coronavirus (MERS-CoV), is distinguished from other coronaviruses by its high pathogenicity and mortality. However, virulence determinants that distinguish MERS-CoV from other HCoVs have yet to be identified. MERS-CoV ORF4b antagonizes the early antiviral response, which may contribute to MERS-CoV pathogenesis. Here, we report the identification of the interferon (IFN) antagonism mechanism of MERS-CoV ORF4b. MERS-CoV ORF4b inhibits the production

  15. Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins.

    PubMed

    Cundiff, Judy K; McConnell, Elizabeth J; Lohe, Kimberly J; Maria, Sarah D; McMahon, Robert J; Zhang, Qiang

    2016-01-01

    Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.

  16. Sequencing and Modification of the Gene Encoding the 42-Kilodalton Protein in the Cytoplasmic Membrane of Synechococcus PCC 7942

    PubMed Central

    Omata, Tatsuo; Carlson, Thomas J.; Ogawa, Teruo; Pierce, John

    1990-01-01

    A 42-kilodalton cytoplasmic membrane protein is synthesized when high CO2-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) are exposed to low CO2. The structural gene for this protein (cmpA) has been cloned and sequenced and shown to encode a 450 amino acid polypeptide with a molecular mass of 49 kilodalton. A deletion mutant lacking the 42-kilodalton protein was obtained by transformation of Synechococcus PCC 7942 following in vitro mutagenesis of the cloned gene. There were no significant differences between the mutant and wild-type cells in their growth rates under either low or high CO2 conditions. The activity of inorganic carbon (Ci) transport in the mutant was as high as that in the wild-type strain. In both types of cells, CO2 was the main species of Ci transported and the activities of CO2 and HCO3− transport increased when high CO2-grown cells were exposed to low CO2. We conclude that the 42-kilodalton protein is not directly involved in the Ci-accumulating mechanism of Synechococcus PCC 7942. Images Figure 3 PMID:16667451

  17. Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation.

    PubMed

    El Asmar, Zeina; Terrand, Jérome; Jenty, Marion; Host, Lionel; Mlih, Mohamed; Zerr, Aurélie; Justiniano, Hélène; Matz, Rachel L; Boudier, Christian; Scholler, Estelle; Garnier, Jean-Marie; Bertaccini, Diego; Thiersé, Danièle; Schaeffer, Christine; Van Dorsselaer, Alain; Herz, Joachim; Bruban, Véronique; Boucher, Philippe

    2016-03-01

    The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFβ-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (β) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 β-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.

  18. Spindle-E cycling between nuage and cytoplasm is controlled by Qin and PIWI proteins

    PubMed Central

    Andress, Arlise; Bei, Yanxia; Fonslow, Bryan R.; Giri, Ritika; Wu, Yilong; Yates, John R.

    2016-01-01

    Transposable elements (TEs) are silenced in germ cells by a mechanism in which PIWI proteins generate and use PIWI-interacting ribonucleic acid (piRNA) to repress expression of TE genes. piRNA biogenesis occurs by an amplification cycle in microscopic organelles called nuage granules, which are localized to the outer face of the nuclear envelope. One cofactor required for amplification is the helicase Spindle-E (Spn-E). We found that the Spn-E protein physically associates with the Tudor domain protein Qin and the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3). Spn-E and Qin proteins are mutually dependent for their exit from nuage granules, whereas Spn-E and both Aub and Ago3 are mutually dependent for their entry or retention in nuage. The result is a dynamic cycling of Spn-E and its associated factors in and out of nuage granules. This implies that nuage granules can be considered to be hubs for active, mobile, and transient complexes. We suggest that this is in some way coupled with the execution of the piRNA amplification cycle. PMID:27091448

  19. Natural variability in abundance of prevalent soybean proteins.

    PubMed

    Natarajan, Savithiry S

    2010-12-01

    Soybean is an inexpensive source of protein for humans and animals. Genetic modifications (GMO) to soybean have become inevitable on two fronts, both quality and yield will need to improve to meet increasing global demand. To ensure the safety of the crop for consumers it is important to determine the natural variation in seed protein constituents as well as any unintended changes that may occur in the GMO as a result of genetic modification. Understanding the natural variation of seed proteins in wild and cultivated soybeans that have been used in conventional soybean breeding programs is critical for determining unintended protein expression in GMO soybeans. In recent years, proteomic technologies have been used as an effective analytical tool for examining modifications of protein profiles. We have standardized and applied these technologies to determine and quantify the spectrum of proteins present in soybean seed. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography mass spectrometry (LC-MS) for the separation, quantification, and identification of different classes of soybean seed proteins. We have observed significant variations in different classes of proteins, including storage, allergen and anti-nutritional protein profiles, between non-GMO cultivated and wild soybean varieties. This information is useful for scientists and regulatory agencies to determine whether the unintended expression of proteins found in transgenic soybean is within the range of natural variation. PMID:20709130

  20. Natural variability in abundance of prevalent soybean proteins.

    PubMed

    Natarajan, Savithiry S

    2010-12-01

    Soybean is an inexpensive source of protein for humans and animals. Genetic modifications (GMO) to soybean have become inevitable on two fronts, both quality and yield will need to improve to meet increasing global demand. To ensure the safety of the crop for consumers it is important to determine the natural variation in seed protein constituents as well as any unintended changes that may occur in the GMO as a result of genetic modification. Understanding the natural variation of seed proteins in wild and cultivated soybeans that have been used in conventional soybean breeding programs is critical for determining unintended protein expression in GMO soybeans. In recent years, proteomic technologies have been used as an effective analytical tool for examining modifications of protein profiles. We have standardized and applied these technologies to determine and quantify the spectrum of proteins present in soybean seed. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography mass spectrometry (LC-MS) for the separation, quantification, and identification of different classes of soybean seed proteins. We have observed significant variations in different classes of proteins, including storage, allergen and anti-nutritional protein profiles, between non-GMO cultivated and wild soybean varieties. This information is useful for scientists and regulatory agencies to determine whether the unintended expression of proteins found in transgenic soybean is within the range of natural variation.

  1. Chloroplast isolation and affinity chromatography for enrichment of low-abundant proteins in complex proteomes.

    PubMed

    Bayer, Roman G; Stael, Simon; Teige, Markus

    2015-01-01

    Detailed knowledge of the proteome is crucial to advance the biological sciences. Low-abundant proteins are of particular interest to many biologists as they include, for example those proteins involved in signal transduction. Recent technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS). However, the dynamic range in protein abundance still forms a fundamental problem that limits the detection of low-abundant proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of other proteins at an abundance several-fold higher in order of magnitude. Therefore, specific enrichment strategies are required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundant proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles. PMID:25820724

  2. HPV16 E1E4 protein is phosphorylated by Cdk2/cyclin A and relocalizes this complex to the cytoplasm

    SciTech Connect

    Davy, Clare E. . E-mail: cdavy@nimr.mrc.ac.uk; Ayub, Mahmood . E-mail: mahmood.ayub@imperial.ac.uk; Jackson, Deborah J. . E-mail: djackso@nimr.mrc.ac.uk; Das, Papia . E-mail: pdas@nimr.mrc.ac.uk; McIntosh, Pauline . E-mail: pmcinto@nimr.mrc.ac.uk; Doorbar, John . E-mail: jdoorba@nimr.mrc.ac.uk

    2006-05-25

    The human papillomavirus type 16 E1E4 protein is expressed abundantly in cells supporting viral DNA amplification, but its expression is lost during malignant progression. In cell culture, 16E1E4 causes G2 cell cycle arrest by associating with and preventing the nuclear entry of Cdk1/cyclin B1 complexes. Here, we show that 16E1E4 is also able to associate with cyclin A and Cdk2 during the G2 phase of the cell cycle. Only a weak association was apparent during S-phase, and progression through S-phase appeared unaffected. As with cyclin B1, the interaction of 16E1E4 with cyclin A is dependent on residues T22/T23 and results in the accumulation of cyclin A in the cytoplasm where it colocalizes with 16E1E4. 16E1E4 serine 32 was found to be phosphorylated by Cdk2/cyclin A. We hypothesize that the interaction of 16E1E4 with cyclin A may serve to increase the efficiency with which 16E1E4 is able to prevent mitotic entry.

  3. Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems.

    PubMed

    Verdurmen, Wouter P R; Luginbühl, Manuel; Honegger, Annemarie; Plückthun, Andreas

    2015-02-28

    Through advances in protein scaffold engineering and selection technologies, highly specific binding proteins, which fold under reducing conditions, can be generated against virtually all targets. Despite tremendous therapeutic opportunities, intracellular applications are hindered by difficulties associated with achieving cytosolic delivery, compounded by even correctly measuring it. Here, we addressed cytosolic delivery systematically through the development of a biotin ligase-based assay that objectively quantifies cytosolic delivery in a generic fashion. We developed modular transport systems that consist of a designed ankyrin repeat protein (DARPin) for receptor targeting and a different DARPin for intracellular recognition and a bacterial toxin-derived component for cytosolic translocation. We show that both anthrax pores and the translocation domain of Pseudomonas exotoxin A (ETA) efficiently deliver DARPins into the cytosol. We found that the cargo must not exceed a threshold thermodynamic stability for anthrax pores, which can be addressed by engineering, while the ETA pathway does not appear to have this restriction.

  4. Cytoplasmic nonpolysomal ribonucleoprotein particles in sea urchin embryos and their relationship to protein synthesis

    PubMed Central

    Dworkin, Mark B.; Rudensey, Lyle M.; Infante, Anthony A.

    1977-01-01

    We have examined the relationship between the newly synthesized mRNA that enters polysomes in sea urchin embryos and the messengerlike RNA that enters the pool of ribosome-free ribonucleoprotein particles (free RNPs or informosomes). Although the RNA in the free RNPs turns over 25% more rapidly than in the polysomes, labeling kinetics indicate that the RNA containing poly(A) [poly(A)(+)RNA] and the RNA not containing poly(A) [poly(A)(-)RNA] within each cytoplasmic compartment have very similar half-lives. The poly(A)(+)RNA from both free RNPs and polysomes binds ribosomes almost equally well in a reticulocyte lysate, and this binding is sensitive to inhibitors of initiation. The poly(A)(-)RNA from polysomes initiates as well as poly(A)(+)RNA; however, poly(A)(-)RNA from free RNPs is only half as efficient in binding to ribosomes, and by this criterion is only 50% mRNA. We have also examined the size and dynamics of shortening of the poly(A) tails of poly(A)(+)RNA from free RNPs and polysomes. Pulse-labeled poly(A) from both free RNPs and polysomes is about 180 nucleotides in length. Poly(A) shortening is very rapid in polysomes; steady-state labeled polysomal RNA is largely devoid of the 180-nucleotide-long poly(A) segments. Poly(A) shortening in free RNPs is slower; half of the poly(A) derived from steady-state free RNPs is still 180 nucleotides long. Despite this difference in the rates of poly(A) shortening, polysomes and free RNPs have very similar half-lives. There is, then, no obvious relationship between poly(A) shortening and turnover of mRNA in these embryos. The data are interpreted to mean that poly(A)(+)RNA from free RNPs is enriched for a class of mRNA that initiates less frequently in vivo than the bulk of the cellular mRNA. PMID:267921

  5. Identification of Differentially Abundant Proteins of Edwardsiella ictaluri during Iron Restriction

    PubMed Central

    Dumpala, Pradeep R.; Peterson, Brian C.; Lawrence, Mark L.; Karsi, Attila

    2015-01-01

    Edwardsiella ictaluri is a Gram-negative facultative anaerobe intracellular bacterium that causes enteric septicemia in channel catfish. Iron is an essential inorganic nutrient of bacteria and is crucial for bacterial invasion. Reduced availability of iron by the host may cause significant stress for bacterial pathogens and is considered a signal that leads to significant alteration in virulence gene expression. However, the precise effect of iron-restriction on E. ictaluri protein abundance is unknown. The purpose of this study was to identify differentially abundant proteins of E. ictaluri during in vitro iron-restricted conditions. We applied two-dimensional difference in gel electrophoresis (2D-DIGE) for determining differentially abundant proteins and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS) for protein identification. Gene ontology and pathway-based functional modeling of differentially abundant proteins was also conducted. A total of 50 unique differentially abundant proteins at a minimum of 2-fold (p ≤ 0.05) difference in abundance due to iron-restriction were detected. The numbers of up- and down-regulated proteins were 37 and 13, respectively. We noted several proteins, including EsrB, LamB, MalM, MalE, FdaA, and TonB-dependent heme/hemoglobin receptor family proteins responded to iron restriction in E. ictaluri. PMID:26168192

  6. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage.

    PubMed

    West, Ewan; Osborne, Craig; Nolan, William; Bate, Clive

    2015-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound "natural Aβ", sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson's disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage. PMID:26043272

  7. Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules.

    PubMed

    Marx, Astrid; Godinez, William J; Tsimashchuk, Vasil; Bankhead, Peter; Rohr, Karl; Engel, Ulrike

    2013-05-01

    Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin-microtubule interactions. PMID:23515224

  8. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

    PubMed Central

    West, Ewan; Osborne, Craig; Nolan, William; Bate, Clive

    2015-01-01

    Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage. PMID:26043272

  9. A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate.

    PubMed

    Bollineni, Ravi Chand; Guldvik, Ingrid J; Grönberg, Henrik; Wiklund, Fredrik; Mills, Ian G; Thiede, Bernd

    2015-12-21

    Depletion of highly abundant proteins is an approved step in blood plasma analysis by mass spectrometry (MS). In this study, we explored a precipitation and differential protein solubility approach as a fractionation strategy for abundant protein removal from plasma. Total proteins from plasma were precipitated with 90% saturated ammonium sulfate, followed by differential solubilization in 55% and 35% saturated ammonium sulfate solutions. Using a four hour liquid chromatography (LC) gradient and an LTQ-Orbitrap XL mass spectrometer, a total of 167 and 224 proteins were identified from the 55% and 35% ammonium sulfate fractions, whereas 235 proteins were found in the remaining protein fractions with at least two unique peptides. SDS-PAGE and exclusive total spectrum counts from LC-MS/MS analyses clearly showed that majority of the abundant plasma proteins were solubilized in 55% and 35% ammonium sulfate solutions, indicating that the remaining protein fraction is of potential interest for identification of less abundant plasma proteins. Serum albumin, serotransferrin, alpha-1-antitrypsin and transthyretin were the abundant proteins that were highly enriched in 55% ammonium sulfate fractions. Immunoglobulins, complement system proteins, and apolipoproteins were among other abundant plasma proteins that were enriched in 35% ammonium sulfate fractions. In the remaining protein fractions a total of 40 unique proteins were identified of which, 32 proteins were identified with at least 10 exclusive spectrum counts. According to PeptideAtlas, 9 of these 32 proteins were estimated to be present at low μg ml(-1) (0.12-1.9 μg ml(-1)) concentrations in the plasma, and 17 at low ng ml(-1) (0.1-55 ng ml(-1)) range.

  10. The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction.

    PubMed

    Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J

    2015-03-01

    Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction.

  11. The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction

    PubMed Central

    Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J.

    2015-01-01

    Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. PMID:25655701

  12. Nuclear relocalisation of cytoplasmic poly(A)-binding proteins PABP1 and PABP4 in response to UV irradiation reveals mRNA-dependent export of metazoan PABPs

    PubMed Central

    Burgess, Hannah M.; Richardson, William A.; Anderson, Ross C.; Salaun, Christine; Graham, Sheila V.; Gray, Nicola K.

    2011-01-01

    Poly(A)-binding protein 1 (PABP1) has a fundamental role in the regulation of mRNA translation and stability, both of which are crucial for a wide variety of cellular processes. Although generally a diffuse cytoplasmic protein, it can be found in discrete foci such as stress and neuronal granules. Mammals encode several additional cytoplasmic PABPs that remain poorly characterised, and with the exception of PABP4, appear to be restricted in their expression to a small number of cell types. We have found that PABP4, similarly to PABP1, is a diffusely cytoplasmic protein that can be localised to stress granules. However, UV exposure unexpectedly relocalised both proteins to the nucleus. Nuclear relocalisation of PABPs was accompanied by a reduction in protein synthesis but was not linked to apoptosis. In examining the mechanism of PABP relocalisation, we found that it was related to a change in the distribution of poly(A) RNA within cells. Further investigation revealed that this change in RNA distribution was not affected by PABP knockdown but that perturbations that block mRNA export recapitulate PABP relocalisation. Our results support a model in which nuclear export of PABPs is dependent on ongoing mRNA export, and that a block in this process following UV exposure leads to accumulation of cytoplasmic PABPs in the nucleus. These data also provide mechanistic insight into reports that transcriptional inhibitors and expression of certain viral proteins cause relocation of PABP to the nucleus. PMID:21940797

  13. Probing short-range protein Brownian motion in the cytoplasm of living cells.

    PubMed

    Di Rienzo, Carmine; Piazza, Vincenzo; Gratton, Enrico; Beltram, Fabio; Cardarelli, Francesco

    2014-12-23

    The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes.

  14. Probing short-range protein Brownian motion in the cytoplasm of living cells

    NASA Astrophysics Data System (ADS)

    di Rienzo, Carmine; Piazza, Vincenzo; Gratton, Enrico; Beltram, Fabio; Cardarelli, Francesco

    2014-12-01

    The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes.

  15. A Novel Function of Human Pumilio Proteins in Cytoplasmic Sensing of Viral Infection

    PubMed Central

    Narita, Ryo; Takahasi, Kiyohiro; Murakami, Etsu; Hirano, Emi; Yamamoto, Seiji P.; Yoneyama, Mitsutoshi; Kato, Hiroki; Fujita, Takashi

    2014-01-01

    RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-β promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2. PMID:25340845

  16. Detection of cytoplasmic glycosylation associated with hydroxyproline.

    PubMed

    West, Christopher M; van der Wel, Hanke; Blader, Ira J

    2006-01-01

    A special class of glycosylation occurs on a proline residue of the cytoplasmic/nuclear protein Skp1 in the social amoeba Dictyostelium. For this glycosylation to occur, the proline must first be hydroxylated by the action of a soluble prolyl 4-hydroxylase acting on the protein. Cytoplasmic prolyl 4-hydroxylases are dioxygen-dependent enzymes that have low affinity for their O2 substrate and, therefore, have been implicated in O2-sensing in Dictyostelium, as well as in vertebrates and invertebrates. The sugar-hydroxyproline linkage has low abundance, is resistant to alkali cleavage and known glycosidases, and does not bind known lectins. However, initial screens for this modification can be made by assessing changes in electrophoretic mobility of candidate proteins after treatment of cells with prolyl hydroxylase inhibitors, and/or by metabolic labeling with [3H]sugar precursors. In addition, cytoplasmic hydroxylation/glycosylation can be assessed by assaying for cytoplasmic glycosyltransferases. Here we describe these methods and examples of their use in analyzing Skp1 glycosylation in Dictyostelium and the apicomplexan Toxoplasma gondii, the causative agent of toxoplasmosis in humans. PMID:17132515

  17. The cytoplasmic membrane is a primary target for the staphylocidal action of thrombin-induced platelet microbicidal protein.

    PubMed Central

    Koo, S P; Yeaman, M R; Nast, C C; Bayer, A S

    1997-01-01

    Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner. PMID:9353067

  18. The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins.

    PubMed

    Davies, Rebecca G; Wagstaff, Kylie M; McLaughlin, Eileen A; Loveland, Kate L; Jans, David A

    2013-12-01

    Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development. PMID:23707952

  19. The Mammalian Proteins MMS19, MIP18, and ANT2 Are Involved in Cytoplasmic Iron-Sulfur Cluster Protein Assembly*

    PubMed Central

    van Wietmarschen, Niek; Moradian, Annie; Morin, Gregg B.; Lansdorp, Peter M.; Uringa, Evert-Jan

    2012-01-01

    Iron-sulfur (Fe-S) clusters are essential cofactors of proteins with a wide range of biological functions. A dedicated cytosolic Fe-S cluster assembly (CIA) system is required to assemble Fe-S clusters into cytosolic and nuclear proteins. Here, we show that the mammalian nucleotide excision repair protein homolog MMS19 can simultaneously bind probable cytosolic iron-sulfur protein assembly protein CIAO1 and Fe-S proteins, confirming that MMS19 is a central protein of the CIA machinery that brings Fe-S cluster donor proteins and the receiving apoproteins into proximity. In addition, we show that mitotic spindle-associated MMXD complex subunit MIP18 also interacts with both CIAO1 and Fe-S proteins. Specifically, it binds the Fe-S cluster coordinating regions in Fe-S proteins. Furthermore, we show that ADP/ATP translocase 2 (ANT2) interacts with Fe-S apoproteins and MMS19 in the CIA complex but not with the individual proteins. Together, these results elucidate the composition and interactions within the late CIA complex. PMID:23150669

  20. Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.

    PubMed

    Sierralta, Walter D; Kohen, Paulina; Castro, Olga; Muñoz, Alex; Strauss, Jerome F; Devoto, Luigi

    2005-10-20

    The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase. PMID:16162390

  1. Long-term memory: disruption by inhibitors of protein synthesis and cytoplasmic flow

    SciTech Connect

    Flood, J.F.; Landry, D.W.; Bennett, E.L.; Jarvik, M.E.

    1981-01-01

    Colchicine (60 ..mu..g/kg), an inhibitor of axoplasmic transport, administered subcutaneously to mice has no detectable effect on retention when given shortly after active avoidance training, nor did a pertaining injection of anisomycin (ANI) have an amnesic effect. However, when ANI was administered shortly prior to training and colchicine was administered after training, retention performance was impaired. The amnesic effect was dependent on the time at which colchicine was administered. The amnesic effect was also obtained when ANI was combined with either vinblastine (6 ..mu..g/kg) or podophyllotoxin (3 ..mu..g/kg), drugs that inhibit axoplasmic transport. Intracerebral injections of colchicine (60 ng to 60 pg) caused amnesia in subjects pretreated with ANI, but not in subjects pretreated with saline. Lumicolchicine, an isomer of colchicine, which has similar central nervous system effects but has a low binding affinity for microtubule protein, did not impair retention in ANI pretreated mice. It is suggested that axonal transport of recently synthesized protein is required for long-term memory storage.

  2. Propeptide of aminopeptidase 1 protein mediates aggregation and vesicle formation in cytoplasm-to-vacuole targeting pathway.

    PubMed

    Morales Quinones, Mariana; Winston, Jared T; Stromhaug, Per E

    2012-03-23

    Misfolded protein aggregation causes disease and aging; autophagy counteracts this by eliminating damaged components, enabling cells to survive starvation. The cytoplasm-to-vacuole targeting pathway in yeast encompasses the aggregation of the premature form of aminopeptidase 1 (prApe1) in cytosol and its sequestration by autophagic proteins into a vesicle for vacuolar transport. We show that the propeptide of Ape1 is important for aggregation and vesicle formation and that it is sufficient for binding to prApe1 and Atg19. Defective aggregation disrupts vacuolar transport, suggesting that aggregate shape is important in vesicle formation, whereas Atg19 binding is not sufficient for vacuolar transport. Aggregation involves hydrophobicity, whereas Atg19 binding requires additional electrostatic interactions. Ape1 dodecamerization may cluster propeptides into trimeric structures, with sufficient affinity to form propeptide hexamers by binding to other dodecamers, causing aggregation. We show that Ape1 aggregates bind Atg19 and Atg8 in vitro; this could be used as a scaffold for an in vitro assay of autophagosome formation to elucidate the mechanisms of autophagy.

  3. The human actin-related protein hArp5: Nucleo-cytoplasmic shuttling and involvement in DNA repair

    SciTech Connect

    Kitayama, Kumiko; Kamo, Mariko; Oma, Yukako; Matsuda, Ryo; Uchida, Takafumi; Ikura, Tsuyoshi; Tashiro, Satoshi; Ohyama, Takashi; Winsor, Barbara; Harata, Masahiko

    2009-01-15

    Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5{delta} yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX ({gamma}-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced {gamma}-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.

  4. Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells

    PubMed Central

    1990-01-01

    The cellular prion protein (PrPC) is a sialoglycoprotein anchored to the external surface of cells by a glycosyl phosphatidylinositol moiety. During scrapie, an abnormal PrP isoform designated PrPSc accumulates, and much evidence argues that it is a major and necessary component of the infectious prion. Based on the resistance of native PrPSc to proteolysis and to digestion with phosphatidylinositol- specific phospholipase C as well as the enhancement of PrPSc immunoreactivity after denaturation, we devised in situ immunoassays for the detection of PrPSc in cultured cells. Using these immunoassays, we identified the sites of PrPSc accumulation in scrapie-infected cultured cells. We also used these immunoassays to isolate PrPSc- producing clones from a new hamster brain cell line (HaB) and found an excellent correlation between their PrPSc content and prion infectivity titers. In scrapie-infected HaB cells as well as in scrapie-infected mouse neuroblastoma cells, most PrPSc was found to be intracellular and most localized with ligands of the Golgi marker wheat germ agglutinin. In one scrapie-infected HaB clone, PrPSc also localized extensively with MG-160, a protein resident of the medial-Golgi stack whereas this colocalization was not observed in another subclone of these cells. Whether the sites of intracellular accumulation of PrPSc are limited to a few subcellular organelles or they are highly variable remains to be determined. If the intracellular accumulation of PrPSc is found in the cells of the central nervous system, then it might be responsible for the neuronal dysfunction and degeneration which are cardinal features of prion diseases. PMID:1693623

  5. Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2)

    SciTech Connect

    Weger, Stefan . E-mail: stefan.weger@charite.de; Hammer, Eva; Goetz, Anne; Heilbronn, Regine

    2007-05-25

    Through yeast two-hybrid analysis and coimmunoprecipitation studies, we have identified a novel cellular AAV-2 Rep78/Rep68 interaction partner located predominantly in the cytoplasm. In public databases, it has been assigned as KCTD5, because of a region of high similarity to the cytoplasmic tetramerization domain of voltage-gated potassium channels. Whereas Rep/KCTD5 interaction relied on the region surrounding the Rep nuclear localization signal, nuclear accumulation of Rep was not required. Wildtype Rep78/Rep68 proteins induced the translocation of large portions of KCTD5 into the nucleus pointing to functional interactions both in the cytoplasm and the nucleus. In line with an anticipated functional interference in the cytoplasm, KCTD5 overexpression completely abrogated Rep68-mediated posttranscriptional activation of a HIV-LTR driven luciferase reporter gene. Our study expands the panel of already identified nuclear Rep interaction partners to a cytoplasmic protein, which raises the awareness that important steps in the AAV life cycle may be regulated in this compartment.

  6. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    SciTech Connect

    Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying; Kirby, Ralph; Lin, Alan

    2013-11-15

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.

  7. Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA.

    PubMed

    Czyzyk-Krzeska, M F; Dominski, Z; Kole, R; Millhorn, D E

    1994-04-01

    Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein. RNase T1 mapping revealed that the protein was bound to a 28 nucleotide long sequence that is located between bases 1551-1579 of TH mRNA. Moreover, protein binding to this fragment was prevented with an antisense oligonucleotide directed against bases 1551-1579 and subsequent RNase H digestion. This fragment of the 3'-untranslated region of TH mRNA is rich in pyrimidine nucleotides, and the binding of cytoplasmic protein to this fragment was reduced by competition with other polypyrimidine sequences including poly(C) but not poly(U) polymers. The binding of the protein to TH mRNA was increased when cytoplasmic proteins were extracted from PC12 cells exposed to hypoxia (5% O2) for 24 h. Electrophoresis of the UV cross-linked RNA-protein complex on SDS-polyacrylamide gel electrophoresis revealed a complex of 74 kDa. The potential role of this protein-TH mRNA interaction in regulation of TH mRNA stability during hypoxia is discussed. PMID:7908289

  8. Protein abundance changes of Zygosaccharomyces rouxii in different sugar concentrations.

    PubMed

    Guo, Hong; Niu, Chen; Liu, Bin; Wei, JianPing; Wang, HuXuan; Yuan, YaHong; Yue, TianLi

    2016-09-16

    Zygosaccharomyces rouxii is a yeast which can cause spoilage in the concentrated juice industries. It exhibits resistance to high sugar concentrations but genome- and proteome-wide studies on Z. rouxii in response to high sugar concentrations have been poorly investigated. Herein, by using a 2-D electrophoresis based workflow, the proteome of a wild strain of Z. rouxii under different sugar concentrations has been analyzed. Proteins were extracted, quantified, and subjected to 2-DE analysis in the pH range 4-7. Differences in growth (lag phase), protein content (13.97-19.23mg/g cell dry weight) and number of resolved spots (196-296) were found between sugar concentrations. ANOVA test showed that 168 spots were different, and 47 spots, corresponding to 40 unique gene products have been identified. These protein species are involved in carbohydrate and energy metabolism, amino acid metabolism, response to stimulus, protein transport and vesicle organization, cell morphogenesis regulation, transcription and translation, nucleotide metabolism, amino-sugar nucleotide-sugar pathways, oxidoreductases balancing, and ribosome biogenesis. The present study provides important information about how Z. rouxii acts to cope with high sugar concentration at molecular levels, which might enhance our global understanding of Z. rouxii's high sugar-tolerance trait. PMID:27322723

  9. Similarity of a 16.5kDa tegumental protein of the human liver fluke Opisthorchis viverrini to nematode cytoplasmic motility protein.

    PubMed

    Labbunruang, Nipawan; Phadungsil, Wansika; Tesana, Smarn; Smooker, Peter M; Grams, Rudi

    2016-05-01

    Opisthorchis viverrini is the causative agent of human opisthorchiasis in Thailand and long lasting infection with the parasite has been correlated with the development of cholangiocarcinoma. In this work we have molecularly characterized the first member of a protein family carrying two DM9 repeats in this parasite (OvDM9-1). InterPro and other protein family databases describe the DM9 repeat as a protein domain of unknown function that has been first noted in Drosophila melanogaster. Two paralogous proteins have been partially characterized in the genus Fasciola, Fasciola hepatica TP16.5, a novel tegumental antigen in human fascioliasis and, recently F. gigantica DM9-1, a parenchymal protein with structural similarity to nematode cytoplasmic motility protein (MFP2). In this study, we show further evidence that this family of trematode proteins is related to MFP2 in sequence and structure. Soluble recombinant OvDM9-1 was used for structural analyses and for production of specific antisera. The native protein was detected in soluble and insoluble crude worm extracts and in seemingly various oligomeric forms in the latter. The potential for oligomerization was supported by cross-linking experiments of recombinant OvDM9-1. Structure prediction suggested a β-rich secondary structure of the protein and this was supported by a circular dichroism analysis. Molecular modeling in Phyre2 identified both MFP2 domains as distant homologs of OvDM9-1. The protein was located in tegumental type tissue and the cecal epithelium in the mature parasite. Recombinant OvDM9-1 was used as target in indirect ELISA but sera from infected hamsters showed only marginal reactivity towards it. It is proposed that OvDM9-1 and other members of this protein family have a role in cellular transport through functions on the cytoskeleton. PMID:27140280

  10. Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos

    SciTech Connect

    Murray, M.T.; Krohne, G.; Franke, W.W. )

    1991-01-01

    To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occur in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.

  11. Membrane structure correlates to function of LLP2 on the cytoplasmic tail of HIV-1 gp41 protein.

    PubMed

    Boscia, Alexander L; Akabori, Kiyotaka; Benamram, Zachary; Michel, Jonathan A; Jablin, Michael S; Steckbeck, Jonathan D; Montelaro, Ronald C; Nagle, John F; Tristram-Nagle, Stephanie

    2013-08-01

    Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (-1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function. PMID:23931314

  12. Expression of the ELAV-like protein HuR in the cytoplasm is associated with endometrial carcinoma progression.

    PubMed

    Wang, Dandan; Wang, Min; Hu, Chang'e; Shuang, Ting; Zhou, Yingying; Yan, Xiaoyu

    2014-12-01

    Human antigen R (HuR) is an mRNA-binding factor that belongs to the embryonic lethal abnormal vision/Hu protein family which may function as a tumor maintenance gene in a variety of carcinomas. However, there is no study to analyze HuR expression in endometrial carcinoma. Here, we investigated the expression of HuR in endometrial carcinoma carcinogenesis and subsequent progression. The expression of HuR and estrogen receptor alpha (ER-α) protein was examined by immunohistochemistry on paraffin embedding specimens containing endometrial carcinoma and adjacent non-cancerous tissues. Short hairpin RNA against HuR was transfected to investigate the role of HuR in regulating the expression of ER-α and progression in endometrial carcinoma. Cell viability and cycle were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. Apoptosis was examined by annexin V apoptosis assay. Our study result show that cytoplasmic HuR expression is more frequent in poorly differentiated carcinomas (p = 0.005), advanced stage (p = 0.020), and positive ER-α expression (p = 0.026). Three HuR short hairpin RNAs (shRNAs) were transfected into Ishikawa cells, and we selected the most effective shRNA for the following experiments. After the transfection, the ER-α protein level was decreased. Further, decreased expression of HuR resulted in the inhibition of proliferation and induced apoptosis in Ishikawa cells. Our results showed that HuR could be a causal factor of ER-α regulation in Ishikawa cells and thus may induce the hormone-dependent endometrial carcinoma.

  13. RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

    PubMed

    Stefanovic, Branko

    2013-01-01

    Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments.

  14. Structure of an invertebrate gene encoding cytoplasmic intermediate filament (IF) proteins: implications for the origin and the diversification of IF proteins.

    PubMed Central

    Dodemont, H; Riemer, D; Weber, K

    1990-01-01

    The structure of the single gene encoding the cytoplasmic intermediate filament (IF) proteins in non-neuronal cells of the gastropod Helix aspersa is described. Genomic and cDNA sequences show that the gene is composed of 10 introns and 11 exons, spanning greater than 60 kb of DNA. Alternative RNA processing accounts for two mRNA families which encode two IF proteins differing only in their C-terminal sequence. The intron/exon organization of the Helix rod domain is identical to that of the vertebrate type III IF genes in spite of low overall protein sequence homology and the presence of an additional 42 residues in coil 1b of the invertebrate sequence. Intron position homology extends to the entire coding sequence comprising both the rod and tail domains when the invertebrate IF gene is compared with the nuclear lamin LIII gene of Xenopus laevis presented in the accompanying report of Döring and Stick. In contrast the intron patterns of the tail domains of the invertebrate IF and the lamin genes differ from those of the vertebrate type III genes. The combined data are in line with an evolutionary descent of cytoplasmic IF proteins from a nuclear lamin-like progenitor and suggest a mechanism for this derivation. The unique position of intron 7 in the Helix IF gene indicates that the archetype IF gene arose by the elimination of the nuclear localization sequence due to the recruitment of a novel splice site. The presumptive structural organization of the archetype IF gene allows predictions with respect to the later diversification of metazoan IF genes. Whereas models proposing a direct derivation of neurofilament genes seem unlikely, the earlier speculation of an mRNA transposition mechanism is compatible with current results. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:2249666

  15. Cytoplasmic sequestration of the tumor suppressor p53 by a heat shock protein 70 family member, mortalin, in human colorectal adenocarcinoma cell lines

    SciTech Connect

    Gestl, Erin E.; Anne Boettger, S.

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer Eight human colorectal cell lines were evaluated for p53 and mortalin localization. Black-Right-Pointing-Pointer Six cell lines displayed cytoplasmic sequestration of the tumor suppressor p53. Black-Right-Pointing-Pointer Direct interaction between mortalin and p53 was shown in five cell lines. Black-Right-Pointing-Pointer Cell lines positive for p53 sequestration yielded elevated p53 expression levels. Black-Right-Pointing-Pointer This study yields the first evidence of cytoplasmic sequestration p53 by mortalin. -- Abstract: While it is known that cytoplasmic retention of p53 occurs in many solid tumors, the mechanisms responsible for this retention have not been positively identified. Since heatshock proteins like mortalin have been associated with p53 inactivation in other tumors, the current study sought to characterize this potential interaction in never before examined colorectal adenocarcinoma cell lines. Six cell lines, one with 3 different fractions, were examined to determine expression of p53 and mortalin and characterize their cellular localization. Most of these cell lines displayed punctate p53 and mortalin localization in the cell cytoplasm with the exception of HCT-8 and HCT116 379.2 cells, where p53 was not detected. Nuclear p53 was only observed in HCT-116 40-16, LS123, and HT-29 cell lines. Mortalin was only localized in the cytoplasm in all cell lines. Co-immunoprecipitation and immunohistochemistry revealed that p53 and mortalin were bound and co-localized in the cytoplasmic fraction of four cell lines, HCT-116 (40-16 and 386; parental and heterozygous fractions respectively of the same cell line), HT-29, LS123 and LoVo, implying that p53 nuclear function is limited in those cell lines by being restricted to the cytoplasm. Mortalin gene expression levels were higher than gene expression levels of p53 in all cell lines. Cell lines with cytoplasmic sequestration of p53, however, also displayed elevated p53

  16. In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques.

    PubMed

    Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne; Røntved, Christine Maria

    2012-09-01

    Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high-speed centrifugation contributed most to detection of low abundant proteins. Hence, prefractionation of colostrum prior to 2D-LC-MS/MS analysis expanded our knowledge on the presence and location of low abundant proteins in bovine colostrum. PMID:22848049

  17. Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility

    PubMed Central

    Huang, Wenchao; Yu, Changchun; Hu, Jun; Wang, Lili; Dan, Zhiwu; Zhou, Wei; He, Chunlan; Zeng, Yafei; Yao, Guoxin; Qi, Jianzhao; Zhang, Zhihong; Zhu, Renshan; Chen, Xuefeng; Zhu, Yingguo

    2015-01-01

    Cytoplasmic male sterility (CMS) has been extensively used for hybrid seed production in many major crops. Honglian CMS (HL-CMS) is one of the three major types of CMS in rice and has contributed greatly to food security worldwide. The HL-CMS trait is associated with an aberrant chimeric mitochondrial transcript, atp6-orfH79, which causes pollen sterility and can be rescued by two nonallelic restorer-of-fertility (Rf) genes, Rf5 or Rf6. Here, we report the identification of Rf6, which encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3–5. RF6 is targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS-associated transcript atp6-orfH79 at nucleotide 1238, which ensures normal pollen development and restores fertility. The duplicated motif 3 of RF6 is essential for RF6-OsHXK6 interactions, processing of the aberrant transcript, and restoration of fertility. Furthermore, reductions in the level of OsHXK6 result in atp6-orfH79 transcript accumulation and male sterility. Together these results reveal a novel mechanism for CMS restoration by which RF6 functions with OsHXK6 to restore HL-CMS fertility. The present study also provides insight into the function of hexokinase 6 in regulating mitochondrial RNA metabolism and may facilitate further exploitation of heterosis in rice. PMID:26578814

  18. Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility.

    PubMed

    Huang, Wenchao; Yu, Changchun; Hu, Jun; Wang, Lili; Dan, Zhiwu; Zhou, Wei; He, Chunlan; Zeng, Yafei; Yao, Guoxin; Qi, Jianzhao; Zhang, Zhihong; Zhu, Renshan; Chen, Xuefeng; Zhu, Yingguo

    2015-12-01

    Cytoplasmic male sterility (CMS) has been extensively used for hybrid seed production in many major crops. Honglian CMS (HL-CMS) is one of the three major types of CMS in rice and has contributed greatly to food security worldwide. The HL-CMS trait is associated with an aberrant chimeric mitochondrial transcript, atp6-orfH79, which causes pollen sterility and can be rescued by two nonallelic restorer-of-fertility (Rf) genes, Rf5 or Rf6. Here, we report the identification of Rf6, which encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3-5. RF6 is targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS-associated transcript atp6-orfH79 at nucleotide 1238, which ensures normal pollen development and restores fertility. The duplicated motif 3 of RF6 is essential for RF6-OsHXK6 interactions, processing of the aberrant transcript, and restoration of fertility. Furthermore, reductions in the level of OsHXK6 result in atp6-orfH79 transcript accumulation and male sterility. Together these results reveal a novel mechanism for CMS restoration by which RF6 functions with OsHXK6 to restore HL-CMS fertility. The present study also provides insight into the function of hexokinase 6 in regulating mitochondrial RNA metabolism and may facilitate further exploitation of heterosis in rice.

  19. Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility.

    PubMed

    Huang, Wenchao; Yu, Changchun; Hu, Jun; Wang, Lili; Dan, Zhiwu; Zhou, Wei; He, Chunlan; Zeng, Yafei; Yao, Guoxin; Qi, Jianzhao; Zhang, Zhihong; Zhu, Renshan; Chen, Xuefeng; Zhu, Yingguo

    2015-12-01

    Cytoplasmic male sterility (CMS) has been extensively used for hybrid seed production in many major crops. Honglian CMS (HL-CMS) is one of the three major types of CMS in rice and has contributed greatly to food security worldwide. The HL-CMS trait is associated with an aberrant chimeric mitochondrial transcript, atp6-orfH79, which causes pollen sterility and can be rescued by two nonallelic restorer-of-fertility (Rf) genes, Rf5 or Rf6. Here, we report the identification of Rf6, which encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3-5. RF6 is targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS-associated transcript atp6-orfH79 at nucleotide 1238, which ensures normal pollen development and restores fertility. The duplicated motif 3 of RF6 is essential for RF6-OsHXK6 interactions, processing of the aberrant transcript, and restoration of fertility. Furthermore, reductions in the level of OsHXK6 result in atp6-orfH79 transcript accumulation and male sterility. Together these results reveal a novel mechanism for CMS restoration by which RF6 functions with OsHXK6 to restore HL-CMS fertility. The present study also provides insight into the function of hexokinase 6 in regulating mitochondrial RNA metabolism and may facilitate further exploitation of heterosis in rice. PMID:26578814

  20. Selenium Deficiency Reduces the Abundance of mRNA for Se-Dependent Glutathione Peroxidase 1 by a UGA-Dependent Mechanism Likely To Be Nonsense Codon-Mediated Decay of Cytoplasmic mRNA

    PubMed Central

    Moriarty, Patrick M.; Reddy, C. Channa; Maquat, Lynne E.

    1998-01-01

    The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an ∼50-fold reduction in Se-GPx1 activity and an ∼20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i

  1. Chloroplast and Cytoplasmic Enzymes

    PubMed Central

    Anderson, Louise E.; Advani, Vimal R.

    1970-01-01

    Three pea (Pisum sativum) leaf chloroplast enzymes—triose phosphate isomerase, glyceric acid 3-phosphate kinase, and fructose 1,6-diphosphate aldolase—have been separated from the corresponding cytoplasmic enzymes by isoelectric focusing. These three enzymes of the reductive pentose phosphate cycle are therefore distinct proteins, not identical with the analogous enzymes of the Embden-Meyerhof-Parnas pathway. PMID:16657347

  2. The cytoplasmic domain is essential for transport function of the integral membrane transport protein SLC4A11.

    PubMed

    Loganathan, Sampath K; Lukowski, Chris M; Casey, Joseph R

    2016-01-15

    Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl(-)/HCO3(-) exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function.

  3. Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

    PubMed

    Saito, Shoko; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

    2016-07-01

    Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system. PMID:27114368

  4. Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

    PubMed

    Saito, Shoko; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

    2016-07-01

    Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system.

  5. Assessment of the natural variation of low abundant metabolic proteins in soybean seeds using proteomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, we investigated the distribution of the low abundant proteins that are involved in soybean seed development in four wild and twelve cultivated soybean genotypes. We found proteomic variation of these proteins within and...

  6. High-grain diets suppress ruminal tissue abundance of angiopoietin-like protein 4 in cattle.

    PubMed

    Li, S; Yuan, K; Mamedova, L K; Penner, G B; Oba, M; Beauchemin, K A; Bradford, B J

    2014-09-01

    Angiopoietin-like protein 4 (ANGPTL4) is expressed in bovine ruminal epithelium, making it possible that dietary components or commensal microbes may influence gastrointestinal ANGPTL4 production via interactions at the mucosal surface. Therefore, we conducted 3 experiments to evaluate the effects of dietary concentrate level and VFA infusions on ANGPTL4 abundance in ruminal tissue. In Exp. 1, we assigned 12 nonlactating Holstein cows to either 8% or 64% concentrate diets; diets were fed 28 d before euthanasia and ruminal tissue collection. Ruminal tissue and plasma ANGPTL4 protein abundance were unaltered by treatment. In Exp. 2, we assigned 8 continental crossbred heifers to either a 45% or 90% concentrate diet; diets were fed for 75 d before euthanasia and ruminal tissue collection. Compared with the 45% concentrate diet, the 90% concentrate diet decreased (P < 0.01) ruminal tissue ANGPTL4 protein abundance. In Exp. 3, we assigned 6 ruminally cannulated lactating Holstein cows to a treatment sequence in replicated 3 × 3 Latin squares and fed a standard lactation diet. Cows were infused with 5 mol/d sodium acetate, sodium propionate, or sodium butyrate for 2 d. Infusions of VFA did not affect (P > 0.10) mRNA or protein abundance of ANGPTL4 in ruminal papillae. Ruminal papillae ANGPTL4 abundance was, however, negatively correlated with (P = 0.01) ruminal VFA concentrations. ANGPTL4 abundance in ruminal tissue decreases in response to very high dietary concentrate and is inversely correlated with ruminal total VFA concentrations.

  7. Changes in the Cytoplasmic Composition of Amino Acids and Proteins Observed in Staphylococcus aureus during Growth under Variable Growth Conditions Representative of the Human Wound Site

    PubMed Central

    Alreshidi, Mousa M.; Dunstan, R. Hugh; Gottfries, Johan; Macdonald, Margaret M.; Crompton, Marcus J.; Ang, Ching-Seng; Williamson, Nicholas A.; Roberts, Tim K.

    2016-01-01

    Staphylococcus aureus is an opportunistic pathogen responsible for a high proportion of nosocomial infections. This study was conducted to assess the bacterial responses in the cytoplasmic composition of amino acids and ribosomal proteins under various environmental conditions designed to mimic those on the human skin or within a wound site: pH6-8, temperature 35–37°C, and additional 0–5% NaCl. It was found that each set of environmental conditions elicited substantial adjustments in cytoplasmic levels of glutamic acid, aspartic acid, proline, alanine and glycine (P< 0.05). These alterations generated characteristic amino acid profiles assessed by principle component analysis (PCA). Substantial alterations in cytoplasmic amino acid and protein composition occurred during growth under conditions of higher salinity stress implemented via additional levels of NaCl in the growth medium. The cells responded to additional NaCl at pH 6 by reducing levels of ribosomal proteins, whereas at pH 8 there was an upregulation of ribosomal proteins compared with the reference control. The levels of two ribosomal proteins, L32 and S19, remained constant across all experimental conditions. The data supported the hypothesis that the bacterium was continually responding to the dynamic environment by modifying the proteome and optimising metabolic homeostasis. PMID:27442022

  8. 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.

  9. Cytoplasmic CopZ-Like Protein and Periplasmic Rusticyanin and AcoP Proteins as Possible Copper Resistance Determinants in Acidithiobacillus ferrooxidans ATCC 23270

    PubMed Central

    Navarro, Claudio A.; von Bernath, Diego; Martínez-Bussenius, Cristóbal; Castillo, Rodrigo A.

    2015-01-01

    Acidophilic organisms, such as Acidithiobacillus ferrooxidans, possess high-level resistance to copper and other metals. A. ferrooxidans contains canonical copper resistance determinants present in other bacteria, such as CopA ATPases and RND efflux pumps, but these components do not entirely explain its high metal tolerance. The aim of this study was to find other possible copper resistance determinants in this bacterium. Transcriptional expression of A. ferrooxidans genes coding for a cytoplasmic CopZ-like copper-binding chaperone and the periplasmic copper-binding proteins rusticyanin and AcoP, which form part of an iron-oxidizing supercomplex, was found to increase when the microorganism was grown in the presence of copper. All of these proteins conferred more resistance to copper when expressed heterologously in a copper-sensitive Escherichia coli strain. This effect was absent when site-directed-mutation mutants of these proteins with altered copper-binding sites were used in this metal sensitivity assay. These results strongly suggest that the three copper-binding proteins analyzed here are copper resistance determinants in this extremophile and contribute to the high-level metal resistance of this industrially important biomining bacterium. PMID:26637599

  10. Cytoplasmic CopZ-Like Protein and Periplasmic Rusticyanin and AcoP Proteins as Possible Copper Resistance Determinants in Acidithiobacillus ferrooxidans ATCC 23270.

    PubMed

    Navarro, Claudio A; von Bernath, Diego; Martínez-Bussenius, Cristóbal; Castillo, Rodrigo A; Jerez, Carlos A

    2016-02-01

    Acidophilic organisms, such as Acidithiobacillus ferrooxidans, possess high-level resistance to copper and other metals. A. ferrooxidans contains canonical copper resistance determinants present in other bacteria, such as CopA ATPases and RND efflux pumps, but these components do not entirely explain its high metal tolerance. The aim of this study was to find other possible copper resistance determinants in this bacterium. Transcriptional expression of A. ferrooxidans genes coding for a cytoplasmic CopZ-like copper-binding chaperone and the periplasmic copper-binding proteins rusticyanin and AcoP, which form part of an iron-oxidizing supercomplex, was found to increase when the microorganism was grown in the presence of copper. All of these proteins conferred more resistance to copper when expressed heterologously in a copper-sensitive Escherichia coli strain. This effect was absent when site-directed-mutation mutants of these proteins with altered copper-binding sites were used in this metal sensitivity assay. These results strongly suggest that the three copper-binding proteins analyzed here are copper resistance determinants in this extremophile and contribute to the high-level metal resistance of this industrially important biomining bacterium. PMID:26637599

  11. Quantitative Analysis of Age Specific Variation in the Abundance of Human Female Parotid Salivary Proteins

    PubMed Central

    Ambatipudi, Kiran S.; Lu, Bingwen; Hagen, Fred K; Melvin, James E.; Yates, John R.

    2010-01-01

    Summary Human saliva is a protein-rich, easily accessible source of potential local and systemic biomarkers to monitor changes that occur under pathological conditions; however little is known about the changes in abundance associated with normal aging. In this study, we performed a comprehensive proteomic profiling of pooled saliva collected from the parotid glands of healthy female subjects, divided into two age groups 1 and 2 (20–30 and 55–65 years old, respectively). Hydrophobic charge interaction chromatography was used to separate high from low abundant proteins prior to characterization of the parotid saliva using multidimensional protein identification technology (MudPIT). Collectively, 532 proteins were identified in the two age groups. Of these proteins, 266 were identified exclusively in one age group, while 266 proteins were common to both groups. The majority of the proteins identified in the two age groups belonged to the defense and immune response category. Of note, several defense related proteins (e.g. lysozyme, lactoferrin and histatin-1) were significantly more abundant in group 2 as determined by G-test. Selected representative mass spectrometric findings were validated by western blot analysis. Our study reports the first quantitative analysis of differentially regulated proteins in ductal saliva collected from young and older female subjects. This study supports the use of high-throughput proteomics as a robust discovery tool. Such results provide a foundation for future studies to identify specific salivary proteins which may be linked to age-related diseases specific to women. PMID:19764810

  12. High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum) by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

    PubMed

    Singh, Amarjeet Kumar; Paritosh, Kumar; Kant, Uma; Burma, Pradeep Kumar; Pental, Deepak

    2016-01-01

    Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp), which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy. PMID:27391960

  13. High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum) by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids

    PubMed Central

    Singh, Amarjeet Kumar; Paritosh, Kumar; Kant, Uma; Burma, Pradeep Kumar; Pental, Deepak

    2016-01-01

    Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp), which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2–2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4–5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy. PMID:27391960

  14. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F. William; Rosenberg, Alan H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

  15. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F.W.; Rosenberg, A.H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

  16. Structural properties of fibrillar proteins isolated from the cell surface and cytoplasm of Streptococcus salivarius (K+) cells and nonadhesive mutants.

    PubMed Central

    Weerkamp, A H; van der Mei, H C; Liem, R S

    1986-01-01

    Most Streptococcus salivarius (K+) cells contain two protein antigens with different adhesive functions. The subcellular distribution and some structural properties of purified proteins were studied. Antigen B (AgB), a protein involved in interbacterial coaggregation with gram-negative bacteria, was present in the cell wall fraction only of the wild-type strain and was absent from the cells of a nonadhesive mutant. Antigen C (AgC), a glycoprotein involved in host-associated adhesive functions, was predominantly associated with the cell wall of the wild-type strain (AgCw), but accumulated in high amounts in the cytoplasmic fraction (AgCin) of mutants lacking the wall-associated form. AgB, AgCw, and AgCin had molecular weights of 380,000, 250,000 to 320,000, and 488,000, respectively, upon gel electrophoresis under nondenaturing conditions. In the presence of sodium dodecyl sulfate and beta-mercaptoethanol the molecular weights were only slightly lower, suggesting that the free, isolated molecules exist as monomers under native conditions. AgCin readily stained with periodate-Schiff reagent, indicating a significant content of carbohydrate, similar to AgCw. Circular dichroism spectra showed that about 45% of the amino acids of AgCw were involved in alpha-helical coiled structures. AgB had a significantly lower proportion of ordered coiled structure. Electron microscopic observations of low-angle-shadowed preparations of purified antigens showed that they were flexible, thin rods with thickened or globular ends. Measurements corrected for shadow thickness showed lengths of 184 nm (AgB), 112 nm (AgCin), and 87 nm (AgCw). Treatment of AgCw with protease destroyed the fibrillar core, but seemed not to affect the globular ends. Comparison of the results with the localization of the antigens in wild-type and specific mutant strains suggested that each antigen molecule may represent a single, characteristic surface fibril with a specific adhesive capacity. Images PMID

  17. Choice of dietary protein of vegetarians and omnivores is reflected in their hair protein 13C and 15N abundance.

    PubMed

    Petzke, Klaus J; Boeing, Heiner; Metges, Cornelia C

    2005-01-01

    Stable isotopic (15N, 13C) composition of tissues depends on isotopic pattern of food sources. We investigated whether the isotopic compositions of human hair protein and amino acids reflect the habitual dietary protein intake. Hair samples were analyzed from 100 omnivores (selected randomly out of the 1987-1988 German nutrition survey VERA), and from 15 ovo-lacto-vegetarians (OLV), and from 6 vegans recruited separately. Hair bulk and amino acid specific isotopic compositions were analyzed by isotope-ratio mass spectrometry (EA/IRMS and GC/C/IRMS, respectively) and the results were correlated with data of the 7 day dietary records. Hair bulk 15N and 13C abundances clearly reflect the particular eating habits. Vegans can be distinguished from OLV and both are significantly distinct from omnivores in both 15N and 13C abundances. 15N and 13C abundances rose with a higher proportion of animal to total protein intake (PAPI). Individual proportions of animal protein consumption (IPAP) were calculated using isotopic abundances and a linear regression model using animal protein consumption data of vegans (PAPI = 0) and omnivores (mean PAPI = 0.639). IPAP values positively correlated with the intake of protein, meat, meat products, and animal protein. Distinct patterns for hair amino acid specific 15N and 13C abundances were measured but with lower resolution between food preference groups compared with bulk values. In conclusion, hair 13C and 15N values both reflected the extent of animal protein consumption. Bulk isotopic abundance of hair can be tested for future use in the validation of dietary assessment methods. PMID:15880664

  18. Choice of dietary protein of vegetarians and omnivores is reflected in their hair protein 13C and 15N abundance.

    PubMed

    Petzke, Klaus J; Boeing, Heiner; Metges, Cornelia C

    2005-01-01

    Stable isotopic (15N, 13C) composition of tissues depends on isotopic pattern of food sources. We investigated whether the isotopic compositions of human hair protein and amino acids reflect the habitual dietary protein intake. Hair samples were analyzed from 100 omnivores (selected randomly out of the 1987-1988 German nutrition survey VERA), and from 15 ovo-lacto-vegetarians (OLV), and from 6 vegans recruited separately. Hair bulk and amino acid specific isotopic compositions were analyzed by isotope-ratio mass spectrometry (EA/IRMS and GC/C/IRMS, respectively) and the results were correlated with data of the 7 day dietary records. Hair bulk 15N and 13C abundances clearly reflect the particular eating habits. Vegans can be distinguished from OLV and both are significantly distinct from omnivores in both 15N and 13C abundances. 15N and 13C abundances rose with a higher proportion of animal to total protein intake (PAPI). Individual proportions of animal protein consumption (IPAP) were calculated using isotopic abundances and a linear regression model using animal protein consumption data of vegans (PAPI = 0) and omnivores (mean PAPI = 0.639). IPAP values positively correlated with the intake of protein, meat, meat products, and animal protein. Distinct patterns for hair amino acid specific 15N and 13C abundances were measured but with lower resolution between food preference groups compared with bulk values. In conclusion, hair 13C and 15N values both reflected the extent of animal protein consumption. Bulk isotopic abundance of hair can be tested for future use in the validation of dietary assessment methods.

  19. Genetics of single-cell protein abundance variation in large yeast populations

    NASA Astrophysics Data System (ADS)

    Albert, Frank W.; Treusch, Sebastian; Shockley, Arthur H.; Bloom, Joshua S.; Kruglyak, Leonid

    2014-02-01

    Variation among individuals arises in part from differences in DNA sequences, but the genetic basis for variation in most traits, including common diseases, remains only partly understood. Many DNA variants influence phenotypes by altering the expression level of one or several genes. The effects of such variants can be detected as expression quantitative trait loci (eQTL). Traditional eQTL mapping requires large-scale genotype and gene expression data for each individual in the study sample, which limits sample sizes to hundreds of individuals in both humans and model organisms and reduces statistical power. Consequently, many eQTL are probably missed, especially those with smaller effects. Furthermore, most studies use messenger RNA rather than protein abundance as the measure of gene expression. Studies that have used mass-spectrometry proteomics reported unexpected differences between eQTL and protein QTL (pQTL) for the same genes, but these studies have been even more limited in scope. Here we introduce a powerful method for identifying genetic loci that influence protein expression in the yeast Saccharomyces cerevisiae. We measure single-cell protein abundance through the use of green fluorescent protein tags in very large populations of genetically variable cells, and use pooled sequencing to compare allele frequencies across the genome in thousands of individuals with high versus low protein abundance. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci that we detected were clustered in `hotspots' that influence multiple proteins, and some hotspots were found to influence more than half of the proteins that we examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell

  20. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution.

    PubMed

    He, Yi-Ming; Ma, Bin-Guang

    2016-01-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions. PMID:27220911

  1. Abundance and Temperature Dependency of Protein-Protein Interaction Revealed by Interface Structure Analysis and Stability Evolution

    NASA Astrophysics Data System (ADS)

    He, Yi-Ming; Ma, Bin-Guang

    2016-05-01

    Protein complexes are major forms of protein-protein interactions and implement essential biological functions. The subunit interface in a protein complex is related to its thermostability. Though the roles of interface properties in thermal adaptation have been investigated for protein complexes, the relationship between the interface size and the expression level of the subunits remains unknown. In the present work, we studied this relationship and found a positive correlation in thermophiles rather than mesophiles. Moreover, we found that the protein interaction strength in complexes is not only temperature-dependent but also abundance-dependent. The underlying mechanism for the observed correlation was explored by simulating the evolution of protein interface stability, which highlights the avoidance of misinteraction. Our findings make more complete the picture of the mechanisms for protein complex thermal adaptation and provide new insights into the principles of protein-protein interactions.

  2. Synaptic abnormalities and cytoplasmic glutamate receptor aggregates in contactin associated protein-like 2/Caspr2 knockout neurons

    PubMed Central

    Varea, Olga; Martin-de-Saavedra, Maria Dolores; Kopeikina, Katherine J.; Schürmann, Britta; Fleming, Hunter J.; Fawcett-Patel, Jessica M.; Bach, Anthony; Jang, Seil; Peles, Elior; Kim, Eunjoon; Penzes, Peter

    2015-01-01

    Central glutamatergic synapses and the molecular pathways that control them are emerging as common substrates in the pathogenesis of mental disorders. Genetic variation in the contactin associated protein-like 2 (CNTNAP2) gene, including copy number variations, exon deletions, truncations, single nucleotide variants, and polymorphisms have been associated with intellectual disability, epilepsy, schizophrenia, language disorders, and autism. CNTNAP2, encoded by Cntnap2, is required for dendritic spine development and its absence causes disease-related phenotypes in mice. However, the mechanisms whereby CNTNAP2 regulates glutamatergic synapses are not known, and cellular phenotypes have not been investigated in Cntnap2 knockout neurons. Here we show that CNTNAP2 is present in dendritic spines, as well as axons and soma. Structured illumination superresolution microscopy reveals closer proximity to excitatory, rather than inhibitory synaptic markers. CNTNAP2 does not promote the formation of synapses and cultured neurons from Cntnap2 knockout mice do not show early defects in axon and dendrite outgrowth, suggesting that CNTNAP2 is not required at this stage. However, mature neurons from knockout mice show reduced spine density and levels of GluA1 subunits of AMPA receptors in spines. Unexpectedly, knockout neurons show large cytoplasmic aggregates of GluA1. Here we characterize, for the first time to our knowledge, synaptic phenotypes in Cntnap2 knockout neurons and reveal a novel role for CNTNAP2 in GluA1 trafficking. Taken together, our findings provide insight into the biological roles of CNTNAP2 and into the pathogenesis of CNTNAP2-associated neuropsychiatric disorders. PMID:25918374

  3. Anti-neutrophil cytoplasmic antibody-enriched IgG induces adhesion of human T lymphocytes to extracellular matrix proteins.

    PubMed

    Tomer, Y; Lider, O; Gilburd, B; Hershkoviz, R; Meroni, P L; Wiik, A; Shoenfeld, Y

    1997-06-01

    Recent studies have shown that anti-neutrophil cytoplasmic antibodies (ANCA) can activate neutrophils to adhere to endothelium, degranulate, and cause endothelial cell injury. These data have lead to the hypothesis that the T cell inflammatory response causing the vasculitis in Wegener's granulomatosis (WG) is secondary to stimulation of neutrophils by ANCA. So far there is no evidence for a direct effect of ANCA on lymphocytes. The present study was designed to examine whether lymphocytes can be directly stimulated by ANCA to adhere to endothelial extracellular matrix (ECM) proteins. Human and mouse ANCA-enriched IgG were tested for their ability to increase adhesion of human T lymphocytes to fibronectin, laminin, and intact ECM. Incubation of human T lymphocytes with human ANCA-enriched IgG increased adhesion of the lymphocytes in a dose-dependent manner to fibronectin, laminin, and intact ECM (the percentage adhesion to intact ECM was 55.7 +/- 3.1 and 45.0 +/- 1.0% for lymphocytes incubated with human IgG containing ANCA or control human IgG, respectively; P = 0.0045). The same induction of adhesion to fibronectin, laminin, and intact ECM was observed when the cells were incubated with the F(ab)2 fragment of ANCA-enriched IgG. Similarly, ANCA-enriched IgG produced in mice increased the adhesion of lymphocytes to fibronectin (the percentage adhesion to fibronectin was 29.7 +/- 4.3 and 16.6 +/- 1.9% for lymphocytes incubated with mouse IgG-ANCA or control mouse IgG, respectively; P = 0.0008). These results may suggest that ANCA can directly stimulate lymphocytes to adhere to endothelial ECM and to induce the vasculitic lesions of WG. It remains to be shown by which mechanisms ANCA stimulate lymphocytes to adhere to ECM. PMID:9175913

  4. Members of the HCMV US12 family of predicted heptaspanning membrane proteins have unique intracellular distributions, including association with the cytoplasmic virion assembly complex

    SciTech Connect

    Das, Subhendu; Pellett, Philip E. . E-mail: pelletp@ccf.org

    2007-05-10

    The human cytomegalovirus (HCMV) US12 gene family is a group of 10 predicted seven-transmembrane domain proteins that have some features in common with G-protein-coupled receptors. Little is known of their patterns of expression, localization, or functional interactions. Here, we studied the intracellular localization of three US12 family members, US14, US17, and US18, with respect to various intracellular markers and the cytoplasmic virion assembly compartment (AC). The three proteins have distinct patterns of expression, which include associations with the AC. US14 is often distributed in a uniform granular manner throughout the cytoplasm, concentrating in the AC in some cells. US17 is expressed in a segmented manner, with its N-terminal domain localizing to the periphery of what we show here to be the AC and the C-terminal domain localizing to nuclei and the cytoplasm [Das, S., Skomorovska-Prokvolit, Y., Wang, F. Z., Pellett, P.E., 2006. Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins. J. Virol. 80, 1191-1203]. Here, we show that the C-terminal domain is present at the center of the AC, in close association with markers of early endosomes; the N-terminal staining corresponds to an area stained by markers for the Golgi and trans-Golgi. US18 is distributed throughout the cytoplasm, concentrating in the AC at later stages of infection; it is localized more to the periphery of the AC than are US14 and US17C, in association with markers of the trans-Golgi. Although not detected in virions, their structures and localization in various zones within the AC suggest possible roles for these proteins in the process of virion maturation and egress.

  5. Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

    PubMed

    Qin, Rong; Zhang, Huaning; Li, Shaoshan; Jiang, Wusheng; Liu, Donghua

    2014-09-01

    Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.

  6. The nuclear protein Sam68 is redistributed to the cytoplasm and is involved in PI3K/Akt activation during EV71 infection.

    PubMed

    Zhang, Hua; Cong, Haolong; Song, Lei; Tien, Po

    2014-02-13

    Nuclear proteins can be triggered to be redistributed to the cytoplasm to assist with EV71 virus replication. This process is frequently involved in cellular signal transduction upon virus infection. In this study, we have demonstrated that a new nuclear protein, 68-kDa Src-associated in mitosis protein (Sam68), was translocated to the cytoplasm and was co-localized with EV71 during virus infection. Confocal microscopy and subcellular fractionation assay confirmed that virus 3C protease triggered the redistribution of Sam68 to the cytoplasm. Knockdown of Sam68 expression using ShRNA significantly inhibited virus replication, suggesting that Sam68 may be a host factor involved in EV71 life cycle. In addition, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Sam68 is known to be an upstream regulator of PI3K and our immunoprecipitation studies confirmed that Sam68 interacted directly with the p85 regulatory subunit of PI3K and mediated PI3K/Akt activation during EV71 infection. On the contrary, silencing of Sam68 dramatically abrogated Akt phosphorylation. These data, plus the fact that Sam68 is known to be a signaling adaptor protein, indicated that Sam68 is a signal molecule with a functional role in the PI3K/Akt signal pathway during EV71 infection.

  7. The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus

    PubMed Central

    Senissar, Meriem; Saux, Agnès Le; Belgareh-Touzé, Naïma; Adam, Céline; Banroques, Josette; Tanner, N. Kyle

    2014-01-01

    The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5′ 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrometry analyses, genetic experiments, sucrose gradients, in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs. PMID:25013175

  8. Natural Genetic Variation Influences Protein Abundances in C. elegans Developmental Signalling Pathways.

    PubMed

    Singh, Kapil Dev; Roschitzki, Bernd; Snoek, L Basten; Grossmann, Jonas; Zheng, Xue; Elvin, Mark; Kamkina, Polina; Schrimpf, Sabine P; Poulin, Gino B; Kammenga, Jan E; Hengartner, Michael O

    2016-01-01

    Complex traits, including common disease-related traits, are affected by many different genes that function in multiple pathways and networks. The apoptosis, MAPK, Notch, and Wnt signalling pathways play important roles in development and disease progression. At the moment we have a poor understanding of how allelic variation affects gene expression in these pathways at the level of translation. Here we report the effect of natural genetic variation on transcript and protein abundance involved in developmental signalling pathways in Caenorhabditis elegans. We used selected reaction monitoring to analyse proteins from the abovementioned four pathways in a set of recombinant inbred lines (RILs) generated from the wild-type strains N2 (Bristol) and CB4856 (Hawaii) to enable quantitative trait locus (QTL) mapping. About half of the cases from the 44 genes tested showed a statistically significant change in protein abundance between various strains, most of these were however very weak (below 1.3-fold change). We detected a distant QTL on the left arm of chromosome II that affected protein abundance of the phosphatidylserine receptor protein PSR-1, and two separate QTLs that influenced embryonic and ionizing radiation-induced apoptosis on chromosome IV. Our results demonstrate that natural variation in C. elegans is sufficient to cause significant changes in signalling pathways both at the gene expression (transcript and protein abundance) and phenotypic levels. PMID:26985669

  9. Visualization and Dissemination of Multidimensional Proteomics Data Comparing Protein Abundance During Caenorhabditis elegans Development

    NASA Astrophysics Data System (ADS)

    Riffle, Michael; Merrihew, Gennifer E.; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N.; Noble, William S.; MacCoss, Michael J.

    2015-11-01

    Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/.

  10. Visualization and dissemination of multidimensional proteomics data comparing protein abundance during Caenorhabditis elegans development.

    PubMed

    Riffle, Michael; Merrihew, Gennifer E; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N; Noble, William S; MacCoss, Michael J

    2015-11-01

    Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/ . Graphical Abstract ᅟ.

  11. Mapping protein abundance patterns in the brain using voxelation combined with liquid chromatography and mass spectrometry

    SciTech Connect

    Petyuk, Vladislav A.; Qian, Weijun; Smith, Richard D.; Smith, Desmond J.

    2010-02-01

    Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps

  12. Structural basis for the phosphorylation-regulated interaction between the cytoplasmic tail of cell polarity protein crumbs and the actin-binding protein moesin.

    PubMed

    Wei, Zhiyi; Li, Youjun; Ye, Fei; Zhang, Mingjie

    2015-05-01

    The type I transmembrane protein crumbs (Crb) plays critical roles in the establishment and maintenance of cell polarities in diverse tissues. As such, mutations of Crb can cause different forms of cancers. The cell intrinsic role of Crb in cell polarity is governed by its conserved, 37-residue cytoplasmic tail (Crb-CT) via binding to moesin and protein associated with Lin7-1 (PALS1). However, the detailed mechanism governing the Crb·moesin interaction and the balance of Crb in binding to moesin and PALS1 are not well understood. Here we report the 1.5 Å resolution crystal structure of the moesin protein 4.1/ezrin/radixin/moesin (FERM)·Crb-CT complex, revealing that both the canonical FERM binding motif and the postsynaptic density protein-95/Disc large-1/Zonula occludens-1 (PDZ) binding motif of Crb contribute to the Crb·moesin interaction. We further demonstrate that phosphorylation of Crb-CT by atypical protein kinase C (aPKC) disrupts the Crb·moesin association but has no impact on the Crb·PALS1 interaction. The above results indicate that, upon the establishment of the apical-basal polarity in epithelia, apical-localized aPKC can actively prevent the Crb·moesin complex formation and thereby shift Crb to form complex with PALS1 at apical junctions. Therefore, Crb may serve as an aPKC-mediated sensor in coordinating contact-dependent cell growth inhibition in epithelial tissues.

  13. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    PubMed Central

    Shi, Tujin; Niepel, Mario; McDermott, Jason E.; Gao, Yuqian; Nicora, Carrie D.; Chrisler, William B.; Markillie, Lye M.; Petyuk, Vladislav A.; Smith, Richard D.; Rodland, Karin D.; Sorger, Peter K.; Qian, Wei-Jun; Wiley, H. Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components—16 core proteins and 10 feedback regulators—of the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  14. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway.

    PubMed

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  15. Genetics of single-cell protein abundance variation in large yeast populations

    PubMed Central

    Albert, Frank W.; Treusch, Sebastian; Shockley, Arthur H.; Bloom, Joshua S.; Kruglyak, Leonid

    2014-01-01

    Variation among individuals arises in part from differences in DNA sequences, but the genetic basis for variation in most traits, including common diseases, remains only partly understood. Many DNA variants influence phenotypes by altering the expression level of one or multiple genes. The effects of such variants can be detected as expression quantitative trait loci (eQTL) 1. Traditional eQTL mapping requires large-scale genotype and gene expression data for each individual in the study sample, which limits sample sizes to hundreds of individuals in both humans and model organisms and reduces statistical power 2–6. Consequently, many eQTL are likely missed, especially those with smaller effects 7. Further, most studies use mRNA rather than protein abundance as the measure of gene expression. Studies that have used mass-spectrometry proteomics 8–13 reported surprising differences between eQTL and protein QTL (pQTL) for the same genes 9,10, but these studies have been even more limited in scope. Here, we introduce a powerful method for identifying genetic loci that influence protein expression in the yeast Saccharomyes cerevisiae. We measure single-cell protein abundance through the use of green-fluorescent-protein tags in very large populations of genetically variable cells, and use pooled sequencing to compare allele frequencies across the genome in thousands of individuals with high vs. low protein abundance. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci cluster at hotspot locations that influence multiple proteins—in some cases, more than half of those examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell physiology between yeast strains. PMID:24402228

  16. Cytoplasmic sensing of viral nucleic acids.

    PubMed

    Habjan, Matthias; Pichlmair, Andreas

    2015-04-01

    Viruses are the most abundant pathogens on earth. A fine-tuned framework of intervening pathways is in place in mammalian cells to orchestrate the cellular defence against these pathogens. Key for this system is sensor proteins that recognise specific features associated with nucleic acids of incoming viruses. Here we review the current knowledge on cytoplasmic sensors for viral nucleic acids. These sensors induce expression of cytokines, affect cellular functions required for virus replication and directly target viral nucleic acids through degradation or sequestration. Their ability to respond to a given nucleic acid is based on both the differential specificity of the individual proteins and the downstream signalling or adaptor proteins. The cooperation of these multiple proteins and pathways plays a key role in inducing successful immunity against virus infections.

  17. Isolation and characterization of hardening-induced proteins in Chlorella vulgaris C-27: identification of late embryogenesis abundant proteins.

    PubMed

    Honjoh, K; Yoshimoto, M; Joh, T; Kajiwara, T; Miyamoto, T; Hatano, S

    1995-12-01

    Hardening-induced soluble proteins of Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27) were isolated and purified by two-dimensional high-performance liquid chromatography (2D-HPLC) on an anion-exchange column, with subsequent reversed-phase chromatography. Some of the proteins were resolved by SDS-PAGE, characterized by amino-terminal sequencing and identified by searching for homologies in databases. Separation of the soluble proteins during the hardening of Chlorella by a combination of 2D-HPLC and SDS-PAGE revealed that at least 31 proteins were induced or increased in abundance. Of particular interest was the induction after 12 h of a 10-kDa protein with the amino-terminal amino acid sequence AGNKPITEQISDAVGAAGQKVG and the induction after 6 h of a 14-kDa protein with the amino-terminal sequence ALGEESLGDKAKNAFEDAKDAVKDAAGNVKEAV. The amino-terminal sequences of these proteins indicated that they were homologous to late embryogenesis abundant (LEA) proteins. Furthermore, the level of a 22-kDa protein also increased after 12 h. The amino-terminal sequence of this protein, AAPLVGGPAPDFTAAAVFD, indicated that it was homologous to thioredoxin peroxidase. PMID:8589927

  18. The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm

    PubMed Central

    1994-01-01

    The origin of the vacuole membrane surrounding the intracellular protozoan parasite Toxoplasma gondii is not known. Although unique secretory organelles, the rhoptries, discharge during invasion of the host cell and may contribute to the formation of this parasitophorous vacuole membrane (PVM), no direct evidence for this hypothesis exists. Using a novel approach we have determined that parasite-encoded proteins are present in the PVM, exposed to the host cell cytoplasm. In infected cells incubated with streptolysin-O or low concentrations of digitonin, the host cell plasma membrane was selectively permeabilized without significantly affecting the integrity of the PVM. Antisera prepared against whole parasites or a parasite fraction enriched in rhoptries and dense granules reacted with the PVM in these permeabilized cells, indicating that parasite-encoded antigens were exposed on the cytoplasmic side of the PVM. Parasite antigens responsible for this staining of the PVM were identified by fractionating total parasite proteins by SDS-PAGE and velocity sedimentation, and then affinity purifying "fraction-specific" antibodies from the crude antisera. Proteins responsible for the PVM- staining, identified with fraction-specific antibodies, cofractionated with known rhoptry proteins. The gene encoding one of the rhoptry proteins, ROP 2, was cloned and sequenced, predicting and integral membrane protein. Antibodies specific for ROP 2 reacted with the intact PVM. These results provide the first direct evidence that rhoptry contents participate in the formation of the PVM of T. gondii and suggest a possible role of ROP 2 in parasite-host cell interactions. PMID:7962077

  19. Hsp90 is involved in the regulation of cytosolic precursor protein abundance in tomato.

    PubMed

    Tillmann, Bodo; Röth, Sascha; Bublak, Daniela; Sommer, Manuel; Stelzer, Ernst H K; Scharf, Klaus-Dieter; Schleiff, Enrico

    2015-02-01

    Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 (Hsp70) and 90 (Hsp90) assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein (Chip), appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response. PMID:25619681

  20. Chlamydomonas axonemal dynein assembly locus ODA8 encodes a conserved flagellar protein needed for cytoplasmic maturation of outer dynein arm complexes.

    PubMed

    Desai, Paurav B; Freshour, Judy R; Mitchell, David R

    2015-01-01

    The Chlamydomonas reinhardtii oda8 mutation blocks assembly of flagellar outer dynein arms (ODAs), and interacts genetically with ODA5 and ODA10, which encode axonemal proteins thought to aid dynein binding onto axonemal docking sites. We positionally cloned ODA8 and identified the gene product as the algal homolog of vertebrate LRRC56. Its flagellar localization depends on ODA5 and ODA10, consistent with genetic interaction studies, but phylogenomics suggests that LRRC56 homologs play a role in intraflagellar transport (IFT)-dependent assembly of outer row dynein arms, not axonemal docking. ODA8 distribution between cytoplasm and flagella is similar to that of IFT proteins and about half of flagellar ODA8 is in the soluble matrix fraction. Dynein extracted in vitro from wild type axonemes will rebind efficiently to oda8 mutant axonemes, without re-binding of ODA8, further supporting a role in dynein assembly or transport, not axonemal binding. Assays comparing preassembled ODA complexes from the cytoplasm of wild type and mutant strains show that dynein in oda8 mutant cytoplasm has not properly preassembled and cannot bind normally onto oda axonemes. We conclude that ODA8 plays an important role in formation and transport of mature dynein complexes during flagellar assembly.

  1. Chlamydomonas Axonemal Dynein Assembly Locus ODA8 Encodes a Conserved Flagellar Protein Needed for Cytoplasmic Maturation of Outer Dynein Arm Complexes

    PubMed Central

    Desai, Paurav B; Freshour, Judy R; Mitchell, David R

    2015-01-01

    The Chlamydomonas reinhardtii oda8 mutation blocks assembly of flagellar outer dynein arms (ODAs), and interacts genetically with ODA5 and ODA10, which encode axonemal proteins thought to aid dynein binding onto axonemal docking sites. We positionally cloned ODA8 and identified the gene product as the algal homolog of vertebrate LRRC56. Its flagellar localization depends on ODA5 and ODA10, consistent with genetic interaction studies, but phylogenomics suggests that LRRC56 homologs play a role in intraflagellar transport (IFT)-dependent assembly of outer row dynein arms, not axonemal docking. ODA8 distribution between cytoplasm and flagella is similar to that of IFT proteins and about half of flagellar ODA8 is in the soluble matrix fraction. Dynein extracted in vitro from wild type axonemes will rebind efficiently to oda8 mutant axonemes, without re-binding of ODA8, further supporting a role in dynein assembly or transport, not axonemal binding. Assays comparing preassembled ODA complexes from the cytoplasm of wild type and mutant strains show that dynein in oda8 mutant cytoplasm has not properly preassembled and cannot bind normally onto oda axonemes. We conclude that ODA8 plays an important role in formation and transport of mature dynein complexes during flagellar assembly. © 2014 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:25558044

  2. GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

    PubMed

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation.

  3. Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase

    PubMed Central

    Amm, Ingo; Norell, Derrick; Wolf, Dieter H.

    2015-01-01

    The Saccharomyces cerevisiae heat shock proteins Hsp31, Hsp32, Hsp33 and Hsp34 belong to the DJ-1/ThiJ/PfpI superfamily which includes the human protein DJ-1 (PARK7) as the most prominent member. Mutations in the DJ-1 gene are directly linked to autosomal recessive, early-onset Parkinson’s disease. DJ-1 acts as an oxidative stress-induced chaperone preventing aggregation and fibrillation of α-synuclein, a critical factor in the development of the disease. In vivo assays in Saccharomyces cerevisiae using the model substrate ΔssCPY*Leu2myc (ΔssCL*myc) as an aggregation-prone misfolded cytoplasmic protein revealed an influence of the Hsp31 chaperone family on the steady state level of this substrate. In contrast to the ubiquitin ligase of the N-end rule pathway Ubr1, which is known to be prominently involved in the degradation process of misfolded cytoplasmic proteins, the absence of the Hsp31 chaperone family does not impair the degradation of newly synthesized misfolded substrate. Also degradation of substrates with strong affinity to Ubr1 like those containing the type 1 N-degron arginine is not affected by the absence of the Hsp31 chaperone family. Epistasis analysis indicates that one function of the Hsp31 chaperone family resides in a pathway overlapping with the Ubr1-dependent degradation of misfolded cytoplasmic proteins. This pathway gains relevance in late growth phase under conditions of nutrient limitation. Additionally, the Hsp31 chaperones seem to be important for maintaining the cellular Ssa Hsp70 activity which is important for Ubr1-dependent degradation. PMID:26466368

  4. The human LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct cytoplasmic foci.

    PubMed Central

    Ingelfinger, Dierk; Arndt-Jovin, Donna J; Lührmann, Reinhard; Achsel, Tilmann

    2002-01-01

    Sm and Sm-like (LSm) proteins form heptameric complexes that are involved in various steps of RNA metabolism. In yeast, the Lsm1-7 complex functions in mRNA degradation and is associated with several enzymes of this pathway, while the complex LSm2-8, the composition of which largely overlaps with that of LSm1-7, has a role in pre-mRNA splicing. A human gene encoding an LSm1 homolog has been identified, but its role in mRNA degradation has yet to be elucidated. We performed subcellular localization studies and found hLSm1 predominantly in the cytoplasm. However, it is not distributed evenly; rather, it is highly enriched in small, discrete foci. The endogenous hLSm4 is similarly localized, as are the overexpressed proteins hLSm1-7, but not hLSm8. The foci also contain two key factors in mRNA degradation, namely the decapping enzyme hDcp1/2 and the exonuclease hXrn1. Moreover, coexpression of wild-type and mutant LSm proteins, as well as fluorescence resonance energy transfer (FRET) studies, indicate that the mammalian proteins hLSm1-7 form a complex similar to the one found in yeast, and that complex formation is required for enrichment of the proteins in the cytoplasmic foci. Therefore, the foci contain a partially or fully assembled machinery for the degradation of mRNA. PMID:12515382

  5. Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

    SciTech Connect

    Bejerman, Nicolás; Giolitti, Fabián; Breuil, Soledad de; Trucco, Verónica; Nome, Claudia; Lenardon, Sergio; Dietzgen, Ralf G.

    2015-09-15

    Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

  6. Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore

    PubMed Central

    Echeverría, Pablo C; Erlejman, Alejandra G; Piwien-Pilipuk, Graciela

    2010-01-01

    In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and MR (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR)-domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor “transformation”) is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin β and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore. PMID:21113270

  7. Using antibody arrays to measure protein abundance and glycosylation: considerations for optimal performance.

    PubMed

    Haab, Brian B; Partyka, Katie; Cao, Zheng

    2013-09-24

    Antibody arrays provide a valuable method for obtaining multiple protein measurements from small volumes of biological samples. Antibody arrays can be designed to target not only core protein abundances (relative or absolute abundances, depending on the availability of standards for calibration), but also posttranslational modifications, provided antibodies or other affinity reagents are available to detect them. Glycosylation is a common modification that has important and diverse functions in both normal and disease biology. Significant progress has been made in developing methods for measuring glycan levels on multiple specific proteins using antibody arrays and glycan-binding reagents. This unit describes practical approaches for developing, optimizing, and using antibody array assays to determine both protein abundance and glycosylation state. Low-volume arrays can be used to reduce sample consumption, and a new way to improve the binding strength of particular glycan-binding reagents through multimerization is discussed. These methods can be useful for a wide range of biological studies in which glycosylation may change and/or affect protein function.

  8. Identification of Proteins of Altered Abundance in Oil Palm Infected with Ganoderma boninense

    PubMed Central

    Al-Obaidi, Jameel R.; Mohd-Yusuf, Yusmin; Razali, Nurhanani; Jayapalan, Jaime Jacqueline; Tey, Chin-Chong; Md-Noh, Normahnani; Junit, Sarni Mat; Othman, Rofina Yasmin; Hashim, Onn Haji

    2014-01-01

    Basal stem rot is a common disease that affects oil palm, causing loss of yield and finally killing the trees. The disease, caused by fungus Ganoderma boninense, devastates thousands of hectares of oil palm plantings in Southeast Asia every year. In the present study, root proteins of healthy oil palm seedlings, and those infected with G. boninense, were analyzed by 2-dimensional gel electrophoresis (2-DE). When the 2-DE profiles were analyzed for proteins, which exhibit consistent significant change of abundance upon infection with G. boninense, 21 passed our screening criteria. Subsequent analyses by mass spectrometry and database search identified caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, enolase, fructokinase, cysteine synthase, malate dehydrogenase, and ATP synthase as among proteins of which abundances were markedly altered. PMID:24663087

  9. Identification of proteins of altered abundance in oil palm infected with Ganoderma boninense.

    PubMed

    Al-Obaidi, Jameel R; Mohd-Yusuf, Yusmin; Razali, Nurhanani; Jayapalan, Jaime Jacqueline; Tey, Chin-Chong; Md-Noh, Normahnani; Junit, Sarni Mat; Othman, Rofina Yasmin; Hashim, Onn Haji

    2014-01-01

    Basal stem rot is a common disease that affects oil palm, causing loss of yield and finally killing the trees. The disease, caused by fungus Ganoderma boninense, devastates thousands of hectares of oil palm plantings in Southeast Asia every year. In the present study, root proteins of healthy oil palm seedlings, and those infected with G. boninense, were analyzed by 2-dimensional gel electrophoresis (2-DE). When the 2-DE profiles were analyzed for proteins, which exhibit consistent significant change of abundance upon infection with G. boninense, 21 passed our screening criteria. Subsequent analyses by mass spectrometry and database search identified caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, enolase, fructokinase, cysteine synthase, malate dehydrogenase, and ATP synthase as among proteins of which abundances were markedly altered. PMID:24663087

  10. The extreme N-terminus of the Caulobacter crescentus surface-layer protein directs export of passenger proteins from the cytoplasm but is not required for secretion of the native protein.

    PubMed

    Bingle, W H; Le, K D; Smit, J

    1996-07-01

    The paracrystalline surface layer (S-layer) of Caulobacter crescentus is composed of a single protein (RsaA, 1026 amino acids) that associates noncovalently with the lipopolysaccharide of the outer membrane. Like many other extracellular proteins of Gram-negative bacteria, the S-layer protein is not processed during transport to the cell surface. To study the secretion of RsaA, several N-terminal deletions of the protein were made by modifying the 5'-region of the rsaA gene. This analysis showed that portions of the N-terminus totalling the first 775 N-terminal amino acids (75% of the protein) could be removed from RsaA without abolishing secretion of the remainder of the protein. Although the RsaA N-terminus was not required for secretion, an N-terminal domain consisting of either 34 or 52 RsaA-derived amino acids promoted export of the alkaline phosphatase reporter (PhoA) and a cellulase reporter (delta CenA) from the cytoplasm; using the cellulase reporter, the efficiency of hybrid protein export was estimated at 9%. No enzyme activity was detected in the cell-free culture fluids as the result of expressing any gene fusion, indicating that no hybrid protein was completely secreted from the cell. RsaA:PhoA hybrid proteins were also exported from the E. coli cytoplasm, a bacterium not expected to contain the necessary machinery for the secretion of RsaA. Taken together, these data indicate that the secretion pathway of RsaA relies on a C-terminal secretion signal and that once separated from the context of the native protein, the extreme N-terminus of RsaA can act as an inefficient cryptic export signal that is not used during native RsaA secretion.

  11. Specificity factors in cytoplasmic polyadenylation.

    PubMed

    Charlesworth, Amanda; Meijer, Hedda A; de Moor, Cornelia H

    2013-01-01

    Poly(A) tail elongation after export of an messenger RNA (mRNA) to the cytoplasm is called cytoplasmic polyadenylation. It was first discovered in oocytes and embryos, where it has roles in meiosis and development. In recent years, however, has been implicated in many other processes, including synaptic plasticity and mitosis. This review aims to introduce cytoplasmic polyadenylation with an emphasis on the factors and elements mediating this process for different mRNAs and in different animal species. We will discuss the RNA sequence elements mediating cytoplasmic polyadenylation in the 3' untranslated regions of mRNAs, including the CPE, MBE, TCS, eCPE, and C-CPE. In addition to describing the role of general polyadenylation factors, we discuss the specific RNA binding protein families associated with cytoplasmic polyadenylation elements, including CPEB (CPEB1, CPEB2, CPEB3, and CPEB4), Pumilio (PUM2), Musashi (MSI1, MSI2), zygote arrest (ZAR2), ELAV like proteins (ELAVL1, HuR), poly(C) binding proteins (PCBP2, αCP2, hnRNP-E2), and Bicaudal C (BICC1). Some emerging themes in cytoplasmic polyadenylation will be highlighted. To facilitate understanding for those working in different organisms and fields, particularly those who are analyzing high throughput data, HUGO gene nomenclature for the human orthologs is used throughout. Where human orthologs have not been clearly identified, reference is made to protein families identified in man.

  12. Unfoldomics of prostate cancer: on the abundance and roles of intrinsically disordered proteins in prostate cancer.

    PubMed

    Landau, Kevin S; Na, Insung; Schenck, Ryan O; Uversky, Vladimir N

    2016-01-01

    Prostatic diseases such as prostate cancer and benign prostatic hyperplasia are highly prevalent among men. The number of studies focused on the abundance and roles of intrinsically disordered proteins in prostate cancer is rather limited. The goal of this study is to analyze the prevalence and degree of disorder in proteins that were previously associated with the prostate cancer pathogenesis and to compare these proteins to the entire human proteome. The analysis of these datasets provides means for drawing conclusions on the roles of disordered proteins in this common male disease. We also hope that the results of our analysis can potentially lead to future experimental studies of these proteins to find novel pathways associated with this disease.

  13. Unfoldomics of prostate cancer: on the abundance and roles of intrinsically disordered proteins in prostate cancer

    PubMed Central

    Landau, Kevin S; Na, Insung; Schenck, Ryan O; Uversky, Vladimir N

    2016-01-01

    Prostatic diseases such as prostate cancer and benign prostatic hyperplasia are highly prevalent among men. The number of studies focused on the abundance and roles of intrinsically disordered proteins in prostate cancer is rather limited. The goal of this study is to analyze the prevalence and degree of disorder in proteins that were previously associated with the prostate cancer pathogenesis and to compare these proteins to the entire human proteome. The analysis of these datasets provides means for drawing conclusions on the roles of disordered proteins in this common male disease. We also hope that the results of our analysis can potentially lead to future experimental studies of these proteins to find novel pathways associated with this disease. PMID:27453073

  14. Mature adipocyte proteome reveals differentially altered protein abundances between lean, overweight and morbidly obese human subjects.

    PubMed

    Benabdelkamel, Hicham; Masood, Afshan; Almidani, Ghaith M; Alsadhan, Abdulmajeed A; Bassas, Abdulelah F; Duncan, Mark W; Alfadda, Assim A

    2015-02-01

    Overweight (OW) and obese individuals are considered to be graded parts of the scale having increasing weight as a common feature. They may not, however, be part of the same continuum and may differ metabolically. In this study we applied an untargeted proteomic approach to compare protein abundances in mature adipocytes derived from the subcutaneous adipose tissue of overweight and morbidly obese female subjects to those of lean age matched controls. Mature adipocytes were isolated from liposuction samples of abdominal subcutaneous adipose tissue collected from both lean (L; n = 7, 23.3 ± 0.4 kg/m(2); mean BMI ± SD), overweight (OW; n = 8, 27.9 ± 0.6 kg/m(2); mean BMI ± SD) and morbidly obese (MOB; n = 7, 44.8 ± 3.8 kg/m(2); mean BMI ± SD) individuals. Total protein extracts were then compared by two-dimensional difference in gel electrophoresis (2D DIGE). One hundred and ten differentially expressed protein spots (i.e., fitting the statistical criteria ANOVA test, p < 0.05; fold-change ≥1.5) were detected, and of these, 89 were identified by MALDI-TOF mass spectrometry. Of these, 66 protein spots were common to both groups whereas 23 were unique to the MOB group. Significant differences were evident in the abundances of key proteins involved in glucose and lipid metabolism, energy regulation, cytoskeletal structure and redox control signaling pathways. Differences in the abundance of some chaperones were also evident. The differentially abundant proteins were investigated using Ingenuity Pathway Analysis (IPA) to establish their associations with known biological functions. The network identified in the OW group with the highest score relates to-: cell-to-cell signaling and interaction; in contrast, in the MOB group the major interacting pathways are associated with lipid metabolism, small molecule biochemistry and cancer. The differences in abundance of the differentially regulated proteins were validated by

  15. Photoreactivation of a Cytoplasmic Virus

    PubMed Central

    Pfefferkorn, E. R.; Boyle, Mary K.

    1972-01-01

    Ultraviolet light-inactivated frog virus 3 is efficiently photoreactivated by chick embryo cells. A cellular enzyme is presumably responsible for this repair of viral deoxyribonucleic acid, for the phenomenon is insensitive to an inhibitor of protein synthesis and is not seen in mammalian cells that are known to lack photoreactivating enzyme. Since frog virus 3 is a cytoplasmic virus, functionally significant amounts of photoreactivating enzyme are probably present in the cytoplasm of chick embryo cells. PMID:5062749

  16. Cytoplasmic polyadenylation element (CPE)- and CPE-binding protein (CPEB)-independent mechanisms regulate early class maternal mRNA translational activation in Xenopus oocytes.

    PubMed

    Charlesworth, Amanda; Cox, Linda L; MacNicol, Angus M

    2004-04-23

    Meiotic cell cycle progression during vertebrate oocyte maturation requires the correct temporal translation of maternal mRNAs encoding key regulatory proteins. The mechanism by which specific mRNAs are temporally activated is unknown, although both cytoplasmic polyadenylation elements (CPE) within the 3'-untranslated region (3'-UTR) of mRNAs and the CPE-binding protein (CPEB) have been implicated. We report that in progesterone-stimulated Xenopus oocytes, the early cytoplasmic polyadenylation and translational activation of multiple maternal mRNAs occur in a CPE- and CPEB-independent manner. We demonstrate that polyadenylation response elements, originally identified in the 3'-UTR of the mRNA encoding the Mos proto-oncogene, direct CPE- and CPEB-independent polyadenylation of an early class of Xenopus maternal mRNAs. Our findings refute the hypothesis that CPE sequences alone account for the range of temporal inductions of maternal mRNAs observed during Xenopus oocyte maturation. Rather, our data indicate that the sequential action of distinct 3'-UTR-directed translational control mechanisms coordinates the complex temporal patterns and extent of protein synthesis during vertebrate meiotic cell cycle progression.

  17. Changes in protein abundance are observed in bacterial isolates from a natural host

    PubMed Central

    Rees, Megan A.; Stinear, Timothy P.; Goode, Robert J. A.; Coppel, Ross L.; Smith, Alexander I.; Kleifeld, Oded

    2015-01-01

    Bacterial proteomic studies frequently use strains cultured in synthetic liquid media over many generations. It is uncertain whether bacterial proteins expressed under these conditions will be the same as the repertoire found in natural environments, or when bacteria are infecting a host organism. Thus, genomic and proteomic characterization of bacteria derived from the host environment in comparison to reference strains grown in the lab, should aid understanding of pathogenesis. Isolates of Corynebacterium pseudotuberculosis were obtained from the lymph nodes of three naturally infected sheep and compared to a laboratory reference strain using bottom-up proteomics, after whole genome sequencing of each of the field isolates. These comparisons were performed following growth in liquid media that allowed us to reach the required protein amount for proteomic analysis. Over 1350 proteins were identified in the isolated strains, from which unique proteome features were revealed. Several of the identified proteins demonstrated a significant abundance difference in the field isolates compared to the reference strain even though there were no obvious differences in the DNA sequence of the corresponding gene or in nearby non-coding DNA. Higher abundance in the field isolates was observed for proteins related to hypoxia and nutrient deficiency responses as well as to thiopeptide biosynthesis. PMID:26528441

  18. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

    PubMed Central

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota. PMID:27042829

  19. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

    PubMed

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota. PMID:27042829

  20. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

    PubMed

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.

  1. Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display

    PubMed Central

    Kushwaha, Rekha; Lloyd, Taylor D.; Schäfermeyer, Kim R.; Kumar, Santosh; Downie, Allan Bruce

    2012-01-01

    Arabidopsis thaliana seeds without functional SEED MATURATION PROTEIN1 (SMP1), a boiling soluble protein predicted to be of intrinsic disorder, presumed to be a LATE EMBRYOGENESIS ABUNDANT (LEA) family protein based on sequence homology, do not enter secondary dormancy after 3 days at 40 °C. We hypothesized that SMP1 may protect a heat labile protein involved in the promotion of secondary dormancy. Recombinant SMP1 and GmPM28, its soybean (Glycine max), LEA4 homologue, protected the labile GLUCOSE-6-PHOSPHATE DEHYROGENASE enzyme from heat stress, as did a known protectant, Bovine Serum Albumin, whether the LEA protein was in solution or attached to the bottom of microtiter plates. Maintenance of a biological function for both recombinant LEA proteins when immobilized encouraged a biopanning approach to screen for potential protein interactors. Phage display with two Arabidopsis seed, T7 phage, cDNA libraries, normalized for transcripts present in the mature, dehydrated, 12-, 24-, or 36-h imbibed seeds, were used in biopans against recombinant SMP1 and GmPM28. Phage titer increased considerably over four rounds of biopanning for both LEA proteins, but not for BSA, at both 25 and at 41 °C, regardless of the library used. The prevalence of multiple, independent clones encoding portions of specific proteins repeatedly retrieved from different libraries, temperatures and baits, provides evidence suggesting these LEA proteins are discriminating which proteins they protect, a novel finding. The identification of putative LEA-interacting proteins provides targets for reverse genetic approaches to further dissect the induction of secondary dormancy in seeds in response to heat stress. PMID:22837651

  2. The cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor

    SciTech Connect

    Leeuw, Frederic de; Zhang Tong; Wauquier, Corinne; Huez, Georges; Kruys, Veronique; Gueydan, Cyril

    2007-12-10

    The cold-inducible RNA-binding protein (CIRP) is a nuclear 18-kDa protein consisting of an amino-terminal RNA Recognition Motif (RRM) and a carboxyl-terminal domain containing several RGG motifs. First characterized for its overexpression upon cold shock, CIRP is also induced by stresses such as UV irradiation and hypoxia. Here, we investigated the expression as well as the subcellular localization of CIRP in response to other stress conditions. We demonstrate that oxidative stress leads to the migration of CIRP to stress granules (SGs) without alteration of expression. Stress granules are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. Relocalization of CIRP into SGs also occurs upon other cytoplasmic stresses (osmotic pressure or heat shock) as well as in response to stresses of the endoplasmic reticulum. CIRP migration into SGs is independent from TIA-1 which has been previously reported to be a general mediator of SG formation, thereby suggesting the existence of multiple pathways leading to SG formation. Moreover, deletion mutants revealed that both RGG and RRM domains can independently promote CIRP migration into SGs. However, the methylation of arginine residues in the RGG domain is necessary for CIRP to exit the nucleus to be further recruited into SGs. By RNA-tethering experiments, we also show that CIRP down-regulates mRNA translation and that this activity is carried by the carboxyl-terminal RG-enriched domain. Altogether, our findings further reveal the diversity of mechanisms by which CIRP is regulated by environmental stresses and provide new insights into CIRP cytoplasmic function.

  3. SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE Directly Interacts with the Cytoplasmic Domain of SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE and Negatively Regulates Leaf Senescence in Arabidopsis.

    PubMed

    Xiao, Dong; Cui, Yanjiao; Xu, Fan; Xu, Xinxin; Gao, Guanxiao; Wang, Yaxin; Guo, Zhaoxia; Wang, Dan; Wang, Ning Ning

    2015-10-01

    Reversible protein phosphorylation mediated by protein kinases and phosphatases plays an important role in the regulation of leaf senescence. We previously reported that the leucine-rich repeat receptor-like kinase SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (AtSARK) positively regulates leaf senescence in Arabidopsis (Arabidopsis thaliana). Here, we report the involvement of a protein serine/threonine phosphatase 2C-type protein phosphatase, SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP), in the negative regulation of Arabidopsis leaf senescence. SSPP transcript levels decreased greatly during both natural senescence and SARK-induced precocious senescence. Overexpression of SSPP significantly delayed leaf senescence in Arabidopsis. Protein pull-down and bimolecular fluorescence complementation assays demonstrated that the cytosol-localized SSPP could interact with the cytoplasmic domain of the plasma membrane-localized AtSARK. In vitro assays showed that SSPP has protein phosphatase function and can dephosphorylate the cytosolic domain of AtSARK. Consistent with these observations, overexpression of SSPP effectively rescued AtSARK-induced precocious leaf senescence and changes in hormonal responses. All our results suggested that SSPP functions in sustaining proper leaf longevity and preventing early senescence by suppressing or perturbing SARK-mediated senescence signal transduction.

  4. SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE Directly Interacts with the Cytoplasmic Domain of SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE and Negatively Regulates Leaf Senescence in Arabidopsis1[OPEN

    PubMed Central

    Xiao, Dong; Cui, Yanjiao; Xu, Fan; Xu, Xinxin; Gao, Guanxiao; Wang, Yaxin; Guo, Zhaoxia; Wang, Dan; Wang, Ning Ning

    2015-01-01

    Reversible protein phosphorylation mediated by protein kinases and phosphatases plays an important role in the regulation of leaf senescence. We previously reported that the leucine-rich repeat receptor-like kinase SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (AtSARK) positively regulates leaf senescence in Arabidopsis (Arabidopsis thaliana). Here, we report the involvement of a protein serine/threonine phosphatase 2C-type protein phosphatase, SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP), in the negative regulation of Arabidopsis leaf senescence. SSPP transcript levels decreased greatly during both natural senescence and SARK-induced precocious senescence. Overexpression of SSPP significantly delayed leaf senescence in Arabidopsis. Protein pull-down and bimolecular fluorescence complementation assays demonstrated that the cytosol-localized SSPP could interact with the cytoplasmic domain of the plasma membrane-localized AtSARK. In vitro assays showed that SSPP has protein phosphatase function and can dephosphorylate the cytosolic domain of AtSARK. Consistent with these observations, overexpression of SSPP effectively rescued AtSARK-induced precocious leaf senescence and changes in hormonal responses. All our results suggested that SSPP functions in sustaining proper leaf longevity and preventing early senescence by suppressing or perturbing SARK-mediated senescence signal transduction. PMID:26304848

  5. A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

    SciTech Connect

    Sharma, Aarti; Lambrechts, Anja; Le thi Hao; Le, Thanh T.; Sewry, Caroline A.; Ampe, Christophe; Burghes, Arthur H.M.; Morris, Glenn E. . E-mail: glenn.morris@rjah.nhs.uk

    2005-09-10

    Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with {beta}-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.

  6. Structure of the cytoplasmic domain of TcpE, the inner membrane core protein required for assembly of the Vibrio cholerae toxin-coregulated pilus.

    PubMed

    Kolappan, Subramaniapillai; Craig, Lisa

    2013-04-01

    Type IV pili are long thin surface-displayed polymers of the pilin subunit that are present in a diverse group of bacteria. These multifunctional filaments are critical to virulence for pathogens such as Vibrio cholerae, which use them to form microcolonies and to secrete the colonization factor TcpF. The type IV pili are assembled from pilin subunits by a complex inner membrane machinery. The core component of the type IV pilus-assembly platform is an integral inner membrane protein belonging to the GspF superfamily of secretion proteins. These proteins somehow convert chemical energy from ATP hydrolysis by an assembly ATPase on the cytoplasmic side of the inner membrane to mechanical energy for extrusion of the growing pilus filament out of the inner membrane. Most GspF-family inner membrane core proteins are predicted to have N-terminal and central cytoplasmic domains, cyto1 and cyto2, and three transmembrane segments, TM1, TM2 and TM3. Cyto2 and TM3 represent an internal repeat of cyto1 and TM1. Here, the 1.88 Å resolution crystal structure of the cyto1 domain of V. cholerae TcpE, which is required for assembly of the toxin-coregulated pilus, is reported. This domain folds as a monomeric six-helix bundle with a positively charged membrane-interaction face at one end and a hydrophobic groove at the other end that may serve as a binding site for partner proteins in the pilus-assembly complex.

  7. Protein abundance of clinically relevant multidrug transporters along the entire length of the human intestine.

    PubMed

    Drozdzik, Marek; Gröer, Christian; Penski, Jette; Lapczuk, Joanna; Ostrowski, Marek; Lai, Yurong; Prasad, Bhagwat; Unadkat, Jashvant D; Siegmund, Werner; Oswald, Stefan

    2014-10-01

    Intestinal transporters are crucial determinants in the oral absorption of many drugs. We therefore studied the mRNA expression (N = 33) and absolute protein content (N = 10) of clinically relevant transporters in healthy epithelium of the duodenum, the proximal and distal jejunum and ileum, and the ascending, transversal, descending, and sigmoidal colon of six organ donors (24-54 years). In the small intestine, the abundance of nearly all studied proteins ranged between 0.2 and 1.6 pmol/mg with the exception of those of OCT3 (<0.1 pmol/mg) and PEPT1 (2.6-4.9 pmol/mg) that accounted for ∼50% of all measured transporters. OATP1A2 was not detected in any intestinal segment. ABCB1, ABCG2, PEPT1, and ASBT were significantly more abundant in jejunum and ileum than in colon. In contrast to this, the level of expression of ABCC2, ABCC3, and OCT3 was found to be highest in colon. Site-dependent differences in the levels of gene and protein expression were observed for ABCB1 and ASBT. Significant correlations between mRNA and protein levels have been found for ABCG2, ASBT, OCT3, and PEPT1 in the small intestine. Our data provide further physiological pieces of the puzzle required to predict intestinal drug absorption in humans.

  8. A Systems Approach to Elucidate Heterosis of Protein Abundances in Yeast*

    PubMed Central

    Blein-Nicolas, Mélisande; Albertin, Warren; da Silva, Telma; Valot, Benoît; Balliau, Thierry; Masneuf-Pomarède, Isabelle; Bely, Marina; Marullo, Philippe; Sicard, Delphine; Dillmann, Christine; de Vienne, Dominique; Zivy, Michel

    2015-01-01

    Heterosis is a universal phenomenon that has major implications in evolution and is of tremendous agro-economic value. To study the molecular manifestations of heterosis and to find factors that maximize its strength, we implemented a large-scale proteomic experiment in yeast. We analyzed the inheritance of 1,396 proteins in 55 inter- and intraspecific hybrids obtained from Saccharomyces cerevisiae and S. uvarum that were grown in grape juice at two temperatures. We showed that the proportion of heterotic proteins was highly variable depending on the parental strain and on the temperature considered. For intraspecific hybrids, this proportion was higher at nonoptimal temperature. Unexpectedly, heterosis for protein abundance was strongly biased toward positive values in interspecific hybrids but not in intraspecific hybrids. Computer modeling showed that this observation could be accounted for by assuming concave relationships between protein abundances and their controlling factors, in line with the metabolic model of heterosis. These results point to nonlinear processes that could play a central role in heterosis. PMID:25971257

  9. Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.

    PubMed

    Rychli, Kathrin; Grunert, Tom; Ciolacu, Luminita; Zaiser, Andreas; Razzazi-Fazeli, Ebrahim; Schmitz-Esser, Stephan; Ehling-Schulz, Monika; Wagner, Martin

    2016-02-01

    The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented. In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains. Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37 °C but also at 10 °C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actin-assembly-inducing protein ActA and the

  10. Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein.

    PubMed Central

    Dastot, F; Sobrier, M L; Duquesnoy, P; Duriez, B; Goossens, M; Amselem, S

    1996-01-01

    The mechanism underlying the generation of soluble growth hormone binding protein (GHBP) probably differs among species. In rats and mice, it involves an alternatively spliced mRNA, whereas in rabbits, it involves limited proteolysis of the membrane-bound growth hormone receptor (GHR). In humans, this latter mechanism is favored, as no transcript coding for a soluble GHR has been detected so far. To test this hypothesis, we analyzed COS-7 cells transiently expressing the full-length human (h) GHR and observed specific GH-binding activity in the cell supernatants. Concomitantly, an alternatively spliced form in the cytoplasmic domain of GHR, hGHR-tr, was isolated from several human tissues. hGHR-tr is identical in sequence to hGHR, except for a 26-bp deletion leading to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor protein. When compared with hGHR, hGHR-tr showed a significantly increased capacity to generate a soluble GHBP. Interestingly, this alternative transcript is also expressed in liver from rabbits, mice, and rats, suggesting that, in these four species, proteolysis of the corresponding truncated transmembrane GHR is a common mechanism leading to GHBP generation. These findings support the hypothesis that GHBP may at least partly result from alternative splicing of the region encoding the intracellular domain and that the absence of a cytoplasmic domain may be involved in increased release of GHBP. Images Fig. 2 Fig. 4 PMID:8855247

  11. The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor.

    PubMed Central

    Rottapel, R; Reedijk, M; Williams, D E; Lyman, S D; Anderson, D M; Pawson, T; Bernstein, A

    1991-01-01

    The W/c-kit and Steel loci respectively encode a receptor tyrosine kinase (Kit) and its extracellular ligand, Steel factor, which are essential for the development of hematopoietic, melanocyte, and germ cell lineages in the mouse. To determine the biochemical basis of the Steel/W developmental pathway, we have investigated the response of the Kit tyrosine kinase and several potential cytoplasmic targets to stimulation with Steel in mast cells derived from normal and mutant W mice. In normal mast cells, Steel induces Kit to autophosphorylate on tyrosine and bind to phosphatidylinositol 3'-kinase (PI3K) and phospholipase C-gamma 1 but not detectably to Ras GTPase-activating protein. Additionally, we present evidence that Kit tyrosine phosphorylation acts as a switch to promote complex formation with PI3K. In mast cells from mice homozygous for the W42 mutant allele, Kit is not tyrosine phosphorylated and fails to bind PI3K following Steel stimulation. In contrast, in the transformed mast cell line P815, Kit is constitutively phosphorylated and binds to PI3K in the absence of ligand. These results suggest that Kit autophosphorylation and its physical association with a unique subset of cytoplasmic signaling proteins are critical for mammalian development. Images PMID:1710023

  12. Changes in Relative Thylakoid Protein Abundance Induced by Fluctuating Light in the Diatom Thalassiosira pseudonana.

    PubMed

    Grouneva, Irina; Muth-Pawlak, Dorota; Battchikova, Natalia; Aro, Eva-Mari

    2016-05-01

    One of the hallmarks of marine diatom biology is their ability to cope with rapid changes in light availability due to mixing of the water column and the lens effect. We investigated how irradiance fluctuations influence the relative abundance of key photosynthetic proteins in the centric diatom Thalassiosira pseudonana by means of mass-spectrometry-based approaches for relative protein quantitation. Most notably, fluctuating-light conditions lead to a substantial overall up-regulation of light-harvesting complex proteins as well as several subunits of photosystems II and I. Despite an initial delay in growth under FL, there were no indications of FL-induced photosynthesis limitation, in contrast to other photosynthetic organisms. Our findings further strengthen the notion that diatoms use a qualitatively different mechanism of photosynthetic regulation in which chloroplast-mitochondria interaction has overtaken crucial regulatory processes of photosynthetic light reactions that are typical for the survival of land plants, green algae, and cyanobacteria. PMID:27025989

  13. Mass Spectrometry and Next-Generation Sequencing Reveal an Abundant and Rapidly Evolving Abalone Sperm Protein

    PubMed Central

    Palmer, Melody R.; McDowall, Margo H.; Stewart, Lia; Ouaddi, Aleena; MacCoss, Michael J.; Swanson, Willie J.

    2014-01-01

    SUMMARY Abalone, a broadcast spawning marine mollusk, is an important model for molecular interactions and positive selection in fertilization, but the focus has previously been on only two sperm proteins, lysin and sp18.We used genomic and proteomic techniques to bring new insights to this model by characterizing the testis transcriptome and sperm proteome of the Red abalone Haliotis rufescens. One pair of homologous, testis-specific proteins contains a secretion signal and is small, abundant, and associated with the acrosome. Comparative analysis revealed that homologs are extremely divergent between species, and show strong evidence for positive selection. The acrosomal localization and rapid evolution of these proteins indicates that they play an important role in fertilization, and could be involved in the species-specificity of sperm-egg interactions in abalone. Our genomic and proteomic characterization of abalone fertilization resulted in the identification of interesting, novel peptides that have eluded detection in this important model system for 20 years. PMID:23585193

  14. Elevated Expression of Pentraxin 3 in Anti-neutrophil Cytoplasmic Antibody-associated Glomerulonephritis with Normal Serum C-reactive Protein.

    PubMed

    Ishida, Risa; Nakai, Kentaro; Fujii, Hideki; Goto, Shunsuke; Hara, Shigeo; Imai, Naofumi; Nishi, Shinichi

    2015-01-01

    A 20-year-old woman was admitted to our hospital with an elevated serum creatinine level of 1.61 mg/dL and a normal C-reactive protein level of less than 0.1 mg/dL. Her myeloperoxidase anti-neutrophil cytoplasmic antibody (ANCA) titer was slightly increased at 9.2 U/mL; a kidney biopsy revealed that 23 of 32 glomeruli had crescents. The expression of pentraxin 3 was detected in her kidney and her plasma pentraxin 3 level was elevated at 63.53 ng/mL. Plasma pentraxin 3 levels may be an activity marker for ANCA-associated glomerulonephritis, particularly when serum C-reactive protein levels are within the normal limits.

  15. Antibody-based analysis reveals “filamentous vs. non-filamentous” and “cytoplasmic vs. nuclear” crosstalk of cytoskeletal proteins

    SciTech Connect

    Kumeta, Masahiro; Hirai, Yuya; Yoshimura, Shige H.; Horigome, Tsuneyoshi; Takeyasu, Kunio

    2013-12-10

    To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus. - Highlights: • A set of monoclonal antibodies were raised against nuclear scaffold proteins. • Helix-based cytoskeletal proteins were involved in nuclear scaffold. • Many cytoskeletal components shuttle into the nucleus in a CRM1-dependent manner. • Sets of antibodies distinguished distinct subcellular localization of a single isoform. • Nuclear keratin is soluble and does not form an obvious filamentous structure.

  16. Phosphorylation of Golgi Peripheral Membrane Protein Grasp65 Is an Integral Step in the Formation of the Human Cytomegalovirus Cytoplasmic Assembly Compartment

    PubMed Central

    Rebmann, G. Michael; Grabski, Robert; Sanchez, Veronica

    2016-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is the largest member of the Herpesviridae and represents a significant cause of disease. During virus replication, HCMV alters cellular functions to facilitate its replication, including significant reorganization of the secretory and endocytic pathways of the infected cell. A defining morphologic change of the infected cell is the formation of a membranous structure in the cytoplasm that is designated the virion assembly compartment (AC), which consists of virion structural proteins surrounded by cellular membranes. The loss of normal Golgi compartment morphology and its relocalization from a juxtanuclear ribbonlike structure to a series of concentric rings on the periphery of the AC represents a readily recognized reorganization of cellular membranes in the HCMV-infected cell. Although trafficking of viral proteins to this compartment is required for the assembly of infectious virions, the functional significance of the reorganization of intracellular membranes like the Golgi membranes into the AC in the assembly of infectious virus remains understudied. In this study, we determined that Golgi membrane ribbon fragmentation increased during the early cytoplasmic phase of virion assembly and that Golgi membrane fragmentation in infected cells was dependent on the phosphorylation of an integral cis-Golgi protein, Grasp65. Inhibition of Golgi membrane fragmentation and of its reorganization into the AC resulted in decreased production of infectious particles and alteration of the incorporation of an essential protein into the envelope of the mature virion. These results demonstrated the complexity of the virus-host cell interactions required for efficient assembly of this large DNA virus. PMID:27703074

  17. Inhibition of cytoplasmic streaming by cytochalasin D is superior to paraformaldehyde fixation for measuring FRET between fluorescent protein-tagged Golgi components.

    PubMed

    Poulsen, Christian Peter; Vereb, György; Geshi, Naomi; Schulz, Alexander

    2013-09-01

    Protein-protein interaction at the organelle level can be analyzed by using tagged proteins and assessing Förster resonance energy transfer (FRET) between fluorescent donor and acceptor proteins. Such studies are able to uncover partners in the regulation of proteins and enzymes. However, any organelle movement is an issue for live FRET microscopy, as the observed organelle must not change position during measurement. One of the mobile organelles in plants is the Golgi apparatus following cytoplasmic streaming. It is involved in the decoration of proteins and processing of complex glycan structures for the cell wall. Understanding of these processes is still limited, but evidence is emerging that protein-protein interaction plays a key role in the function of this organelle. In the past, mobile organelles were usually immobilized with paraformaldehyde (PFA) for FRET-based interaction studies. Here, we show that the actin inhibitor Cytochalasin D (CytD) is superior to PFA for immobilization of Golgi stacks in plant cells. Two glycosyltransferases known to interact were tagged with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, coexpressed in Nicotiana benthamiana leaves and analyzed using confocal microscopy and spectral imaging. Fixation with PFA leads to reduced emission intensity when compared to CytD treatment. Furthermore, the calculated FRET efficiency was significantly higher with CytD than with PFA. The documented improvements are beneficial for all methods measuring FRET, where immobilization of the investigated molecules is necessary. It can be expected that FRET measurement in organelles of animal cells will also benefit from the use of inhibitors acting on the cytoskeleton.

  18. Interaction of cellular proteins with BCL-xL targeted to cytoplasmic inclusion bodies in adenovirus infected cells.

    PubMed

    Subramanian, T; Vijayalingam, S; Kuppuswamy, M; Chinnadurai, G

    2015-09-01

    Adenovirus-mediated apoptosis was suppressed when cellular anti-apoptosis proteins (BCL-2 and BCL-xL) were substituted for the viral E1B-19K. For unbiased proteomic analysis of proteins targeted by BCL-xL in adenovirus-infected cells and to visualize the interactions with target proteins, BCL-xL was targeted to cytosolic inclusion bodies utilizing the orthoreovirus µNS protein sequences. The chimeric protein was localized in non-canonical cytosolic factory-like sites and promoted survival of virus-infected cells. The BCL-xL-associated proteins were isolated from the cytosolic inclusion bodies in adenovirus-infected cells and analyzed by LC-MS. These proteins included BAX, BAK, BID, BIK and BIM as well as mitochondrial proteins such as prohibitin 2, ATP synthase and DNA-PKcs. Our studies suggested that in addition to the interaction with various pro-apoptotic proteins, the association with certain mitochondrial proteins such as DNA-PKcs and prohibitins might augment the survival function of BCL-xL in virus infected cells.

  19. RcLEA, a late embryogenesis abundant protein gene isolated from Rosa chinensis, confers tolerance to Escherichia coli and Arabidopsis thaliana and stabilizes enzyme activity under diverse stresses.

    PubMed

    Zhang, Xuan; Lu, Songchong; Jiang, Changhua; Wang, Yaofeng; Lv, Bo; Shen, Jiabin; Ming, Feng

    2014-07-01

    The late embryogenesis abundant (LEA) protein family is a large protein family that is closely associated with resistance to abiotic stresses in many organisms, such as plants, bacteria and animals. In this study, we isolated a LEA gene, RcLEA, which was cytoplasm-localized, from Rosa chinensis. RcLEA was found to be induced by high temperature through RT-PCR. Overexpression of RcLEA in Escherichia coli improved its growth performance compared with the control under high temperature, low temperature, NaCl and oxidative stress conditions. RcLEA was also overexpressed in Arabidopsis thaliana. The transgenic Arabidopsis showed better growth after high and low temperature treatment and exhibited less peroxide according to 3, 3-diaminobenzidine staining. However, RcLEA did not improve the tolerance to NaCl or osmotic stress in Arabidopsis. In vitro analysis showed that RcLEA was able to prevent the freeze-thaw-induced inactivation or heat-induced aggregation of various substrates, such as lactate dehydrogenase and citrate synthase. It also protected the proteome of E. coli from denaturation when the proteins were heat-shocked or subjected to acidic conditions. Furthermore, bimolecular fluorescence complementation assays suggested that RcLEA proteins function in a complex manner by making the form of homodimers. PMID:24760474

  20. Nanomagnetic competition assay for low-abundance protein biomarker quantification in unprocessed human sera.

    PubMed

    Li, Yuanpeng; Srinivasan, Balasubramanian; Jing, Ying; Yao, Xiaofeng; Hugger, Marie A; Wang, Jian-Ping; Xing, Chengguo

    2010-03-31

    A novel giant magnetoresistive sensor and uniform high-magnetic-moment FeCo nanoparticles (12.8 nm)-based detecting platform with minimized detecting distance was developed for rapid biomolecule quantification from body fluids. Such a system demonstrates specific, accurate, and quick detection and quantification of interleukin-6, a low-abundance protein and a potential cancer biomarker, directly in 4 muL of unprocessed human sera. This platform is expected to facilitate the identification and validation of disease biomarkers. It may eventually lead to a low-cost personal medical device for chronic disease early detection, diagnosis, and prognosis.

  1. Low-abundant protein extraction from complex protein sample using a novel continuous aqueous two-phase systems device.

    PubMed

    Vázquez-Villegas, Patricia; Espitia-Saloma, Edith; Rito-Palomares, Marco; Aguilar, Oscar

    2013-01-01

    The present work describes the application of a novel continuous aqueous two-phase system prototype for the recovery of biomolecules. The prototype is an alternative platform for protein recovery and α-amylase from soybean extracts was used as a model system. The system was selected as an example of low-abundant protein present in complex mixtures. Compared with batch systems, continuous operation in this prototype seems to increase partition coefficient with higher recovery efficiencies. Processing time is reduced at least three times in the continuous system when compared to batch mode, while hold up (volumetric quantity of the opposing phase in a determined phase sample) decreases with decreasing phases flow. Furthermore, similar partition coefficient (Kp > 4) with a higher top phase enzyme recovery (81%) is also obtained in this system probably due to better contact surface between phases, compared with that obtained in batch (79%). A continuous aqueous two-phase system process with purification factor 40-fold higher than batch experiments was achieved. These preliminary results exhibit the potential of continuous systems for the recovery of low-abundant proteins from complex mixtures. The promising performance of this prototype can raise the attention of the industry for the adoption of aqueous two-phase system processes.

  2. Selected Reaction Monitoring to Determine Protein Abundance in Arabidopsis Using the Arabidopsis Proteotypic Predictor1[W][OPEN

    PubMed Central

    Taylor, Nicolas L.; Fenske, Ricarda; Castleden, Ian; Tomaz, Tiago; Nelson, Clark J.; Millar, A. Harvey

    2014-01-01

    In reverse genetic knockout (KO) studies that aim to assign function to specific genes, confirming the reduction in abundance of the encoded protein will often aid the link between genotype and phenotype. However, measuring specific protein abundance is particularly difficult in plant research, where only a limited number of antibodies are available. This problem is enhanced when studying gene families or different proteins derived from the same gene (isoforms), as many antibodies cross react with more than one protein. We show that utilizing selected reaction monitoring (SRM) mass spectrometry allows researchers to confirm protein abundance in mutant lines, even when discrimination between very similar proteins is needed. Selecting the best peptides for SRM analysis to ensure that protein- or gene-specific information can be obtained requires a series of steps, aids, and interpretation. To enable this process in Arabidopsis (Arabidopsis thaliana), we have built a Web-based tool, the Arabidopsis Proteotypic Predictor, to select candidate SRM transitions when no previous mass spectrometry evidence exists. We also provide an in-depth analysis of the theoretical Arabidopsis proteome and its use in selecting candidate SRM peptides to establish assays for use in determining protein abundance. To test the effectiveness of SRM mass spectrometry in determining protein abundance in mutant lines, we selected two enzymes with multiple isoforms, aconitase and malate dehydrogenase. Selected peptides were quantified to estimate the abundance of each of the two mitochondrial isoforms in wild-type, KO, double KO, and complemented plant lines. We show that SRM protein analysis is a sensitive and rapid approach to quantify protein abundance differences in Arabidopsis for specific and highly related enzyme isoforms. PMID:24296071

  3. General anesthetic action at an internal protein site involving the S4-S5 cytoplasmic loop of a neuronal K(+) channel.

    PubMed

    Harris, T; Shahidullah, M; Ellingson, J S; Covarrubias, M

    2000-02-18

    The structural bases of general anesthetic action on a neuronal K(+) channel were investigated using the series of homologous 1-alkanols, electrophysiology, and mutational analysis. Domain swapping between dShaw2 (alkanol-sensitive) and hKv3.4 (alkanol-resistant) and site-directed mutagenesis demonstrated that a 13-amino acid cytoplasmic loop (S4-S5) determines the selective inhibition of native dShaw2 channels by 1-alkanols. The S4-S5 loop may contribute to a receptor for both 1-alkanols and the inactivation particle, because the enhanced 1-alkanol sensitivity of hKv3.4 channels hosting S4-S5 mutations correlates directly with disrupted channel inactivation. Evidence of a discrete protein site was also obtained from the analysis of the relationship between potency and alkyl chain length, which begins to level off after 1-hexanol. Rapid application to the cytoplasmic side of inside-out membrane patches shows that the interaction between dShaw2 channels and 1-alkanols equilibrates in <200 ms. By contrast, the equilibration time is >1000-fold slower when the drug is applied externally to outside-out membrane patches. The data strongly favor a mechanism of inhibition involving a discrete internal site for 1-alkanols in dShaw2 K(+) channels. A new working hypothesis proposes that 1-alkanols lock dShaw2 channels in their closed conformation by a direct interaction at a crevice formed by the S4-S5 loop.

  4. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    SciTech Connect

    Kumari, Gita; Mahalingam, S.

    2009-10-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  5. Cold-Regulated Cereal Chloroplast Late Embryogenesis Abundant-Like Proteins. Molecular Characterization and Functional Analyses

    PubMed Central

    NDong, Christian; Danyluk, Jean; Wilson, Kenneth E.; Pocock, Tessa; Huner, Norman P.A.; Sarhan, Fathey

    2002-01-01

    Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed. PMID:12114590

  6. Dual-nanogold-linked bio-barcodes with superstructures for in situ amplified electronic detection of low-abundance proteins.

    PubMed

    Zhou, Jun; Zhuang, Junyang; Tang, Juan; Li, Qunfang; Tang, Dianping; Chen, Guonan

    2013-04-01

    A novel and nonenzyme immunosensing protocol is proposed for ultrahighly sensitive detection of low-abundance-proteins (carcinoembryonic antigen as a model) using dual nanogold-linked complementary bio-barcodes with superstructures for in situ amplified electronic signals.

  7. ADP1 affects abundance and endocytosis of PIN-FORMED proteins in Arabidopsis.

    PubMed

    Li, Jieru; Li, Ruixi; Jiang, Zhaoyun; Gu, Hongya; Qu, Li-Jia

    2015-01-01

    Auxin, as a vital plant hormone, regulates almost every aspect of plant growth and development. We previously identified a dominant mutant, adp1-D, displaying loss of apical dominance. We also demonstrated that down-regulation of local auxin biosynthesis in adp1-D was responsible for the bushy phenotype of this mutant. Consistent with the reduction of local auxin biosynthesis, we recently discovered that protein abundance of PIN1, PIN3, and PIN7 was reduced in adp1-D without accompanying transcription level changes. Additionally, subcellular analysis revealed that over-expression of ADP1 inhibited endocytosis of PIN proteins. Taken together, we conclude that ADP1 regulates plant architecture through the fine-tuning of local auxin biosynthesis and through post-transcriptional regulation of auxin transporters. PMID:25482774

  8. Structure, dynamics and biophysics of the cytoplasmic protein–protein complexes of the bacterial phosphoenolpyruvate: Sugar phosphotransferase system

    SciTech Connect

    Clore, G. Marius; Venditti, Vincenzo

    2013-10-01

    The bacterial phosphotransferase system (PTS) couples phosphoryl transfer, via a series of bimolecular protein–protein interactions, to sugar transport across the membrane. The multitude of complexes in the PTS provides a paradigm for studying protein interactions, and for understanding how the same binding surface can specifically recognize a diverse array of targets. Fifteen years of work aimed at solving the solution structures of all soluble protein–protein complexes of the PTS has served as a test bed for developing NMR and integrated hybrid approaches to study larger complexes in solution and to probe transient, spectroscopically invisible states, including encounter complexes. We review these approaches, highlighting the problems that can be tackled with these methods, and summarize the current findings on protein interactions.

  9. LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana

    PubMed Central

    Hundertmark, Michaela; Hincha, Dirk K

    2008-01-01

    Background LEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown. Results We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE) and/or low temperature response (LTRE) elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded. Conclusion The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for future efforts to elucidate

  10. Making sense of nuclear localization: A zinc-finger protein encoded by a cytoplasmically replicating plant RNA virus acts a transcription factor

    PubMed Central

    Lukhovitskaya, Nina I.; Gushchin, Vladimir A.; Solovyev, Andrey G.; Savenkov, Eugene I.

    2013-01-01

    Recent studies have uncovered numerous nucleus-localized proteins encoded by plant RNA viruses. Whereas for some of these viruses nuclear (or, more specifically, nucleolar) passage of the proteins is needed for the virus movement within the plant or suppression of host defense, the nuclear function of these proteins remains largely unknown. Recently, the situation has been clarified for one group of plant RNA viruses, the Carlaviruses. Being positive-stranded RNA viruses, carlaviruses multiply exclusively in the cytoplasm. Chrysanthemum virus B (CVB, a carlavirus) encodes a zinc-finger protein p12 targeted to the nucleus in a nuclear localization signal-dependent manner. In a recent work, we demonstrated that p12 directly interacts with chromatin and plant promoters, thus, acts as a eukaryotic transcription factor (TF) and activates expression of a host TF involved in regulation of cell size and proliferation to favor virus infection. Therefore our studies identified a novel nuclear stage of in CVB infection involving modulation of host gene expression and plant development. Whereas it is well established that any RNA virus actively replicating in the cell causes changes in the transcriptome, our study expanded this view by showing that some positive-stranded RNA viruses can directly manipulate host transcription by encoding eukaryotic TFs. PMID:23759549

  11. Spatial Mapping of Protein Abundances in the Mouse Brain by Voxelation Integrated with High-Throughput Liquid Chromatography - Mass Spectrometry

    SciTech Connect

    Petyuk, Vladislav A; Qian, Weijun; Chin, Mark H; Wang, Haixing H; Livesay, Eric A; Monroe, Matthew E; Adkins, Joshua N; Jaitly, Navdeep; Anderson, David J; Camp, David G; Smith, Desmond J; Smith, Richard D

    2007-01-25

    Temporally and spatially resolved mapping of protein abundance patterns within the mammalian brain is of significant interest for understanding brain function and molecular etiologies of neurodegenerative diseases; however, such imaging efforts have been greatly challenged by complexity of the proteome, throughput and sensitivity of applied analytical methodologies, and accurate quantitation of protein abundances across the brain. Here, we describe a methodology for comprehensive spatial proteome mapping that addresses these challenges by employing voxelation integrated with automated microscale sample processing, high-throughput LC system coupled with high resolution Fourier transform ion cyclotron mass spectrometer and a “universal” stable isotope labeled reference sample approach for robust quantitation. We applied this methodology as a proof-of-concept trial for the analysis of protein distribution within a single coronal slice of a C57BL/6J mouse brain. For relative quantitation of the protein abundances across the slice, an 18O-isotopically labeled reference sample, derived from a whole control coronal slice from another mouse, was spiked into each voxel sample and stable isotopic intensity ratios were used to obtain measures of relative protein abundances. In total, we generated maps of protein abundance patterns for 1,028 proteins. The significant agreement of the protein distributions with previously reported data supports the validity of this methodology, which opens new opportunities for studying the spatial brain proteome and its dynamics during the course of disease progression and other important biological and associated health aspects in a discovery-driven fashion.

  12. Presence of cytoplasmic factors functional in peroxisomal protein import implicates organelle-associated defects in several human peroxisomal disorders.

    PubMed Central

    Wendland, M; Subramani, S

    1993-01-01

    Cells from patients with peroxisome-deficient disorders contain membrane ghosts devoid of most matrix contents instead of normal peroxisomes indicating that the underlying molecular defects impair the import of matrix proteins into these peroxisome ghosts. Genetic heterogeneity for the molecular defects was inferred from the assignment of patients with peroxisome-deficient disorders into nine complementation groups. The aim of our studies was to analyze cell lines from six different complementation groups in a systematic manner for the presence of peroxisome ghosts, the ability to import Ser-Lys-Leu-containing proteins into peroxisome ghosts and for the presence of cytosolic factors required for peroxisomal protein import. We show that each of the cell lines analyzed contains peroxisome ghosts, but is unable to import matrix proteins as judged by a peroxisomal import assay using permeabilized cells. The addition of wild type cytosol did not restore the capacity to import matrix proteins but cytosol prepared from these cell lines was functional in stimulation of peroxisomal protein import in a heterologous system. These results implicate organelle-associated molecular defects in each of the six cell lines analyzed. Images PMID:7693762

  13. Physical properties of cytoplasmic intermediate filaments.

    PubMed

    Block, Johanna; Schroeder, Viktor; Pawelzyk, Paul; Willenbacher, Norbert; Köster, Sarah

    2015-11-01

    Intermediate filaments (IFs) constitute a sophisticated filament system in the cytoplasm of eukaryotes. They form bundles and networks with adapted viscoelastic properties and are strongly interconnected with the other filament types, microfilaments and microtubules. IFs are cell type specific and apart from biochemical functions, they act as mechanical entities to provide stability and resilience to cells and tissues. We review the physical properties of these abundant structural proteins including both in vitro studies and cell experiments. IFs are hierarchical structures and their physical properties seem to a large part be encoded in the very specific architecture of the biopolymers. Thus, we begin our review by presenting the assembly mechanism, followed by the mechanical properties of individual filaments, network and structure formation due to electrostatic interactions, and eventually the mechanics of in vitro and cellular networks. This article is part of a Special Issue entitled: Mechanobiology.

  14. A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state.

    PubMed

    Tolleter, Dimitri; Hincha, Dirk K; Macherel, David

    2010-10-01

    Late embryogenesis abundant (LEA) proteins are a highly diverse group of polypeptides expected to play important roles in desiccation tolerance of plant seeds. They are also found in other plant tissues and in some anhydrobotic invertebrates, fungi, protists and prokaryotes. The LEA protein LEAM accumulates in the matrix space of pea (Pisum sativum) mitochondria during late seed maturation. LEAM is an intrinsically disordered protein folding into amphipathic alpha-helix upon desiccation. This suggests that it could interact with the inner mitochondrial membrane, providing structural protection in dry seeds. Here, we have used Fourier-transform infrared and fluorescence spectroscopy to gain insight into the molecular details of interactions of LEAM with phospholipid bilayers in the dry state and their effects on liposome stability. LEAM interacted specifically with negatively charged phosphate groups in dry phospholipids, increasing fatty acyl chain mobility. This led to an enhanced stability of liposomes during drying and rehydration, but also upon freezing. Protection depended on phospholipid composition and was strongly enhanced in membranes containing the mitochondrial phospholipid cardiolipin. Collectively, the results provide strong evidence for a function of LEAM as a mitochondrial membrane protectant during desiccation and highlight the role of lipid composition in the interactions between LEA proteins and membranes.

  15. Cytoplasmic Delivery of Liposomes into MCF-7 Breast Cancer Cells Mediated by Cell-Specific Phage Fusion Coat Protein

    PubMed Central

    Wang, Tao; Yang, Shenghong; Petrenko, Valery A; Torchilin, Vladimir P

    2010-01-01

    Earlier, we have shown that doxorubicin-loaded liposomes (Doxil) modified with a chimeric phage fusion coat protein specific towards MCF-7 breast cancer cells identified from a phage landscape library demonstrated a significantly enhanced association with target cells and an increased cytotoxicity. Based on some structural similarities between the N-terminus of the phage potein and known fusogenic peptides, we hypothesized that, in addition to the specific targeting, the phage protein may possess endosome-escaping potential and an increased cytotoxicity of drug-loaded phage protein-targeted liposomes may be explained by an advantageous combination of both, cell targeting and endosomal escape of drug-loaded nanocarrier. The use of the fluorescence resonance energy transfer (FRET) technique allowed us to clearly demonstrate the pH-dependent membrane fusion activity of the phage protein. Endosomal escape and cytosolic delivery of phage-liposomes was visualized with fluorescence microscopy. Endosome acidification inhibition by bafilomycin A 1 resulted in decreased cytotoxicity of the phage-Doxil, while the endosome disruption by chloroquine had a negligible effect on efficacy of phage-Doxil, confirming its endosomal escape. Our results demonstrated an endosome-escaping property of the phage protein and provided an insight on mechanism of the enhanced cytotoxicity of phage-Doxil. PMID:20438086

  16. Protein synthesis by brain-cortex mitochondria. Characterization of a 55S mitochondrial ribosome as the functional unit in protein synthesis by cortex mitochondria and its distinction from a contaminant cytoplasmic protein-synthesizing system

    PubMed Central

    Hernandez, A.; Burdett, I.; Work, T. S.

    1971-01-01

    Homogenates of rat brain cortex were fractionated by conventional methods of velocity sedimentation and separated into a microsomal and a washed mitochondrial fraction. By electron microscopy the mitochondrial fraction was shown to be rich in synaptosomes. The mitochondria–synaptosome fraction synthesized protein in vitro by a route that was partially inhibited by cycloheximide and partly by chloramphenicol. The relative effectiveness of the two inhibitors varied greatly with the medium used. In the mitochondria–synaptosome fraction active 80S cytoplasmic ribosomes and active 55S mitochondrial ribosomes were detected; these were also seen in the electron microscope. Mild osmotic shock of the mitochondria–synaptosome fraction followed by velocity sedimentation in sucrose–EDTA allowed isolation of a mitochondrial fraction free of synaptosomes. Protein synthesis in this fraction was entirely inhibited by chloramphenicol, but was completely resistant to cycloheximide both in a medium promoting oxidative phosphorylation and in ATP-generating medium. Ouabain had no inhibitory effect on protein synthesis in a purified mitochondrial preparation. It is concluded that brain-cortex mitochondria synthesize protein entirely on 55S mitochondrial ribosomes. ImagesPLATE 4PLATE 1PLATE 2PLATE 3 PMID:5158500

  17. Cytoplasmic and nuclear localizations are important for the hypersensitive response conferred by maize autoactive Rp1-D21 protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Disease resistance (R-) genes have been isolated from many plant species. Most encode nucleotide binding leucine-rich-repeat (NLR) proteins that trigger a rapid localized programmed cell death termed the hypersensitive response (HR) upon pathogen recognition. Despite their structural similarities, d...

  18. Centrosomal Protein DZIP1 Regulates Hedgehog Signaling by Promoting Cytoplasmic Retention of Transcription Factor GLI3 and Affecting Ciliogenesis*

    PubMed Central

    Wang, Chengbing; Low, Wee-Chuang; Liu, Aimin; Wang, Baolin

    2013-01-01

    The primary cilium is required for Hedgehog signaling. So far, all known ciliogenic proteins regulate Hedgehog signaling through their role in ciliogenesis. Here we show that the mouse DZIP1 regulates Hedgehog signaling through two mechanisms. First, DZIP1 interacts with GLI3, a transcriptional regulator for Hedgehog signaling, and prevents GLI3 from entering the nucleus. Second, DZIP1 is required for ciliogenesis. We show that DZIP1 colocalizes and interacts with CEP164, a protein localizing at appendages of the mother centrioles, and IFT88, a component of the intraflagellar transport (IFT) machinery. Functionally, both CEP164 and Ninein appendage proteins fail to localize to ciliary appendages in Dzip1 mutant cells; IFT components are not recruited to the basal body of cilia. Importantly, the accumulation of GLI3 in the nucleus is independent of loss of primary cilia in Dzip1 mutant cells. Therefore, DZIP1 is the first known ciliogenic protein that regulates Hedgehog signaling through a dual mechanism and that biochemically links IFT machinery with Hedgehog pathway components. PMID:23955340

  19. Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair.

    PubMed

    Panigrahi, Sunil K; Hopkins, Kevin M; Lieberman, Howard B

    2015-05-19

    RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9(-/-) relative to Rad9(+/+) embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9(-/-) mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells.

  20. Proteome analysis of abundant proteins extracted from the leaf of Gynura procumbens (Lour.) Merr.

    PubMed

    Hew, Chaw-Sen; Gam, Lay-Harn

    2011-12-01

    Gynura procumbens (Lour.) Merr. is a traditionally used medicinal plant to decrease cholesterol level, reduce high blood pressure, control diabetics, and for treatment of cancer. In our present study, a proteomic approach was applied to study the proteome of the plant that had never analyzed before. We have identified 92 abundantly expressed proteins from the leaves of G. procumbens (Lour.) Merr. Amongst the identified proteins was miraculin, a taste-masking agent with high commercial value. Miraculin made up ∼0.1% of the total protein extracted; the finding of miraculin gave a great commercial value to G. procumbens (Lour.) Merr. as miraculin's natural source is limited while the production of recombinant miraculin faced challenges of not being able to exhibit the taste-masking effect as in the natural miraculin. We believe the discovery of miraculin in G. procumbens (Lour.) Merr., provides commercial feasibility of miraculin in view of the availability of G. procumbens (Lour.) Merr. that grow wildly and easily in tropical climate. PMID:21938418

  1. An in vivo detection system for transient and low-abundant protein interactions and their kinetics in budding yeast.

    PubMed

    Brezovich, Andrea; Schuschnig, Martina; Ammerer, Gustav; Kraft, Claudine

    2015-03-01

    Methylation tracking (M-Track) is a protein-proximity assay in Saccharomyces cerevisiae, allowing the detection of transient protein-protein interactions in living cells. The bait protein is fused to a histone lysine methyl transferase and the prey protein to a methylation acceptor peptide derived from histone 3. Upon interaction, the histone 3 fragment is stably methylated on lysine 9 and can be detected by methylation-specific antibodies. Since methylation marking is irreversible in budding yeast and only takes place in living cells, the occurrence of artifacts during cell lysate preparation is greatly reduced, leading to a more accurate representation of native interactions. So far, this method has been limited to highly abundant or overexpressed proteins. However, many proteins of interest are low-abundant, and overexpression of proteins may interfere with their function, leading to an artificial situation. Here we report the generation of a toolbox including a novel cleavage-enrichment system for the analysis of very low-abundant proteins at their native expression levels. In addition, we developed a system for the parallel analysis of two prey proteins in a single cell, as well as an inducible methylation system. The inducible system allows precise control over the time during which the interaction is detected and can be used to determine interaction kinetics. Furthermore, we generated a set of constructs facilitating the cloning-free genomic tagging of proteins at their endogenous locus by homologous recombination, and their expression from centromeric plasmids.

  2. Measurement of Cytoplasmic Streaming in Drosophila Melanogaster

    NASA Astrophysics Data System (ADS)

    Ganguly, Sujoy; Williams, Lucy; Palacios, Isabel; Goldstein, Raymond

    2010-11-01

    During stage 9 of Drosophila melanogastor oogenesis flow of the oocyte cytoplasm, driven by kinesin 1 motor protein is observed. This cytoplasmic streaming is analyzed by PIV in both wild type and kinesin light chain mutants, revealing striking statistical differences. Further measurements of the rheology of the oocyte allow for estimations of the mechanical energy needed to generate the observed flows.

  3. The Respiratory Syncytial Virus Fusion Protein Targets to the Perimeter of Inclusion Bodies and Facilitates Filament Formation by a Cytoplasmic Tail-Dependent Mechanism

    PubMed Central

    Baviskar, Pradyumna S.; Hotard, Anne L.; Moore, Martin L.

    2013-01-01

    The human respiratory syncytial virus (HRSV) fusion (F) protein cytoplasmic tail (CT) and matrix (M) protein are key mediators of viral assembly, but the underlying mechanisms are poorly understood. A complementation assay was developed to systematically examine the role of the F protein CT in infectious virus production. The ability of F mutants with alanine substitutions in the CT to complement an F-null virus in generating infectious progeny was quantitated by flow cytometry. Two CT regions with impact on infectious progeny production were identified: residues 557 to 566 (CT-R1) and 569 to 572 (CT-R2). Substitutions in CT-R1 decreased infectivity by 40 to 85% and increased the level of F-induced cell-cell fusion but had little impact on assembly of viral surface filaments, which are believed to be virions. Substitutions in CT-R2, as well as deletion of the entire CT, abrogated infectious progeny production and impaired viral filament formation. However, CT-R2 mutations did not block but rather delayed the formation of viral filaments, which continued to form at a low rate and contained the viral M protein and nucleoprotein (N). Microscopy analysis revealed that substitutions in CT-R2 but not CT-R1 led to accumulation of M and F proteins within and at the perimeter of viral inclusion bodies (IBs), respectively. The accumulation of M and F at IBs and coincident strong decrease in filament formation and infectivity upon CT-R2 mutations suggest that F interaction with IBs is an important step in the virion assembly process and that CT residues 569 to 572 act to facilitate release of M-ribonucleoprotein complexes from IBs. PMID:23903836

  4. Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of B and T lymphocyte attenuator required for association with protein tyrosine phosphatases SHP-1 and SHP-2.

    PubMed

    Gavrieli, Maya; Watanabe, Norihiko; Loftin, Susan K; Murphy, Theresa L; Murphy, Kenneth M

    2003-12-26

    B and T lymphocytes express receptors providing positive and negative co-stimulatory signals. We recently identified a novel co-stimulatory molecule, B and T lymphocyte attenuator (BTLA), which exerts inhibitory effects on B and T lymphocytes. The cytoplasmic domain of murine and human BTLA share three conserved tyrosine-based signaling motifs, a Grb-2 recognition consensus, and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of the cytoplasmic domain of BTLA induced the association with the protein tyrosine phosphatases SHP-1 and SHP-2. Association of SHP-1 and SHP-2 to other receptors can involve recruitment to either a single receptor ITIM or to two receptor ITIMs. Here, we analyzed the requirements of BTLA interaction with SHP-1 and SHP-2 in a series of murine and human BTLA mutants. For human BTLA, mutations of either Y257 or Y282, but not Y226, abrogated association with both SHP-1 and SHP-2. For murine BTLA, mutation of either Y274 or Y299, but not Y245, also abrogated association with both SHP-1 and SHP-2. These results indicate that for both murine and human BTLA, association with SHP-1 or SHP-2 requires both of conserved ITIM motifs and does not involve the conserved Grb-2 consensus. Thus, similar to the bisphosphoryl tyrosine-based activation motif (BTAM) by which the Grb-2 associated binder (Gab1), PDGF receptor, and PECAM-1 recruit SHP-2, BTLA also relies on dual ITIMs for its association with the phosphatases SHP-1 and SHP-2. PMID:14652006

  5. Iron regulates cytoplasmic levels of a novel iron-responsive element-binding protein without aconitase activity.

    PubMed

    Guo, B; Yu, Y; Leibold, E A

    1994-09-30

    Iron-responsive element-binding proteins (IRE-BPs) are cytosolic proteins that bind to a conserved RNA stem-loop, termed the iron-responsive element (IRE), that is located in the 5'- or 3'-untranslated regions of mRNAs involved in iron metabolism. Binding of the IRE-BP to 5'-IREs represses translation, whereas binding to 3'-IREs stabilizes the mRNA. The previously identified IRE-BP (BP1) contains a 4Fe-4S cluster and has sequence homology to mitochondrial aconitase. The 4Fe-4S cluster is important for iron-dependent regulation: BP1 containing iron has low affinity for the IRE and contains aconitase activity, whereas BP1 lacking iron has high affinity for the IRE, but lacks aconitase activity. A second IRE-BP (BP2) has been identified in rat tissues and cells and exhibits many of the hallmarks of an IRE-BP, including binding to the IRE and functioning as a translational repressor of IRE-containing RNAs. BP1 and BP2 RNA binding activities are decreased in extracts from cells treated with iron, indicating that BP1 and BP2 are negatively regulated by iron. Although BP1 and BP2 share similar characteristics, they differ in two significant ways. Unlike BP1 levels, which do not change when RNA binding activity decreases in response to iron, BP2 decreases to undetectable levels in extracts from cells treated with iron; and unlike BP1, BP2 does not have aconitase activity. These data indicate that BP1 and BP2 are distinct proteins that have similar specificity for IRE binding and that function similarly in translation, but are regulated by iron via different mechanisms. PMID:7523370

  6. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

    PubMed Central

    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

  7. Three-dimensional structure of the bifunctional protein PCD/DCoH, a cytoplasmic enzyme interacting with transcription factor HNF1.

    PubMed Central

    Ficner, R; Sauer, U H; Stier, G; Suck, D

    1995-01-01

    The bifunctional protein pterin-4a-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF1 (DCoH) is a cytoplasmic enzyme involved in the tetrahydrobiopterin regeneration and is found in complex with the transcription factor HNF1 in liver cell nuclei. An atypical hyperphenylalaninemia and the depigmentation disorder vitiligo are related to a deficiency of PCD/DCoH activity. The crystal structure of PCD/DCoH was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.5% at 2.7 A resolution. The single domain monomer comprises three alpha-helices packed against one side of a four-stranded, antiparallel beta-sheet. The functional enzyme is a homo-tetramer of 222 symmetry where each of the monomers contributes one helix to a central four helix bundle. In the tetramer two monomers form an eight-stranded, antiparallel beta-sheet with six helices packing against it from one side. The concave, hydrophobic surface of the eight-stranded beta-sheet with its two protruding loops at either end is reminiscent of the saddle-like shape seen in the TATA-box binding protein. PCD/DCoH binds as a dimer to the helical dimerization domain of dimeric HNF1 forming a hetero-tetramer possibly through a mixed four helix bundle. Images PMID:7744010

  8. Lyar, a cell growth-regulating zinc finger protein, was identified to be associated with cytoplasmic ribosomes in male germ and cancer cells.

    PubMed

    Yonezawa, Kahori; Sugihara, Yoshihiko; Oshima, Kenzi; Matsuda, Tsukasa; Nadano, Daita

    2014-10-01

    Translational control is a basic mechanism for gene regulation in cells and important for tissue growth and development in mammals. Deregulation of the mechanism thus causes diseases such as cancer. Considering the importance of the ribosome as a factory of polypeptide synthesis, some new factors have been expected to be associated with the ribosome and involved in translational control. Our proteomic survey for these factors identified a zinc finger protein, Lyar, in cytoplasmic ribosomes of the rodent testis. Subcellular fractionation of the testis provided data supporting association of Lyar with ribosomes. Lyar was then suggested to be included in the 60S large subunit, but not in polysomes, by ultracentrifugation of testicular ribosomes. While analysis of tissue distribution of Lyar has indicated its testis-predominant expression, Lyar mRNA was expressed in the cancer cells originated from tissues other than testis, and Lyar promoted proliferation of NIH-3T3 cells. Furthermore, translation was increased by Lyar in vitro, pointing out the first experimental link between this protein and translation. Taken together, Lyar seems to be a new player in translational control and a potential target for cancer therapy.

  9. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    SciTech Connect

    Lawrence, Paul; Schafer, Elizabeth A.; Rieder, Elizabeth

    2012-03-30

    Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C{sup pro} induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5 Prime non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C{sup pro}.

  10. Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding.

    PubMed

    Windheim, Mark; Höning, Stefan; Leppard, Keith N; Butler, Larissa; Seed, Christina; Ponnambalam, Sreenivasan; Burgert, Hans-Gerhard

    2016-03-25

    The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved, resulting in secretion of its large ectodomain (sec49K). sec49K binds to CD45 on leukocytes, impairing activation and functions of natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the early endosomes, and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes, but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YXXΦ (where Φ represents a bulky hydrophobic amino acid) and LL, that are important for binding the clathrin adaptor proteins AP-1 and AP-2in vitro Surprisingly, mutating the LL motif, either alone or together with YXXΦ, did not prevent proteolytic processing but increased cell surface expression and secretion. Upon brefeldin A treatment, cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix metalloproteases.

  11. Gossypol Increases Expression of the Pro-apoptotic BH3-only Protein NOXA through a Novel Mechanism Involving Phospholipase A2, Cytoplasmic Calcium, and Endoplasmic Reticulum Stress*

    PubMed Central

    Soderquist, Ryan S.; Danilov, Alexey V.; Eastman, Alan

    2014-01-01

    Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We demonstrated previously that gossypol failed to directly inhibit BCL2 in cells or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL, respectively, for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pretreatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress, and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity, indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154+ stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells, suggesting that a combination of these drugs may be efficacious in the clinic. PMID:24778183

  12. Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding.

    PubMed

    Windheim, Mark; Höning, Stefan; Leppard, Keith N; Butler, Larissa; Seed, Christina; Ponnambalam, Sreenivasan; Burgert, Hans-Gerhard

    2016-03-25

    The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved, resulting in secretion of its large ectodomain (sec49K). sec49K binds to CD45 on leukocytes, impairing activation and functions of natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the early endosomes, and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes, but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YXXΦ (where Φ represents a bulky hydrophobic amino acid) and LL, that are important for binding the clathrin adaptor proteins AP-1 and AP-2in vitro Surprisingly, mutating the LL motif, either alone or together with YXXΦ, did not prevent proteolytic processing but increased cell surface expression and secretion. Upon brefeldin A treatment, cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix metalloproteases. PMID:26841862

  13. Abscisic acid and late embryogenesis abundant protein profile changes in winter wheat under progressive drought stress.

    PubMed

    Vaseva, I I; Grigorova, B S; Simova-Stoilova, L P; Demirevska, K N; Feller, U

    2010-09-01

    Three varieties (cv. Pobeda, Katya and Sadovo) of winter wheat (Triticum aestivum), differing in their agronomic characteristics, were analysed during progressive soil water stress and recovery at early vegetation stages. Changes in abscisic acid content, SDS-PAGE and immunoblot profiles of proteins that remained soluble upon heating were monitored. Initially higher ABA content in control Pobeda and Katya corresponded to earlier expression of the studied late embryogenesis abundant (LEA) proteins. A combination of higher ABA content, early immunodetection of dehydrins, and a significant increase of WZY2 transcript levels were observed in drought-stressed leaves of the tolerant variety Katya. One-step RT-PCR analyses of some acidic dehydrin genes (WCOR410b, TADHN) documented their relatively constant high expression levels in leaves under drought stress during early vegetative development. Neutral WZY2 dehydrin, TaLEA2 and TaLEA3 transcripts accumulated gradually with increasing water deficit. Delayed expression of TaLEA2 and TaLEA3 genes was found in the least drought-tolerant wheat, Sadovo. The expression profile of WZY2 revealed two distinct and separate bands, suggesting alternative splicing, which altered as water stress increased.

  14. Ubiquitous Autofragmentation of Fluorescent Proteins Creates Abundant Defective Ribosomal Products (DRiPs) for Immunosurveillance*

    PubMed Central

    Wei, Jiajie; Gibbs, James S.; Hickman, Heather D.; Cush, Stephanie S.; Bennink, Jack R.; Yewdell, Jonathan W.

    2015-01-01

    broadly, given the wide use of fluorescent proteins, their ubiquitous and abundant fragmentation must be considered when interpreting experiments using these extremely useful probes. PMID:25971973

  15. Pvlea-18, a Member of a New Late-Embryogenesis-Abundant Protein Family That Accumulates during Water Stress and in the Growing Regions of Well-Irrigated Bean Seedlings1

    PubMed Central

    Colmenero-Flores, José M.; Moreno, Liz P.; Smith, Claudia E.; Covarrubias, Alejandra A.

    1999-01-01

    Pvlea-18 is a novel stress gene whose transcript is present in the dry embryo and the endosperm from bean (Phaseolus vulgaris) seeds. It accumulates in vegetative tissues in response to water deficit and abscisic acid application (J.M. Colmenero-Flores, F. Campos, A. Garciarrubio, A.A. Covarrubias [1997] Plant Mol Biol 35: 393–405). We show that the Pvlea-18 gene encodes a 14-kD protein that accumulates during late embryogenesis. Related proteins have been detected in both monocots and dicots, indicating that PvLEA-18 is a member of a new family of LEA (Late Embryogenesis Abundant) proteins. We also show that the PvLEA-18 transcript and protein accumulate not only in different organs of the bean seedlings during water stress but also in well-irrigated seedlings. This accumulation occurs in seedling regions with more negative values of water and osmotic potentials, such as the growing region of the hypocotyl. This phenomenon has not previously been described for LEA proteins. Immunohistochemical localization showed that the PvLEA-18 protein is present in the nucleus and cytoplasm of all cell types, with a higher accumulation in the epidermis and vascular cylinder tissues, particularly in protoxylem cells and root meristematic tissues. We found a similar localization but a higher abundance in water-stressed seedlings. PMID:10318687

  16. Comparative Study of Human and Mouse Postsynaptic Proteomes Finds High Compositional Conservation and Abundance Differences for Key Synaptic Proteins

    PubMed Central

    Bayés, Àlex; Collins, Mark O.; Croning, Mike D. R.; van de Lagemaat, Louie N.; Choudhary, Jyoti S.; Grant, Seth G. N.

    2012-01-01

    Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease. PMID:23071613

  17. Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria

    PubMed Central

    Jia, Qiong; Hu, Xinling; Shi, Dawei; Zhang, Yan; Sun, Meihao; Wang, Jianwei; Mi, Kaixia; Zhu, Guofeng

    2016-01-01

    The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells. PMID:27762279

  18. Molecular cloning and expression of hardening-induced genes in Chlorella vulgaris C-27: the most abundant clone encodes a late embryogenesis abundant protein.

    PubMed

    Joh, T; Honjoh, K; Yoshimoto, M; Funabashi, J; Miyamoto, T; Hatano, S

    1995-01-01

    To investigate the effects of hardening on gene expression in Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27), a frost-hardy strain, 17 cDNA clones corresponding to hardening-induced Chlorella (hiC) genes were isolated by differential screening of a cDNA library from 6-h hardened cells. Northern blot analysis of transcripts of hiC genes showed that these genes are specifically induced by hardening and that their patterns of induction vary. Southern blots of genomic DNAs from two strains (Chlorella ellipsoidea Gerneck IAM C-102, chilling-sensitive; and C. vulgaris C-27, frost-hardy) of Chlorella indicated that ten hiC clones out of 17 hybridized only with DNA of strain C-27 and the other seven clones hybridized with DNA of both strains. However, of these seven clones, transcripts corresponding to six clones did not accumulate in strain C-102 at low temperatures. The sequence of a deduced protein encoded by the most abundant clone, hiC6, exhibited homology to sequences of Group III LEA (late embryogenesis abundant) proteins and had an amino-terminal amino acid sequence that was similar to the sequences of chloroplast transit peptides. PMID:7719632

  19. Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli

    PubMed Central

    Koprowski, Piotr; Grajkowski, Wojciech; Balcerzak, Marcin; Filipiuk, Iwona; Fabczak, Hanna; Kubalski, Andrzej

    2015-01-01

    Bacterial mechano-sensitive (MS) channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS) family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics. PMID:25996836

  20. Mass spectrometry in cancer biomarker research: a case for immunodepletion of abundant blood-derived proteins from clinical tissue specimens

    PubMed Central

    Prieto, DaRue A; Johann, Donald J; Wei, Bih-Rong; Ye, Xiaoying; Chan, King C; Nissley, Dwight V; Simpson, R Mark; Citrin, Deborah E; Mackall, Crystal L; Linehan, W Marston; Blonder, Josip

    2014-01-01

    The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g., serum/plasma) for the removal of highly abundant proteins is a reasonable and reproducible solution. Often overlooked, clinical tissue specimens also contain a formidable amount of highly abundant blood-derived proteins present in tissue-embedded networks of blood/lymph capillaries and interstitial fluid. Hence, the dynamic range impediment to biomarker discovery remains a formidable obstacle, regardless of clinical sample type (solid tissue and/or body fluid). Thus, we optimized and applied simultaneous immunodepletion of blood-derived proteins from solid tissue and peripheral blood, using clear cell renal cell carcinoma as a model disease. Integrative analysis of data from this approach and genomic data obtained from the same type of tumor revealed concordant key pathways and protein targets germane to clear cell renal cell carcinoma. This includes the activation of the lipogenic pathway characterized by increased expression of adipophilin (PLIN2) along with 'cadherin switching', a phenomenon indicative of transcriptional reprogramming linked to renal epithelial dedifferentiation. We also applied immunodepletion of abundant blood-derived proteins to various tissue types (e.g., adipose tissue and breast tissue) showing unambiguously that the removal of abundant blood-derived proteins represents a powerful tool for the reproducible profiling of tissue proteomes. Herein, we show that the removal of abundant blood-derived proteins from solid tissue specimens is of equal importance to depletion of body fluids and recommend its routine use in the context of biological discovery and

  1. Membrane-type 1 matrix metalloproteinase cytoplasmic tail-binding protein-1 is a new member of the Cupin superfamily. A possible multifunctional protein acting as an invasion suppressor down-regulated in tumors.

    PubMed

    Uekita, Takamasa; Gotoh, Isamu; Kinoshita, Takeshi; Itoh, Yoshifumi; Sato, Hiroshi; Shiomi, Takayuki; Okada, Yasunori; Seiki, Motoharu

    2004-03-26

    Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) is an enzyme that promotes tumor cell invasion in tissues. Although the proteolytic activity of MT1-MMP is indispensable for invasion, it is also regulated by functions of the cytoplasmic tail. In this study we obtained a new human gene whose product binds to the tail sequence in yeast. The product, MTCBP-1, is a 19-kDa protein that belongs to the newly proposed Cupin superfamily composed of proteins with diverse functions. MTCBP-1 expressed in cells formed a complex with MT1-MMP and co-localized at the membrane. It was also detected in both the cytoplasm and nucleus, where MT1-MMP does not exist. In human tumor cell lines MTCBP-1 expression was significantly low compared with non-transformed fibroblasts, and enforced expression of MTCBP-1 inhibited the activity of MT1-MMP in promoting cell migration and invasion. MTCBP-1 showed significant homology to the bacterial aci-reductone dioxygenase, which is an enzyme in methionine metabolism. The C-terminal part of MTCBP-1 is identical to Sip-L, which is reported to be important for human hepatitis C virus replication. Thus, MTCBP-1 may have multiple functions other than the regulation of MT1-MMP, which presumably depends on the subcellular compartment.

  2. Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry.

    PubMed

    Lee, Hae-Won; Roh, Seong Woon; Cho, Kichul; Kim, Kil-Nam; Cha, In-Tae; Yim, Kyung June; Song, Hye Seon; Nam, Young-Do; Oda, Tatsuya; Chung, Young-Ho; Kim, Soo Jung; Choi, Jong-Soon; Kim, Daekyung

    2015-01-01

    The blooms of toxic phototrophic microorganisms, such as microalgae and cyanobacteria, which are typically found in freshwater and marine environments, are becoming more frequent and problematic in aquatic systems. Due to accumulation of toxic algae, harmful algal blooms (HABs) exert negative effects on aquatic systems. Therefore, rapid detection of harmful microalgae is important for monitoring the occurrence of HABs. Mass spectrometry-based methods have become sensitive, specific techniques for the identification and characterization of microorganisms. Matrix-assisted laser desorption/ionization (MALDI) with time-of-flight (TOF) mass spectrometry (MS) allows us to measure a unique molecular fingerprint of highly abundant proteins in a microorganism and has been used for the rapid, accurate identification of bacteria and fungi in clinical microbiology. Here, we tested the specificity of MALDI-TOF MS using microalgal strains (Heterocapsa, Alexandrium, Nannochloropsis, Chaetoceros, Chlorella, and Dunaliella spp.). Our research suggested that this method was comparable in terms of the rapid identification of microalgea to conventional methods based on genetic information and morphology. Thus, this efficient mass spectrometry-based technique may have applications in the rapid identification of harmful microorganisms from aquatic environmental samples.

  3. Brugia malayi abundant larval transcript 2 protein treatment attenuates experimentally-induced colitis in mice.

    PubMed

    Khatri, Vishal; Amdare, Nitin; Yadav, Ravi Shankar; Tarnekar, Aaditya; Goswami, Kalyan; Reddy, Maryada Venkata Rami

    2015-11-01

    Helminths are known to modulate host's immunity by suppressing host protective pro-inflammatory responses. Such immunomodulatory effects have been experimentally shown to have therapeutic implications in immune mediated disorders. In the present study, we have explored a filarial protein i.e. Brugia malayi recombinant abundant larval transcript 2 (rBmALT2) for its therapeutic effect in dextran sodium sulfate (DSS) induced colitis in mouse model. The immunomodulatory activity of rBmALT-2 was initially confirmed by demonstrating that it suppressed the lipopolysaccharide (LPS) induced nitric oxide synthesis and down-regulated the expression of pro-inflammatory cytokines in vitro by peritoneal exudate cells of mice. Treatment with rBmALT2 reduced severity of colitis associated with significant reduction in weight loss, disease activity, colon damage, mucosal edema and histopathological score including myeloperoxidase activity in colon tissues. rBmALT2 was comparatively more effective in attenuation of colitis when used in the preventive mode than when used for curative purpose. The therapeutic effect of rBmALT2 was found to be associated with downregulation of IFN-γ, IL-6, IL-17 and upregulation of IL-10 cytokines. These results provide strong experimental evidence that BmALT2 could be a potential alternative therapeutic agent in colitis. PMID:26669016

  4. Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry.

    PubMed

    Lee, Hae-Won; Roh, Seong Woon; Cho, Kichul; Kim, Kil-Nam; Cha, In-Tae; Yim, Kyung June; Song, Hye Seon; Nam, Young-Do; Oda, Tatsuya; Chung, Young-Ho; Kim, Soo Jung; Choi, Jong-Soon; Kim, Daekyung

    2015-01-01

    The blooms of toxic phototrophic microorganisms, such as microalgae and cyanobacteria, which are typically found in freshwater and marine environments, are becoming more frequent and problematic in aquatic systems. Due to accumulation of toxic algae, harmful algal blooms (HABs) exert negative effects on aquatic systems. Therefore, rapid detection of harmful microalgae is important for monitoring the occurrence of HABs. Mass spectrometry-based methods have become sensitive, specific techniques for the identification and characterization of microorganisms. Matrix-assisted laser desorption/ionization (MALDI) with time-of-flight (TOF) mass spectrometry (MS) allows us to measure a unique molecular fingerprint of highly abundant proteins in a microorganism and has been used for the rapid, accurate identification of bacteria and fungi in clinical microbiology. Here, we tested the specificity of MALDI-TOF MS using microalgal strains (Heterocapsa, Alexandrium, Nannochloropsis, Chaetoceros, Chlorella, and Dunaliella spp.). Our research suggested that this method was comparable in terms of the rapid identification of microalgea to conventional methods based on genetic information and morphology. Thus, this efficient mass spectrometry-based technique may have applications in the rapid identification of harmful microorganisms from aquatic environmental samples. PMID:25476355

  5. Effects of elevated circulating cortisol levels on hydromineral status and gill tight junction protein abundance in the stenohaline goldfish.

    PubMed

    Chasiotis, Helen; Kelly, Scott P

    2012-01-15

    A role for cortisol in the regulation of hydromineral balance and gill tight junction (TJ) protein transcript abundance in the stenohaline freshwater goldfish was investigated. Intraperitoneal cortisol implants (50, 100, 200, 400 μg cortisol/g body weight) were used to dose-dependently elevate circulating cortisol levels over a 4 day period. Elevated cortisol did not significantly alter serum osmolality, serum Na(+) or muscle water content, however serum glucose and gill Na(+)-K(+)-ATPase activity were significantly increased and serum Cl(-) levels were significantly reduced when compared to control groups. Transcript levels for glucocorticoid receptor 1 (GR1) and mineralocorticoid receptor (MR) in the gill remained unchanged by cortisol treatment, however glucocorticoid receptor 2 (GR2) mRNA abundance was significantly down-regulated. Conversely, cortisol treatment significantly increased transcript and protein abundance of the TJ protein occludin in goldfish gill tissue, as well as mRNA abundance for claudin e, 7 and 8d. Goldfish tissue expression profiles demonstrated that transcripts encoding for these claudins are particularly abundant in the gill. Overall, results suggest a 'tightened' gill epithelium in response to elevated cortisol levels in goldfish. However, negative autoregulation of gill GR2 transcript suggests a lessened capacity to respond to cortisol and thus a potentially 'dampened' corticosteroid-mediated effect in the gill. Reduced systemic Cl(-) levels also suggest that sustained cortisol elevation in goldfish may have a detrimental effect on other ionoregulatory tissues.

  6. Rearrangements in the Relative Orientation of Cytoplasmic Domains Induced by a Membrane-anchored Protein Mediate Modulations in Kv Channel Gating*

    PubMed Central

    Lvov, Anatoli; Greitzer, Dafna; Berlin, Shai; Chikvashvili, Dodo; Tsuk, Sharon; Lotan, Ilana; Michaelevski, Izhak

    2009-01-01

    Interdomain interactions between intracellular N and C termini have been described for various K+ channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the “docking loop” at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels. PMID:19690160

  7. Increased levels of reactive oxygen species and expression of a cytoplasmic aconitase/iron regulatory protein 1 homolog during the early response of maize pulvini to gravistimulation.

    PubMed

    Clore, A M; Doore, S M; Tinnirello, S M N

    2008-01-01

    The maize (Zea mays L.) stem pulvinus is a disc of tissue located apical to each node that functions to return a tipped stem to a more upright position via increased cell elongation on its lower side. We investigated the possibility that reactive oxygen species (ROS) and hydrogen peroxide (H2O2), in particular, are involved in the gravitropic response of the pulvinus prior to initiation of the growth response by employing the cytochemical stain 3,3'-diaminobenzidine (DAB). DAB polymers were found in the bundle sheath cells of gravistimulated pulvini in association with amyloplasts after 1 min of gravistimulation, and the signal spread throughout the cytosol of these cells by 30 min. Furthermore, treatment of maize stem explants containing pulvini with 1 mm H2O2 on their upper sides caused reversal of bending polarity. Similar, though less dramatic, results were obtained via application of 1 mm ascorbic acid to the lower side of the explants. In addition, we determined that a maize cytoplasmic aconitase/iron regulatory protein 1 (IRP1) homolog is up-regulated in the pulvinus bundle sheath cells after gravistimulation using suppressive subtractive hybridization PCR (SSH PCR), real-time RT-PCR and in situ hybridization. Although we do not yet know the role of the IRP1 homolog in the pulvinus, the protein is known to be a redox sensor in other systems. Collectively, our results point to an increase in ROS quite early in the gravitropic signalling pathway and its possible role in determining the direction of bending of the pulvini. We speculate that an ROS burst may serve to link the physical phenomenon of amyloplast sedimentation to the changes in cellular biochemistry and gene expression that facilitate directional growth. PMID:18004982

  8. An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation.

    PubMed

    Ta, Duy Tien; Redeker, Erik Steen; Billen, Brecht; Reekmans, Gunter; Sikulu, Josephine; Noben, Jean-Paul; Guedens, Wanda; Adriaensens, Peter

    2015-10-01

    In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications. PMID:26243885

  9. Regulation of the Abundance of Kaposi’s Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2

    PubMed Central

    Chang, Tzu-Hsuan; Chen, Lee-Wen; Shih, Ying-Ju; Chang, Li-Kwan; Liu, Shih-Tung; Chang, Pey-Jium

    2016-01-01

    The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50. PMID:27698494

  10. Intergenic sequence between Arabidopsis caseinolytic protease B-cytoplasmic/heat shock protein100 and choline kinase genes functions as a heat-inducible bidirectional promoter.

    PubMed

    Mishra, Ratnesh Chandra; Grover, Anil

    2014-11-01

    In Arabidopsis (Arabidopsis thaliana), the At1g74310 locus encodes for caseinolytic protease B-cytoplasmic (ClpB-C)/heat shock protein100 protein (AtClpB-C), which is critical for the acquisition of thermotolerance, and At1g74320 encodes for choline kinase (AtCK2) that catalyzes the first reaction in the Kennedy pathway for phosphatidylcholine biosynthesis. Previous work has established that the knockout mutants of these genes display heat-sensitive phenotypes. While analyzing the AtClpB-C promoter and upstream genomic regions in this study, we noted that AtClpB-C and AtCK2 genes are head-to-head oriented on chromosome 1 of the Arabidopsis genome. Expression analysis showed that transcripts of these genes are rapidly induced in response to heat stress treatment. In stably transformed Arabidopsis plants harboring this intergenic sequence between head-to-head oriented green fluorescent protein and β-glucuronidase reporter genes, both transcripts and proteins of the two reporters were up-regulated upon heat stress. Four heat shock elements were noted in the intergenic region by in silico analysis. In the homozygous transfer DNA insertion mutant Salk_014505, 4,393-bp transfer DNA is inserted at position -517 upstream of ATG of the AtClpB-C gene. As a result, AtCk2 loses proximity to three of the four heat shock elements in the mutant line. Heat-inducible expression of the AtCK2 transcript was completely lost, whereas the expression of AtClpB-C was not affected in the mutant plants. Our results suggest that the 1,329-bp intergenic fragment functions as a heat-inducible bidirectional promoter and the region governing the heat inducibility is possibly shared between the two genes. We propose a model in which AtClpB-C shares its regulatory region with heat-induced choline kinase, which has a possible role in heat signaling.

  11. Rhodobacter sphaeroides rdxA, a homolog of Rhizobium meliloti fixG, encodes a membrane protein which may bind cytoplasmic [4Fe-4S] clusters.

    PubMed Central

    Neidle, E L; Kaplan, S

    1992-01-01

    In the photosynthetic bacterium Rhodobacter sphaeroides, a chromosomal gene, rdxA, which encodes a 52-kDa protein, was found to be homologous to fixG, the first gene of a Rhizobium meliloti nitrogen fixation operon on the pSym plasmid (D. Kahn, M. David, O. Domergue, M.-L. Daveran, J. Ghai, P. R. Hirsch, and J. Batut, J. Bacteriol. 171:929-939, 1989). The deduced amino acid sequences of RdxA and FixG are 53% identical and 73% similar; sequence analyses suggested that each has five transmembrane helices and a central region resembling bacterial-type ferredoxins. Translational fusion proteins with an alkaline phosphatase reporter group were expressed in both R. sphaeroides and Escherichia coli and were used to assess the membrane topology of RdxA. Its ferredoxinlike sequence, which may bind two [4Fe-4S] centers, was found to be cytoplasmically located. Genetic disruptions showed that rdxA is not essential for nitrogen fixation in R. sphaeroides. Immediately downstream of rdxA, an open reading frame (ORFT2) that encoded a 48-kDa protein was found. This DNA sequence was not homologous to any region of the R. meliloti fixG operon. The N-terminal sequence of the ORFT2 gene product resembled amino acid sequences found in members of the GntR family of regulatory proteins (D. J. Haydon and J. R. Guest, FEMS Microbiol. Lett. 79:291-296, 1991). The rdxA gene was localized to the smaller of two R. sphaeroides chromosomes, upstream of and divergently transcribed from hemT, which encodes one of two 5-aminolevulinate synthase isozymes. The rdxA and hemT genes may share a transcriptional regulatory region. Southern hybridization analysis demonstrated the presence of an rdxA homolog on the R. sphaeroides large chromosome. The functions of this homolog, like those of rdxA, remain to be determined, but roles in oxidation-reduction processes are likely. Images PMID:1400197

  12. The membrane-tubulating potential of amphiphysin 2/BIN1 is dependent on the microtubule-binding cytoplasmic linker protein 170 (CLIP-170).

    PubMed

    Meunier, Brigitte; Quaranta, Muriel; Daviet, Laurent; Hatzoglou, Anastassia; Leprince, Corinne

    2009-02-01

    Amphiphysins are BIN-amphiphysin-RVS (BAR) domain-containing proteins that influence membrane curvature in sites such as T-tubules in muscular cells, endocytic pits in neuronal as well as non-neuronal cells, and possibly cytoplasmic endosomes. This effect on lipid membranes is fulfilled by diverse amphiphysin 2/BIN1 isoforms, generated by alternative splicing and showing distinct structural and functional properties. In this study, our goal was to characterize the functional role of a ubiquitously expressed amphiphysin 2/BIN1 by the characterization of new molecular partners. We performed a two-hybrid screen with an isoform of amphiphysin 2/BIN1 expressed in HeLa cells. We identified CLIP-170 as an amphiphysin 2/BIN1-interacting molecule. CLIP-170 is a plus-end tracking protein involved in microtubule (MT) stability and recruitment of dynactin. The binding between amphiphysin 2/BIN1 and CLIP-170 is dependent on the N-terminal part of amphiphysin 2 (mostly the BAR domain) and an internal coiled-coil region of CLIP-170. This partnership was confirmed by GST pull-down assay and by co-immunoprecipitation in HeLa cells that express endogenous amphiphysin 2 (mostly isoforms 6, 9 and 10). When overexpressed in HeLa cells, amphiphysin 2/BIN1 leads to the formation of intracellular tubules which can closely align with MTs. After MT depolymerization by nocodazole, amphiphysin 2-stained tubules disappear, and reappear after nocodazole washout. Furthermore, depletion of CLIP-170 by RNAi induced a decrease in the proportion of cells with amphiphysin 2-stained tubules and an increase in the proportion of cells with no tubules. This result suggests the existence of a mechanistic link between the two types of tubules, which is likely to involve the +TIP protein, CLIP-170. Amphiphysin 2/BIN1 may be an anchoring point on membranes for CLIP-170, and consequently for MT. Then, the pushing force of polymerizing MT could help amphiphysin 2/BIN1 in its tubulation potential. We propose

  13. Alteration in abundance of specific membrane proteins of Aggregatibacter actinomycetemcomitans is attributed to deletion of the inner membrane protein MorC

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an important pathogen in the etiology of human periodontal and systemic diseases. Inactivation of the gene coding for the inner membrane protein, morphogenesis protein C (MorC), results is pleotropic effects pertaining to the membrane structure and function of this bacterium. The role of this protein in membrane biogenesis is unknown. To begin to understand the role of this conserved protein, stable isotope dimethyl labeling in conjunction with mass spectrometry was used to quantitatively analyze differences in the membrane proteomes of the isogenic mutant and wild-type strain. A total of 613 proteins were quantified and 601 of these proteins were found to be equal in abundance between the two strains. The remaining 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain compared with the wild-type strain. The 12 proteins were ascribed functions associated with protein quality control systems, oxidative stress responses, and protein secretion. The potential relationship between these proteins and the phenotypes of the morC mutant strain is discussed. PMID:25684173

  14. Assessment of 24-hours Aldosterone Administration on Protein Abundances in Fluorescence-Sorted Mouse Distal Renal Tubules by Mass Spectrometry

    PubMed Central

    Jensen, Thomas B; Pisitkun, Trairak; Hoffert, Jason D; Jensen, Uffe B; Fenton, Robert A; Praetorius, Helle A; Knepper, Mark A; Praetorius, Jeppe

    2013-01-01

    Background/Aims Aldosterone exerts multiple long-term effects in the distal renal tubules. The aim of this study was to establish a method for identifying proteins in these tubules that change in abundance by only 24-hours aldosterone administration. Methods Mice endogenously expressing green fluorescent protein (eGFP) in the connecting tubule and cortical collecting ducts were treated with a subcutaneous injection of 2.0 mg/kg aldosterone or vehicle (n=5), and sacrificed 24 hours later. Suspensions of single cells were obtained enzymatically, and eGFP positive cells were isolated by fluorescence activated cell sorting (FACS). Samples of 100 μg proteins were digested with trypsin and labeled with 8-plex iTRAQ reagents and processed for liquid chromatography tandem mass spectrometry (LC-MS/MS). Results FACS yielded 1.4 million cells per mouse. The LC-MS/MS spectra were matched to peptides by the SEQUEST search algorithm, which identified 3002 peptides corresponding to 506 unique proteins of which 20 significantly changed abundance 24-hours after aldosterone injection. Conclusion We find the method suitable and useful for studying hormonal effects on protein abundance in distal tubular segments. PMID:23428628

  15. Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at Thr668.

    PubMed

    De, Soumya; Greenwood, Alexander I; Rogals, Monique J; Kovrigin, Evgenii L; Lu, Kun Ping; Nicholson, Linda K

    2012-10-30

    Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.

  16. Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein

    PubMed Central

    1993-01-01

    In the intestinal brush border, the mechanoenzyme myosin-I links the microvillus core actin filaments with the plasma membrane. Previous immunolocalization shows that myosin-I is associated with vesicles in mature enterocytes (Drenckhahn, D., and R. Dermietzel. 1988. J. Cell Biol. 107:1037-1048) suggesting a potential role mediating vesicle motility. We now report that myosin-I is associated with Golgi-derived vesicles isolated from cells that are rapidly assembling brush borders in intestinal crypts. Crypt cells were isolated in hyperosmotic buffer, homogenized, and fractionated using differential- and equilibrium- density centrifugation. Fractions containing 50-100-nm vesicles, a similar size to those observed in situ, were identified by EM and were shown to contain myosin-I as demonstrated by immunoblotting and immunolabel negative staining. Galactosyltransferase, a marker enzyme for trans-Golgi membranes was present in these fractions, as was alkaline phosphatase, which is an apical membrane targeted enzyme. Galactosyltransferase was also present in vesicles immuno-purified with antibodies to myosin-I. Villin, a marker for potential contamination from fragmented microvilli, was absent. Myosin-I was found to reside on the vesicle "outer" or cytoplasmic surface for it was accessible to exogenous proteases and intact vesicles could be immunolabeled with myosin-I antibodies in solution. The bound myosin-I could be extracted from the vesicles using NaCl, KI and Na2CO3, suggesting that it is a vesicle peripheral membrane protein. These vesicles were shown to bundle actin filaments in an ATP-dependent manner. These results are consistent with a role for myosin-I as an apically targeted motor for vesicle translocation in epithelial cells. PMID:8416982

  17. The adapter protein CD2AP binds to p53 protein in the cytoplasm and can discriminate its polymorphic variants P72R.

    PubMed

    Panni, Simona; Salvioli, Stefano; Santonico, Elena; Langone, Francesca; Storino, Francesca; Altilia, Serena; Franceschi, Claudio; Cesareni, Gianni; Castagnoli, Luisa

    2015-02-01

    Proline-rich motifs are widely distributed in eukaryotic proteomes and are usually involved in the assembly of functional complexes through interaction with specific binding modules. The tumour-suppressor p53 protein presents a proline-rich region that is crucial for regulating apoptosis by connecting the p53 with a complex protein network. In humans, a common polymorphism determines the identity of residue 72, either proline or arginine, and affects the features of the motifs present in the polyproline domain. The two isoforms have different biochemical properties and markedly influence cancer onset and progression. In this article, we analyse the binding of the p53 proline-rich region with a pool of selected polyproline binding domains (i.e. SH3 and WW), and we present the first demonstration that the purified SH3 domains of the CD2AP/Cin85 protein family are able to directly bind the p53 protein, and to discriminate between the two polymorphic variants P72R.

  18. The adapter protein CD2AP binds to p53 protein in the cytoplasm and can discriminate its polymorphic variants P72R.

    PubMed

    Panni, Simona; Salvioli, Stefano; Santonico, Elena; Langone, Francesca; Storino, Francesca; Altilia, Serena; Franceschi, Claudio; Cesareni, Gianni; Castagnoli, Luisa

    2015-02-01

    Proline-rich motifs are widely distributed in eukaryotic proteomes and are usually involved in the assembly of functional complexes through interaction with specific binding modules. The tumour-suppressor p53 protein presents a proline-rich region that is crucial for regulating apoptosis by connecting the p53 with a complex protein network. In humans, a common polymorphism determines the identity of residue 72, either proline or arginine, and affects the features of the motifs present in the polyproline domain. The two isoforms have different biochemical properties and markedly influence cancer onset and progression. In this article, we analyse the binding of the p53 proline-rich region with a pool of selected polyproline binding domains (i.e. SH3 and WW), and we present the first demonstration that the purified SH3 domains of the CD2AP/Cin85 protein family are able to directly bind the p53 protein, and to discriminate between the two polymorphic variants P72R. PMID:25261582

  19. The ubiquitous distribution of late embryogenesis abundant proteins across cell compartments in Arabidopsis offers tailored protection against abiotic stress.

    PubMed

    Candat, Adrien; Paszkiewicz, Gaël; Neveu, Martine; Gautier, Romain; Logan, David C; Avelange-Macherel, Marie-Hélène; Macherel, David

    2014-07-01

    Late embryogenesis abundant (LEA) proteins are hydrophilic, mostly intrinsically disordered proteins, which play major roles in desiccation tolerance. In Arabidopsis thaliana, 51 genes encoding LEA proteins clustered into nine families have been inventoried. To increase our understanding of the yet enigmatic functions of these gene families, we report the subcellular location of each protein. Experimental data highlight the limits of in silico predictions for analysis of subcellular localization. Thirty-six LEA proteins localized to the cytosol, with most being able to diffuse into the nucleus. Three proteins were exclusively localized in plastids or mitochondria, while two others were found dually targeted to these organelles. Targeting cleavage sites could be determined for five of these proteins. Three proteins were found to be endoplasmic reticulum (ER) residents, two were vacuolar, and two were secreted. A single protein was identified in pexophagosomes. While most LEA protein families have a unique subcellular localization, members of the LEA_4 family are widely distributed (cytosol, mitochondria, plastid, ER, and pexophagosome) but share the presence of the class A α-helix motif. They are thus expected to establish interactions with various cellular membranes under stress conditions. The broad subcellular distribution of LEA proteins highlights the requirement for each cellular compartment to be provided with protective mechanisms to cope with desiccation or cold stress. PMID:25005920

  20. The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins

    PubMed Central

    Shinn-Thomas, Jessica H.; del Campo, Jacob J.; Wang, Jianjun; Mohler, William A.

    2016-01-01

    Background Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform’s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. Methodology/Principal Findings Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. Conclusions/Significance Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However

  1. Gender-specific association between the cytoplasmic poly(A) binding protein 4 rs4660293 single nucleotide polymorphism and serum lipid levels

    PubMed Central

    WU, JIAN; YIN, RUI-XING; GUO, TAO; LIN, QUAN-ZHEN; SHEN, SHAO-WEN; SUN, JIA-QI; SHI, GUANG-YUAN; WU, JIN-ZHEN; YANG, DE-ZHAI; LIN, WEI-XIONG

    2015-01-01

    Cytoplasmic poly(A) binding protein 4 (PABPC4) is an RNA-processing protein which has an important role in regulating gene expression. The association of the PABPC4 rs4660293 single nucleotide polymorphism (SNP) and serum lipid profiles has, to the best of our knowledge, not previously been studied in the Chinese population. The present study aimed to investigate the association between the PABPC4 rs4660293 SNP and several environmental factors with serum lipid levels in the Mulao and Han populations. A total of 727 individuals of Mulao nationality and 729 individuals of Han nationality were randomly selected from stratified randomized samples from a previous study by our group. Genotypes of the PABPC4 rs4660293 SNP were determined via polymerase chain reaction and restriction fragment length polymorphism analyses and subsequently confirmed by direct sequencing. Serum levels of low-density lipoprotein cholesterol (LDL-C) and apolipoprotein (Apo) B were higher in the Mulao group than those in the Han group (P<0.01 for each). The genotypic and allelic frequencies of the PABPC4 rs4660293 SNP were significantly different between males and females in the Mulao population (P<0.05 for each), while no significant difference was detected between those of males and females amongst the Han population. The frequency of the G allele was higher in Mulao males than in Mulao females (22.12 vs. 13.44%). The G allele carriers were found to have higher total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and ApoAI levels in Han females but not in Han males, and lower TC and HDL-C levels in Mulao females but not in Mulao males than those of the G allele non-carriers (P<0.05 for all). These associations were confirmed by multiple linear regression analysis (P<0.05–0.001). Serum lipid parameters were also correlated with multiple environmental factors (P<0.05–0.001). The PABPC4 rs4660293 SNP was associated with serum TC, HDL-C, LDL-C and ApoAI levels in these study

  2. Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycosphingolipid composition.

    PubMed Central

    Levi, M; Shayman, J A; Abe, A; Gross, S K; McCluer, R H; Biber, J; Murer, H; Lötscher, M; Cronin, R E

    1995-01-01

    Glucocorticoids are important regulators of renal phosphate transport. This study investigates the role of alterations in renal brush border membrane (BBM) sodium gradient-dependent phosphate transport (Na-Pi cotransporter) mRNA and protein abundance in the dexamethasone induced inhibition of Na-Pi cotransport in the rat. Dexamethasone administration for 4 d caused a 1.5-fold increase in the Vmax of Na-Pi cotransport (1785 +/- 119 vs. 2759 +/- 375 pmol/5 s per mg BBM protein in control, P < 0.01), which was paralleled by a 2.5-fold decrease in the abundance of Na-Pi mRNA and Na-Pi protein. There was also a 1.7-fold increase in BBM glucosylceramide content (528 +/- 63 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02). To determine whether the alteration in glucosylceramide content per se played a functional role in the decrease in Na-Pi cotransport, control rats were treated with the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol (PDMP). The resultant 1.5-fold decrease in BBM glucosylceramide content (199 +/- 19 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02) was associated with a 1.4-fold increase in Na-Pi cotransport activity (1422 +/- 73 vs. 1048 +/- 85 pmol/5 s per mg BBM protein in control, P < 0.01), and a 1.5-fold increase in BBM Na-Pi protein abundance. Thus, dexamethasone-induced inhibition of Na-Pi cotransport is associated with a decrease in BBM Na-Pi cotransporter abundance, and an increase in glucosylceramide. Since primary alteration in BBM glucosylceramide content per se directly and selectively modulates BBM Na-Pi cotransport activity and Na-Pi protein abundance, we propose that the increase in BBM glucosylceramide content plays an important role in mediating the inhibitory effect of dexamethasone on Na-Pi cotransport activity. Images PMID:7615789

  3. Absolute quantification of protein and post-translational modification abundance with stable isotope–labeled synthetic peptides

    PubMed Central

    Kettenbach, Arminja N; Rush, John; Gerber, Scott A

    2013-01-01

    In the analysis of biological systems, it is of interest to identify the components of the system and to monitor their changes in abundance under different conditions. The AQUA (for ‘absolute quantification’) method allows sensitive and specific targeted quantification of protein and post-translational modifications in complex protein mixtures using stable isotope–labeled peptides as internal standards. Each AQUA experiment is composed of two stages: method development and application to a biological scenario. In the method development stage, peptides from the protein of interest are chosen and then synthesized with stable isotopes such as 13C, 2H or 15N. The abundance of these internal standards and their endogenous counterparts can be measured by mass spectrometry with selected reaction monitoring or selected ion monitoring methods. Once an AQUA method is established, it can be rapidly applied to a wide range of biological samples, from tissue culture cells to human plasma and tissue. After AQUA peptide synthesis, the development, optimization and application of AQUA analyses to a specific biological problem can be achieved in ~1 week. Here we demonstrate the usefulness of this method by monitoring both Polo-like kinase 1 (Plk1) protein abundance in multiple lung cancer cell lines and the extent of Plk1 activation loop phosphorylation (pThr-210) during release from S phase. PMID:21293459

  4. Region-Specific Protein Abundance Changes in the Brain of MPTP-induced Parkinson’s Disease Mouse Model

    SciTech Connect

    Zhang, Xu; Zhou, Jianying; Chin, Mark H; Schepmoes, Athena A; Petyuk, Vladislav A; Weitz, Karl K; Petritis, Brianne O; Monroe, Matthew E; Camp, David G; Wood, Stephen A; Melega, William P; Bigelow, Diana J; Smith, Desmond J; Qian, Weijun; Smith, Richard D

    2010-02-15

    Parkinson’s disease (PD) is characterized by dopaminergic neurodegeneration in the nigrostriatal region of the brain; however, the neurodegeneration extends well beyond dopaminergic neurons. To gain a better understanding of the molecular changes relevant to PD, we applied two-dimensional LC-MS/MS to comparatively analyze the proteome changes in four brain regions (striatum, cerebellum, cortex, and the rest of brain) using a MPTP-induced PD mouse model with the objective to identify nigrostriatal-specific and other region-specific protein abundance changes. The combined analyses resulted in the identification of 4,895 non-redundant proteins with at least two unique peptides per protein. The relative abundance changes in each analyzed brain region were estimated based on the spectral count information. A total of 518 proteins were observed with significant MPTP-induced changes across different brain regions. 270 of these proteins were observed with specific changes occurring either only in the striatum and/or in the rest of the brain region that contains substantia nigra, suggesting that these proteins are associated with the underlying nigrostriatal pathways. Many of the proteins that exhibit significant abundance changes were associated with dopamine signaling, mitochondrial dysfunction, the ubiquitin system, calcium signaling, the oxidative stress response, and apoptosis. A set of proteins with either consistent change across all brain regions or with changes specific to the cortex and cerebellum regions were also detected. One of the interesting proteins is ubiquitin specific protease (USP9X), a deubiquination enzyme involved in the protection of proteins from degradation and promotion of the TGF-β pathway, which exhibited altered abundances in all brain regions. Western blot validation showed similar spatial changes, suggesting that USP9X is potentially associated with neurodegeneration. Together, this study for the first time presents an overall picture of

  5. An Arabidopsis Ran-binding protein, AtRanBP1c, is a co-activator of Ran GTPase-activating protein and requires the C-terminus for its cytoplasmic localization

    NASA Technical Reports Server (NTRS)

    Kim, Soo-Hwan; Roux, Stanley J.

    2003-01-01

    Ran-binding proteins (RanBPs) are a group of proteins that bind to Ran (Ras-related nuclear small GTP-binding protein), and thus either control the GTP/GDP-bound states of Ran or help couple the Ran GTPase cycle to a cellular process. AtRanBP1c is a Ran-binding protein from Arabidopsis thaliana (L.) Heynh. that was recently shown to be critically involved in the regulation of auxin-induced mitotic progression [S.-H. Kim et al. (2001) Plant Cell 13:2619-2630]. Here we report that AtRanBP1c inhibits the EDTA-induced release of GTP from Ran and serves as a co-activator of Ran-GTPase-activating protein (RanGAP) in vitro. Transient expression of AtRanBP1c fused to a beta-glucuronidase (GUS) reporter reveals that the protein localizes primarily to the cytosol. Neither the N- nor C-terminus of AtRanBP1c, which flank the Ran-binding domain (RanBD), is necessary for the binding of PsRan1-GTP to the protein, but both are needed for the cytosolic localization of GUS-fused AtRanBP1c. These findings, together with a previous report that AtRanBP1c is critically involved in root growth and development, imply that the promotion of GTP hydrolysis by the Ran/RanGAP/AtRanBP1c complex in the cytoplasm, and the resulting concentration gradient of Ran-GDP to Ran-GTP across the nuclear membrane could be important in the regulation of auxin-induced mitotic progression in root tips of A. thaliana.

  6. M-phase-specific phosphorylation and structural rearrangement of the cytoplasmic cross-linking protein plectin involve p34cdc2 kinase.

    PubMed Central

    Foisner, R; Malecz, N; Dressel, N; Stadler, C; Wiche, G

    1996-01-01

    Plectin, a widespread and abundant cytoskeletal cross-linking protein, serves as a target for protein kinases throughout the cell cycle, without any significant variation in overall phosphorylation level. One of the various phosphorylation sites of the molecule was found to be phosphorylated preferentially during mitosis. By in vivo phosphorylation of ectopically expressed plectin domains in stably transfected Chinese hamster ovary cells, this site was mapped to the C-terminal repeat 6 domain of the polypeptide. The same site has been identified as an in vitro target for p34cdc2 kinase. Mitosis-specific phosphorylation of plectin was accompanied by a rearrangement of plectin structures, changing from a filamentous, largely vimentin-associated state in interphase to a diffuse vimentin-independent distribution in mitosis as visualized by immunofluorescence microscopy. Subcellular fractionation studies showed that in interphase cells up to 80% of cellular plectin was found associated with an insoluble cell fraction mostly consisting of intermediate filaments, while during mitosis the majority of plectin (> 75%) became soluble. Furthermore, phosphorylation of purified plectin by p34cdc2 kinase decreased plectin's ability to interact with preassembled vimentin filaments in vitro. Together, our data suggest that a mitosis-specific phosphorylation involving p34cdc2 kinase regulates plectin's cross-linking activities and association with intermediate filaments during the cell cycle. Images PMID:8688558

  7. Method optimization for proteomic analysis of soybean leaf: Improvements in identification of new and low-abundance proteins

    PubMed Central

    Mesquita, Rosilene Oliveira; de Almeida Soares, Eduardo; de Barros, Everaldo Gonçalves; Loureiro, Marcelo Ehlers

    2012-01-01

    The most critical step in any proteomic study is protein extraction and sample preparation. Better solubilization increases the separation and resolution of gels, allowing identification of a higher number of proteins and more accurate quantitation of differences in gene expression. Despite the existence of published results for the optimization of proteomic analyses of soybean seeds, no comparable data are available for proteomic studies of soybean leaf tissue. In this work we have tested the effects of modification of a TCA-acetone method on the resolution of 2-DE gels of leaves and roots of soybean. Better focusing was obtained when both mercaptoethanol and dithiothreitol were used in the extraction buffer simultaneously. Increasing the number of washes of TCA precipitated protein with acetone, using a final wash with 80% ethanol and using sonication to ressuspend the pellet increased the number of detected proteins as well the resolution of the 2-DE gels. Using this approach we have constructed a soybean protein map. The major group of identified proteins corresponded to genes of unknown function. The second and third most abundant groups of proteins were composed of photosynthesis and metabolism related genes. The resulting protocol improved protein solubility and gel resolution allowing the identification of 122 soybean leaf proteins, 72 of which were not detected in other published soybean leaf 2-DE gel datasets, including a transcription factor and several signaling proteins. PMID:22802721

  8. Abundant class III acidic chitinase homologue in tamarind (Tamarindus indica) seed serves as the major storage protein.

    PubMed

    Rao, Devavratha H; Gowda, Lalitha R

    2008-03-26

    The phyla Leguminosae contains protease inhibitors, lectins, chitinases, and glycohydrolases as major defense proteins in their seeds. Electrophoretic analysis of the seed proteins of tamarind ( Tamarindus indica L.), an agri-waste material, indicated the unusual presence of two major proteins comparable to overexpression of recombinant proteins. These proteins were identified by amino-terminal analysis to be (1) Kunitz-type trypsin inhibitor and (2) class III endochitinase (34000 Da). These two proteins were purified to apparent homogeneity by a single-step chitin bead affinity chromatography and characterized. The Kunitz inhibitor was specific toward inhibiting trypsin with a stoichiometry of 1:1. The 33000 +/- 1000 Da protein, accounting for >50% of the total seed protein, is an acidic glycoprotein exhibiting a very low endotype hydrolytic activity toward chitin derivatives. SDS-PAGE followed by densitometry of tamarind seed germination indicates the disappearance of the chitinase with the concomitant appearance of a cysteine endopeptidase. On the basis of its abundance, accumulation without any pathogenesis-related stimulus, temporal regulation, amino acid composition, and very low enzyme activity, this 34000 Da protein designated "tamarinin" physiologically serves as the major storage protein.

  9. Proteomics reveals differences in protein abundance and highly similar antigenic profiles between Besnoitia besnoiti and Besnoitia tarandi.

    PubMed

    García-Lunar, P; Regidor-Cerrillo, J; Ortega-Mora, L M; Gutiérrez-Expósito, D; Alvarez-García, G

    2014-10-15

    Besnoitia besnoiti and Besnoitia tarandi are two cyst-forming apicomplexan parasites of the genus Besnoitia. B. besnoiti uses cattle as an intermediate host, in which it causes a disease that progresses in two sequential phases: the acute anasarca stage and the chronic scleroderma stage. Reindeer and caribou act as intermediate hosts for B. tarandi, which causes clinical signs similar to those caused by B. besnoiti. Previous studies demonstrated high molecular similarity, as determined by 18S and ITS-1 RNA sequences, between these Besnoitia spp., and strong serological cross-reactivity between these species has recently been demonstrated. Thus, a difference gel electrophoresis approach and mass spectrometry analysis were used to describe the proteomes and explore differences in protein abundance between B. besnoiti and B. tarandi in tachyzoite extracts. Immunoproteomes were also compared using 2-DE immunoblotting with polyclonal sera from experimentally infected rabbits. From approximately 1400 spots detected in DIGE-gels, 28 and 29 spots were differentially abundant in B. besnoiti and B. tarandi tachyzoites, respectively (± 1.5-fold, p<0.05). Four and 13 spots were exclusively detected in B. besnoiti and B. tarandi, respectively. Of the 32 differentially abundant spots analyzed by MALDI-TOF/MS, 6 up-regulated B. besnoiti proteins (LDH; HSP90; purine nucleoside phosphorylase and 3 hypothetical proteins) and 6 up-regulated B. tarandi proteins (G3PDH; LDH; PDI; mRNA decapping protein and 2 hypothetical proteins) were identified. Interestingly, no specific antigen spots were recognized by sera on any of the Besnoitia species studied and a similar antigen profile has been observed for B. tarandi and B. besnoiti sera when cross reactions were studied. This fact corroborates the difficulty in discerning Besnoitia infections using current serological assays. The present study underscores the importance of sequencing the B. besnoiti genome for species diversity studies of

  10. Major cytoplasmic membrane protein of Legionella pneumophila, a genus common antigen and member of the hsp 60 family of heat shock proteins, induces protective immunity in a guinea pig model of Legionnaires' disease.

    PubMed Central

    Blander, S J; Horwitz, M A

    1993-01-01

    We have examined the capacity of the major cytoplasmic membrane protein (MCMP) of Legionella pneumophila, a genus common antigen and member of the hsp 60 family of heat shock proteins, to induce protective immunity in a guinea pig model of Legionnaires' disease. We purified MCMP to homogeneity from L. pneumophila by buffer extraction, ion-exchange chromatography, and molecular sieve chromatography. Guinea pigs immunized with MCMP developed a strong cell-mediated immune response to the immunogen manifest by marked cutaneous delayed-type hypersensitivity. Guinea pigs immunized with MCMP and then challenged with a lethal aerosol dose of L. pneumophila exhibited a high level of protective immunity. Altogether, in four independent experiments, 55 of 64 (86%) animals immunized three times with 0.6-40 micrograms MCMP including 11 of 11 (100%) animals immunized three times with 40 micrograms MCMP survived aerosol challenge with L. pneumophila compared with 1 of 29 (3%) sham-immunized control animals (P < 0.0001, Cochran-Mantel-Haenszel X2 statistic for pooled data). To our knowledge, MCMP is the first member of the hsp 60 family of proteins shown to induce protective immunity to a microbial pathogen. MCMP has potential as a vaccine against Legionnaires' disease. Since MCMP is a genus common antigen, vaccination with a combination of MCMPs derived from different Legionella species has the potential of inducing protective immunity against all the major Legionella species causing human disease. Images PMID:8432872

  11. Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.

    PubMed Central

    Knehr, M; Poppe, M; Schroeter, D; Eickelbaum, W; Finze, E M; Kiesewetter, U L; Enulescu, M; Arand, M; Paweletz, N

    1996-01-01

    Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control. Images Fig. 1 Fig. 2 Fig. 4 PMID:8816782

  12. An endocytic YXXΦ (YRRF) cargo sorting motif in the cytoplasmic tail of murine cytomegalovirus AP2 'adapter adapter' protein m04/gp34 antagonizes virus evasion of natural killer cells.

    PubMed

    Fink, Annette; Blaum, Franziska; Babic Cac, Marina; Ebert, Stefan; Lemmermann, Niels A W; Reddehase, Matthias J

    2015-06-01

    Viruses have evolved proteins that bind immunologically relevant cargo molecules at the cell surface for their downmodulation by internalization. Via a tyrosine-based sorting motif YXXΦ in their cytoplasmic tails, they link the bound cargo to the cellular adapter protein-2 (AP2), thereby sorting it into clathrin-triskelion-coated pits for accelerated endocytosis. Downmodulation of CD4 molecules by lentiviral protein NEF represents the most prominent example. Based on connecting cargo to cellular adapter molecules, such specialized viral proteins have been referred to as 'connectors' or 'adapter adapters.' Murine cytomegalovirus glycoprotein m04/gp34 binds stably to MHC class-I (MHC-I) molecules and suspiciously carries a canonical YXXΦ endocytosis motif YRRF in its cytoplasmic tail. Disconnection from AP2 by motif mutation ARRF should retain m04-MHC-I complexes at the cell surface and result in an enhanced silencing of natural killer (NK) cells, which recognize them via inhibitory receptors. We have tested this prediction with a recombinant virus in which the AP2 motif is selectively destroyed by point mutation Y248A, and compared this with the deletion of the complete protein in a Δm04 mutant. Phenotypes were antithetical in that loss of AP2-binding enhanced NK cell silencing, whereas absence of m04-MHC-I released them from silencing. We thus conclude that AP2-binding antagonizes NK cell silencing by enhancing endocytosis of the inhibitory ligand m04-MHC-I. Based on a screen for tyrosine-based endocytic motifs in cytoplasmic tail sequences, we propose here the new hypothesis that most proteins of the m02-m16 gene family serve as 'adapter adapters,' each selecting its specific cell surface cargo for clathrin-assisted internalization.

  13. Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome

    PubMed Central

    2012-01-01

    Background The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process. Results To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this study (M.W. 7,367). Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for housekeeping functions, particularly

  14. Cytoplasmic Viral Replication Complexes

    PubMed Central

    den Boon, Johan A.; Diaz, Arturo; Ahlquist, Paul

    2010-01-01

    Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bounded RNA replication complexes and their close coordination with virion assembly and budding. The structure, function and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions, and more general similarities with the replication factories of cytoplasmic DNA viruses. PMID:20638644

  15. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  16. Application of an Improved Proteomics Method for Abundant Protein Cleanup: Molecular and Genomic Mechanisms Study in Plant Defense*

    PubMed Central

    Zhang, Yixiang; Gao, Peng; Xing, Zhuo; Jin, Shumei; Chen, Zhide; Liu, Lantao; Constantino, Nasie; Wang, Xinwang; Shi, Weibing; Yuan, Joshua S.; Dai, Susie Y.

    2013-01-01

    High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery. PMID:23943779

  17. Application of an improved proteomics method for abundant protein cleanup: molecular and genomic mechanisms study in plant defense.

    PubMed

    Zhang, Yixiang; Gao, Peng; Xing, Zhuo; Jin, Shumei; Chen, Zhide; Liu, Lantao; Constantino, Nasie; Wang, Xinwang; Shi, Weibing; Yuan, Joshua S; Dai, Susie Y

    2013-11-01

    High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery.

  18. Analysis of the c-src gene product structure, abundance, and protein kinase activity in human neuroblastoma and glioblastoma cells.

    PubMed

    O'Shaughnessy, J; Deseau, V; Amini, S; Rosen, N; Bolen, J B

    1987-01-01

    We have compared in different human neuroblastoma cell lines and human glioblastoma cells the expression level, structure, and tyrosine-specific protein kinase activity of pp60c-src. Our results show that not all human neuroblastoma cell lines express pp60c-src molecules with amino-terminal structural alterations. In neuroblastoma cells which possess pp60c-src with altered gel migration, the diminished polyacrylamide gel mobility of pp60c-src was found not to be dependent upon amino-terminal phosphorylations since extensive treatment of these molecules with phosphatase did not significantly change their gel migration properties. Similar differences in gel migration were observed when RNA from the various neuroblastoma and glioblastoma cells was translated in vitro using either rabbit reticulocyte or wheat germ lysates. White the level of c-src mRNA in the different cells analyzed was found to be similar, the abundance of pp60c-src in these same cells was found to vary by as much as 12-fold. This suggests that the abundance of pp60c-src in human neuroendocrine tumors is regulated through post-transcriptional and/or post-translational events which may be related to the stage of neuronal differentiation of the cells. Based upon determination of pp60c-src abundance by immunoblot analysis, we demonstrate that pp60c-src molecules derived from human neuroblastoma and glioblastoma cells have very similar in vitro protein kinase activities.

  19. Comparison of amino acids physico-chemical properties and usage of late embryogenesis abundant proteins, hydrophilins and WHy domain.

    PubMed

    Jaspard, Emmanuel; Hunault, Gilles

    2014-01-01

    Late Embryogenesis Abundant proteins (LEAPs) comprise several diverse protein families and are mostly involved in stress tolerance. Most of LEAPs are intrinsically disordered and thus poorly functionally characterized. LEAPs have been classified and a large number of their physico-chemical properties have been statistically analyzed. LEAPs were previously proposed to be a subset of a very wide family of proteins called hydrophilins, while a domain called WHy (Water stress and Hypersensitive response) was found in LEAP class 8 (according to our previous classification). Since little is known about hydrophilins and WHy domain, the cross-analysis of their amino acids physico-chemical properties and amino acids usage together with those of LEAPs helps to describe some of their structural features and to make hypothesis about their function. Physico-chemical properties of hydrophilins and WHy domain strongly suggest their role in dehydration tolerance, probably by interacting with water and small polar molecules. The computational analysis reveals that LEAP class 8 and hydrophilins are distinct protein families and that not all LEAPs are a protein subset of hydrophilins family as proposed earlier. Hydrophilins seem related to LEAP class 2 (also called dehydrins) and to Heat Shock Proteins 12 (HSP12). Hydrophilins are likely unstructured proteins while WHy domain is structured. LEAP class 2, hydrophilins and WHy domain are thus proposed to share a common physiological role by interacting with water or other polar/charged small molecules, hence contributing to dehydration tolerance. PMID:25296175

  20. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR)

    PubMed Central

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J.; Laclette, Juan P.; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-01-01

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest. PMID:25989346

  1. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR).

    PubMed

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J; Laclette, Juan P; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-05-19

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest.

  2. Uses of phage display in agriculture: sequence analysis and comparative modeling of late embryogenesis abundant client proteins suggest protein-nucleic acid binding functionality.

    PubMed

    Kushwaha, Rekha; Downie, A Bruce; Payne, Christina M

    2013-01-01

    A group of intrinsically disordered, hydrophilic proteins-Late Embryogenesis Abundant (LEA) proteins-has been linked to survival in plants and animals in periods of stress, putatively through safeguarding enzymatic function and prevention of aggregation in times of dehydration/heat. Yet despite decades of effort, the molecular-level mechanisms defining this protective function remain unknown. A recent effort to understand LEA functionality began with the unique application of phage display, wherein phage display and biopanning over recombinant Seed Maturation Protein homologs from Arabidopsis thaliana and Glycine max were used to retrieve client proteins at two different temperatures, with one intended to represent heat stress. From this previous study, we identified 21 client proteins for which clones were recovered, sometimes repeatedly. Here, we use sequence analysis and homology modeling of the client proteins to ascertain common sequence and structural properties that may contribute to binding affinity with the protective LEA protein. Our methods uncover what appears to be a predilection for protein-nucleic acid interactions among LEA client proteins, which is suggestive of subcellular residence. The results from this initial computational study will guide future efforts to uncover the protein protective mechanisms during heat stress, potentially leading to phage-display-directed evolution of synthetic LEA molecules.

  3. Exploiting the multiplexing capabilities of tandem mass tags for high-throughput estimation of cellular protein abundances by mass spectrometry.

    PubMed

    Ahrné, Erik; Martinez-Segura, Amalia; Syed, Afzal Pasha; Vina-Vilaseca, Arnau; Gruber, Andreas J; Marguerat, Samuel; Schmidt, Alexander

    2015-09-01

    The generation of dynamic models of biological processes critically depends on the determination of precise cellular concentrations of biomolecules. Measurements of system-wide absolute protein levels are particularly valuable information in systems biology. Recently, mass spectrometry based proteomics approaches have been developed to estimate protein concentrations on a proteome-wide scale. However, for very complex proteomes, fractionation steps are required, increasing samples number and instrument analysis time. As a result, the number of full proteomes that can be routinely analyzed is limited. Here we combined absolute quantification strategies with the multiplexing capabilities of isobaric tandem mass tags to determine cellular protein abundances in a high throughput and proteome-wide scale even for highly complex biological systems, such as a whole human cell line. We generated two independent data sets to demonstrate the power of the approach regarding sample throughput, dynamic range, quantitative precision and accuracy as well as proteome coverage in comparison to existing mass spectrometry based strategies.

  4. Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha.

    PubMed

    Hatanaka, Rie; Furuki, Takao; Shimizu, Tempei; Takezawa, Daisuke; Kikawada, Takahiro; Sakurai, Minoru; Sugawara, Yasutake

    2014-11-28

    Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha; preculture treatment of these cells with 0.5M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation-rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a "molecular shield". Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins. PMID:25450698

  5. Simultaneous cytoplasmic and nuclear protein expression of melanoma antigen-A family and NY-ESO-1 cancer-testis antigens represents an independent marker for poor survival in head and neck cancer.

    PubMed

    Laban, Simon; Atanackovic, Djordje; Luetkens, Tim; Knecht, Rainald; Busch, Chia-Jung; Freytag, Marcus; Spagnoli, Giulio; Ritter, Gerd; Hoffmann, Thomas K; Knuth, Alexander; Sauter, Guido; Wilczak, Waldemar; Blessmann, Marco; Borgmann, Kerstin; Muenscher, Adrian; Clauditz, Till S

    2014-09-01

    The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p < 0.0001), 46.6 versus 50.0 versus 109.0 for MAGE-A3/A4 (p = 0.0074) and 13.3 versus 50.0 versus 100.2 months for NY-ESO-1 (p = 0.0019). By multivariate analysis, these factors were confirmed as independent markers for poor survival. HNSCC patients showing protein expression of MAGE-A family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients.

  6. Pro-inflammatory flagellin proteins of prevalent motile commensal bacteria are variably abundant in the intestinal microbiome of elderly humans.

    PubMed

    Neville, B Anne; Sheridan, Paul O; Harris, Hugh M B; Coughlan, Simone; Flint, Harry J; Duncan, Sylvia H; Jeffery, Ian B; Claesson, Marcus J; Ross, R Paul; Scott, Karen P; O'Toole, Paul W

    2013-01-01

    Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute "cell motility" category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ(28). The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13-4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved

  7. Effects of heat stress on proliferation, protein turnover, and abundance of heat shock protein messenger ribonucleic acid in cultured porcine muscle satellite cells.

    PubMed

    Kamanga-Sollo, E; Pampusch, M S; White, M E; Hathaway, M R; Dayton, W R

    2011-11-01

    It is well established that heat stress (HS) negatively affects growth rate in swine. Although reduced feed intake undoubtedly plays a significant role in this reduction, studies in laboratory animals and other nonswine species indicate muscle growth also is affected by HS-related alterations in muscle physiology. Evidence is now emerging that heat shock proteins (Hsp), produced in response to HS and other types of cellular stress, may play an important role in regulating the rate and efficiency of muscle growth. Because muscle satellite cells play a crucial role in postnatal muscle growth, the effects of HS on rates of satellite cell proliferation, protein synthesis, and protein degradation play an important role in determining the rate and extent of muscle growth. Consequently, in the current study we have examined the effects of mild HS (40.5°C for 48 h) on the rates of proliferation, protein synthesis, and protein degradation and on quantities of Hsp90, Hsp70, and Hsp25/27 mRNA and protein in cultured porcine muscle satellite cells (PSC). Mild HS of PSC cultures resulted in 2.5-, 1.4-, and 6.5-fold increases (P < 0.05) in the abundance of Hsp90, Hsp70, and Hsp25/27 mRNA, respectively, relative to control cultures. Abundance of Hsp 90, 70, and 25/27 proteins was also increased in HS PSC cultures compared with those in control cultures. Proliferation rates in HS PSC cultures were 35% less (P < 0.05) than those in control cultures. Protein synthesis rates in HS-fused PSC cultures were 85% greater (P < 0.05) than those in control cultures, and protein degradation rates in HS-fused PSC were 23% less (P < 0.05) than those in control cultures. In light of the crucial role satellite cells play in postnatal muscle growth, the HS-induced changes we have observed in rates of proliferation, protein turnover, and abundance of Hsp mRNA and Hsp protein in PSC cultures indicate that mild HS affects the physiology of PSC in ways that could affect muscle growth in swine.

  8. An odorant-binding protein is abundantly expressed in the nose and in the seminal fluid of the rabbit.

    PubMed

    Mastrogiacomo, Rosa; D'Ambrosio, Chiara; Niccolini, Alberto; Serra, Andrea; Gazzano, Angelo; Scaloni, Andrea; Pelosi, Paolo

    2014-01-01

    We have purified an abundant lipocalin from the seminal fluid of the rabbit, which shows significant similarity with the sub-class of pheromone carriers "urinary" and "salivary" and presents an N-terminal sequence identical with that of an odorant-binding protein (rabOBP3) expressed in the nasal tissue of the same species. This protein is synthesised in the prostate and found in the seminal fluid, but not in sperm cells. The same protein is also expressed in the nasal epithelium of both sexes, but is completely absent in female reproductive organs. It presents four cysteines, among which two are arranged to form a disulphide bridge, and is glycosylated. This is the first report of an OBP identified at the protein level in the seminal fluid of a vertebrate species. The protein purified from seminal fluid is bound to some organic chemicals whose structure is currently under investigation. We reasonably speculate that, like urinary and salivary proteins reported in other species of mammals, this lipocalin performs a dual role, as carrier of semiochemicals in the seminal fluid and as detector of chemical signals in the nose. PMID:25391153

  9. Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 1. High abundance proteins, acute phase proteins and peptidomics.

    PubMed

    Thomas, Funmilola Clara; Mullen, William; Tassi, Riccardo; Ramírez-Torres, Adela; Mudaliar, Manikhandan; McNeilly, Tom N; Zadoks, Ruth N; Burchmore, Richard; David Eckersall, P

    2016-08-16

    A peptidomic investigation of milk from an experimental model of Streptococcus uberis mastitis in dairy cows has incorporated a study of milk high abundance and acute phase (APP) proteins as well as analysis of low molecular weight peptide biomarkers. Intramammary infection (IMI) with S. uberis caused a shift in abundance from caseins, β-lactoglobulin and α-lactalbumin to albumin, lactoferrin and IgG with the increase in lactoferrin occurring last. The APP response of haptoglobin, mammary associated serum amyloid A3 and C-reactive protein occurred between 30-48 hours post challenge with peak concentrations of APPs at 72-96 hours post challenge and declined thereafter at a rate resembling the fall in bacterial count rather than the somatic cell count. A peptide biomarker panel for IMI based on capillary electrophoresis and mass spectrometry was developed. It comprised 77 identified peptides (IMI77) composed mainly of casein derived peptides but also including peptides of glycosylation dependent cell adhesion molecule and serum amyloid A. The panel had a biomarker classification score that increased from 36 hour to 81 hour post challenge, significantly differentiating infected from non-infected milk, thus suggesting potential as a peptide biomarker panel of bovine mastitis and specifically that of S. uberis origin. The use of omic technology has shown a multifactorial cross system reaction in high and low abundance proteins and their peptide derivatives with changes of over a thousand fold in analyte levels in response to S. uberis infection. PMID:27412456

  10. Ferritin acts as the most abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers (Nilaparvata lugens).

    PubMed

    Du, J; Foissac, X; Carss, A; Gatehouse, A M; Gatehouse, J A

    2000-04-01

    The mannose-specific snowdrop lectin [Galanthus nivalis agglutinin (GNA)] displays toxicity to the rice brown planthopper Nilaparvata lugens. A 26kDa GNA-binding polypeptide from N. lugens midgut was identified by lectin blotting and affinity chromatography, and characterized by N-terminal sequencing. This polypeptide is the most abundant binding protein for GNA in the N. lugens midgut. A cDNA (fersub2) encoding this protein was isolated from an N. lugens cDNA library. The deduced amino acid sequence shows significant homology to ferritin subunits from Manduca sexta and other arthropods, plants and vertebrates, and contains a putative N-glycosylation site. Native ferritin was purified from whole insects as a protein of more than 400kDa in size and characterized biochemically. Three subunits of 20, 26 and 27kDa were released from the native complex. The 26kDa subunit binds GNA, and its N-terminal sequence was identical to that of fersub2. A second cDNA (fersub1), exhibiting strong homology with dipteran ferritin, was identified as an abundant cDNA in an N. lugens midgut-specific cDNA library, and could encode the larger ferritin subunit. The fersub1 cDNA carries a stem-loop structure (iron-responsive element) upstream from the start codon, similar to structures that have been shown to play a role in the control of ferritin synthesis in other insects.

  11. Effects of Tamarindus indica fruit pulp extract on abundance of HepG2 cell lysate proteins and their possible consequential impact on metabolism and inflammation.

    PubMed

    Chong, Ursula R W; Abdul-Rahman, Puteri S; Abdul-Aziz, Azlina; Hashim, Onn H; Mat-Junit, Sarni

    2013-01-01

    The fruit pulp extract of Tamarindus indica has been reported for its antioxidant and hypolipidemic properties. In this study, the methanol extract of T. indica fruit pulp was investigated for its effects on the abundance of HepG2 cell lysate proteins. Cell lysate was extracted from HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp. Approximately 2500 spots were resolved using two-dimensional gel electrophoresis and the abundance of 20 cellular proteins was found to be significantly reduced. Among the proteins of reduced abundance, fourteen, including six proteins involved in metabolism (including ethanolamine phosphate cytidylyltransferase), four mitochondrial proteins (including prohibitin and respiratory chain proteins), and four proteins involved in translation and splicing, were positively identified by mass spectrometry and database search. The identified HepG2 altered abundance proteins, when taken together and analyzed by Ingenuity Pathways Analysis (IPA) software, are suggestive of the effects of T. indica fruit pulp extract on metabolism and inflammation, which are modulated by LXR/RXR. In conclusion, the methanol fruit pulp extract of T. indica was shown to cause reduced abundance of HepG2 mitochondrial, metabolic, and regulatory proteins involved in oxidative phosphorylation, protein synthesis, and cellular metabolism.

  12. Effects of Tamarindus indica Fruit Pulp Extract on Abundance of HepG2 Cell Lysate Proteins and Their Possible Consequential Impact on Metabolism and Inflammation

    PubMed Central

    Chong, Ursula R. W.; Abdul-Rahman, Puteri S.; Abdul-Aziz, Azlina; Hashim, Onn H.; Mat-Junit, Sarni

    2013-01-01

    The fruit pulp extract of Tamarindus indica has been reported for its antioxidant and hypolipidemic properties. In this study, the methanol extract of T. indica fruit pulp was investigated for its effects on the abundance of HepG2 cell lysate proteins. Cell lysate was extracted from HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp. Approximately 2500 spots were resolved using two-dimensional gel electrophoresis and the abundance of 20 cellular proteins was found to be significantly reduced. Among the proteins of reduced abundance, fourteen, including six proteins involved in metabolism (including ethanolamine phosphate cytidylyltransferase), four mitochondrial proteins (including prohibitin and respiratory chain proteins), and four proteins involved in translation and splicing, were positively identified by mass spectrometry and database search. The identified HepG2 altered abundance proteins, when taken together and analyzed by Ingenuity Pathways Analysis (IPA) software, are suggestive of the effects of T. indica fruit pulp extract on metabolism and inflammation, which are modulated by LXR/RXR. In conclusion, the methanol fruit pulp extract of T. indica was shown to cause reduced abundance of HepG2 mitochondrial, metabolic, and regulatory proteins involved in oxidative phosphorylation, protein synthesis, and cellular metabolism. PMID:24455694

  13. Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase.

    PubMed

    Krishnan, Hari B; Chen, Ming-Hsuan

    2013-06-01

    Rice, the staple food of south and east Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16, 26, 33, and 56 kDa have been identified as allergens. Recently, it was documented that the 56 kDa rice allergen was responsible for rice-induced anaphylaxis. The 14-16 kDa allergens have been identified as α-amylase inhibitors; the 26 kDa protein has been identified as α-globulin; and the 33 kDa protein has been identified as glyoxalase I. However, the identity of the 56 kDa rice allergen has not yet been determined. In this study, we demonstrate that serum from patients allergic to maize shows IgE binding to a 56 kDa protein that was present in both maize and rice but not in the oil seeds soybean and peanut. The 56 kDa IgE-binding protein was abundant in the rice endosperm. We have purified this protein from rice endosperm and demonstrated its reactivity to IgE antibodies from the serum of maize-allergic patients. The purified protein was subjected to matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry analysis, resulting in identification of this rice allergen as granule-bound starch synthase, a product of the Waxy gene. Immunoblot analysis using protein extracts from a waxy mutant of rice revealed the absence of the 56 kDa IgE-binding protein. Our results demonstrate that the 56 kDa rice allergen is granule-bound starch synthase and raise the possibility of using waxy mutants of rice as a potential source of the hypoallergenic diet for patients sensitized to the 56 kDa rice allergen.

  14. A High-Content Imaging Screen for Cellular Regulators of β-Catenin Protein Abundance.

    PubMed

    Zeng, Xin; Montoute, Monica; Bee, Tiger W; Lin, Hong; Kallal, Lorena A; Liu, Yan; Agarwal, Pankaj; Wang, Dayuan; Lu, Quinn; Morrow, Dwight; Pope, Andrew J; Wu, Zining

    2016-03-01

    Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential. PMID:26656867

  15. Cytoplasmic Z-RNA

    SciTech Connect

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-09-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.

  16. Direct Correlation between Motile Behavior and Protein Abundance in Single Cells.

    PubMed

    Dufour, Yann S; Gillet, Sébastien; Frankel, Nicholas W; Weibel, Douglas B; Emonet, Thierry

    2016-09-01

    Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage. PMID:27599206

  17. Direct Correlation between Motile Behavior and Protein Abundance in Single Cells

    PubMed Central

    Gillet, Sébastien; Frankel, Nicholas W.; Weibel, Douglas B.

    2016-01-01

    Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage. PMID:27599206

  18. Functional characterization of the late embryogenesis abundant (LEA) protein gene family from Pinus tabuliformis (Pinaceae) in Escherichia coli

    PubMed Central

    Gao, Jie; Lan, Ting

    2016-01-01

    Late embryogenesis abundant (LEA) proteins are a large and highly diverse gene family present in a wide range of plant species. LEAs are proposed to play a role in various stress tolerance responses. Our study represents the first-ever survey of LEA proteins and their encoding genes in a widely distributed pine (Pinus tabuliformis) in China. Twenty–three LEA genes were identified from the P. tabuliformis belonging to seven groups. Proteins with repeated motifs are an important feature specific to LEA groups. Ten of 23 pine LEA genes were selectively expressed in specific tissues, and showed expression divergence within each group. In addition, we selected 13 genes representing each group and introduced theses genes into Escherichia coli to assess the protective function of PtaLEA under heat and salt stresses. Compared with control cells, the E. coli cells expressing PtaLEA fusion protein exhibited enhanced salt and heat resistance and viability, indicating the protein may play a protective role in cells under stress conditions. Furthermore, among these enhanced tolerance genes, a certain extent of function divergence appeared within a gene group as well as between gene groups, suggesting potential functional diversity of this gene family in conifers. PMID:26781930

  19. Functional characterization of the late embryogenesis abundant (LEA) protein gene family from Pinus tabuliformis (Pinaceae) in Escherichia coli.

    PubMed

    Gao, Jie; Lan, Ting

    2016-01-01

    Late embryogenesis abundant (LEA) proteins are a large and highly diverse gene family present in a wide range of plant species. LEAs are proposed to play a role in various stress tolerance responses. Our study represents the first-ever survey of LEA proteins and their encoding genes in a widely distributed pine (Pinus tabuliformis) in China. Twenty-three LEA genes were identified from the P. tabuliformis belonging to seven groups. Proteins with repeated motifs are an important feature specific to LEA groups. Ten of 23 pine LEA genes were selectively expressed in specific tissues, and showed expression divergence within each group. In addition, we selected 13 genes representing each group and introduced theses genes into Escherichia coli to assess the protective function of PtaLEA under heat and salt stresses. Compared with control cells, the E. coli cells expressing PtaLEA fusion protein exhibited enhanced salt and heat resistance and viability, indicating the protein may play a protective role in cells under stress conditions. Furthermore, among these enhanced tolerance genes, a certain extent of function divergence appeared within a gene group as well as between gene groups, suggesting potential functional diversity of this gene family in conifers. PMID:26781930

  20. New genetic regulators question relevance of abundant yolk protein production in C. elegans

    PubMed Central

    Rompay, Liesbeth Van; Borghgraef, Charline; Beets, Isabel; Caers, Jelle; Temmerman, Liesbet

    2015-01-01

    Vitellogenesis or maternal yolk formation is considered critical to the reproduction of egg-laying animals. In invertebrates, however, most of its regulatory genes are still unknown. Via a combined mapping and whole-genome sequencing strategy, we performed a forward genetic screen to isolate novel regulators of yolk production in the nematode model system Caenorhabditis elegans. In addition to isolating new alleles of rab-35, rab-10 and M04F3.2, we identified five mutant alleles corresponding to three novel regulatory genes potently suppressing the expression of a GFP-based yolk reporter. We confirmed that mutations in vrp-1, ceh-60 and lrp-2 disrupt endogenous yolk protein synthesis at the transcriptional and translational level. In contrast to current beliefs, our discovered set of mutants with strongly reduced yolk proteins did not show serious reproduction defects. This raises questions as to whether yolk proteins per se are needed for ultimate reproductive success. PMID:26553710

  1. Chernobyl seed project. Advances in the identification of differentially abundant proteins in a radio-contaminated environment

    PubMed Central

    Rashydov, Namik M.; Hajduch, Martin

    2015-01-01

    Plants have the ability to grow and successfully reproduce in radio-contaminated environments, which has been highlighted by nuclear accidents at Chernobyl (1986) and Fukushima (2011). The main aim of this article is to summarize the advances of the Chernobyl seed project which has the purpose to provide proteomic characterization of plants grown in the Chernobyl area. We present a summary of comparative proteomic studies on soybean and flax seeds harvested from radio-contaminated Chernobyl areas during two successive generations. Using experimental design developed for radio-contaminated areas, altered abundances of glycine betaine, seed storage proteins, and proteins associated with carbon assimilation into fatty acids were detected. Similar studies in Fukushima radio-contaminated areas might complement these data. The results from these Chernobyl experiments can be viewed in a user-friendly format at a dedicated web-based database freely available at http://www.chernobylproteomics.sav.sk. PMID:26217350

  2. Chernobyl seed project. Advances in the identification of differentially abundant proteins in a radio-contaminated environment.

    PubMed

    Rashydov, Namik M; Hajduch, Martin

    2015-01-01

    Plants have the ability to grow and successfully reproduce in radio-contaminated environments, which has been highlighted by nuclear accidents at Chernobyl (1986) and Fukushima (2011). The main aim of this article is to summarize the advances of the Chernobyl seed project which has the purpose to provide proteomic characterization of plants grown in the Chernobyl area. We present a summary of comparative proteomic studies on soybean and flax seeds harvested from radio-contaminated Chernobyl areas during two successive generations. Using experimental design developed for radio-contaminated areas, altered abundances of glycine betaine, seed storage proteins, and proteins associated with carbon assimilation into fatty acids were detected. Similar studies in Fukushima radio-contaminated areas might complement these data. The results from these Chernobyl experiments can be viewed in a user-friendly format at a dedicated web-based database freely available at http://www.chernobylproteomics.sav.sk.

  3. Cytoplasmic anchoring of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs) is required for meiotic arrest of porcine full-grown and growing oocytes.

    PubMed

    Nishimura, Takanori; Fujii, Wataru; Sugiura, Koji; Naito, Kunihiko

    2014-03-01

    Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. We previously hypothesized that the meiotic incompetence of porcine GOs was attributed to complex spatial-temporal regulation of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs), but found that AKAP1 is not involved in the meiotic incompetence of porcine GOs. In the present study, we cloned porcine cDNAs of AKAP5 and AKAP7alpha, and found that inhibiting the expression of these AKAPs induced PKA translocation into the nucleus and promoted meiotic resumption of porcine GOs without affecting the total PKA activity of GOs, whereas overexpressing these AKAPs had no effect. Because AKAPs regulate PKA localization through binding with regulatory subunits of PKA (PKA-Rs), PKA-R binding with AKAPs was inhibited by AKAP-binding inhibition peptides or PKA-R expression inhibition by antisense RNAs. We found that the expression inhibition and binding inhibition of PRKAR1A, an isoform of mammalian PKA-R, promoted meiotic resumption of porcine GOs, whereas these inhibitions of PRKAR2A, another PKA-R isoform, had no effect. In contrast, the expression inhibition and binding inhibition of PRKAR2A had higher effects than those of PRKAR1A on meiotic resumption of porcine full-grown oocytes. These results suggest that cytoplasmic anchoring of PKA by AKAPs is required for meiotic arrest of oocytes and that the PKA-R isoform working for the maintenance of meiotic arrest changed from PRKAR1A to PRKAR2A during the acquisition of meiotic competence. PMID:24501172

  4. Cytoplasmic and Periplasmic Proteomic Signatures of Exponentially Growing Cells of the Psychrophilic Bacterium Pseudoalteromonas haloplanktis TAC125 ▿ †

    PubMed Central

    Wilmes, Boris; Kock, Holger; Glagla, Susanne; Albrecht, Dirk; Voigt, Birgit; Markert, Stephanie; Gardebrecht, Antje; Bode, Rüdiger; Danchin, Antoine; Feller, Georges; Hecker, Michael; Schweder, Thomas

    2011-01-01

    The psychrophilic model bacterium Pseudoalteromonas haloplanktis is characterized by remarkably fast growth rates under low-temperature conditions in a range from 5°C to 20°C. In this study the proteome of cellular compartments, the cytoplasm and periplasm, of P. haloplanktis strain TAC125 was analyzed under exponential growth conditions at a permissive temperature of 16°C. By means of two-dimensional protein gel electrophoresis and mass spectrometry, a first inventory of the most abundant cytoplasmic and periplasmic proteins expressed in a peptone-supplemented minimal medium was established. By this approach major enzymes of the amino acid catabolism of this marine bacterium could be functionally deduced. The cytoplasmic proteome showed a predominance of amino acid degradation pathways and tricarboxylic acid (TCA) cycle enzymes but also the protein synthesis machinery. Furthermore, high levels of cold acclimation and oxidative stress proteins could be detected at this moderate growth temperature. The periplasmic proteome was characterized by a significant abundance of transporters, especially of highly expressed putative TonB-dependent receptors. This high capacity for protein synthesis, efficient amino acid utilization, and substrate transport may contribute to the fast growth rates of the copiotrophic bacterium P. haloplanktis in its natural environments. PMID:21183643

  5. Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2.

    PubMed

    Smyllie, Nicola J; Pilorz, Violetta; Boyd, James; Meng, Qing-Jun; Saer, Ben; Chesham, Johanna E; Maywood, Elizabeth S; Krogager, Toke P; Spiller, David G; Boot-Handford, Raymond; White, Michael R H; Hastings, Michael H; Loudon, Andrew S I

    2016-07-25

    Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5-7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock. PMID:27374340

  6. Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins.

    PubMed

    Warner, Alden H; Guo, Zhi-hao; Moshi, Sandra; Hudson, John W; Kozarova, Anna

    2016-01-01

    Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally

  7. Bioinformatics Analysis Reveals Abundant Short Alpha-Helices as a Common Structural Feature of Oomycete RxLR Effector Proteins

    PubMed Central

    Ye, Wenwu; Wang, Yang; Wang, Yuanchao

    2015-01-01

    RxLR effectors represent one of the largest and most diverse effector families in oomycete plant pathogens. These effectors have attracted enormous attention since they can be delivered inside the plant cell and manipulates host immunity. With the exceptions of a signal peptide and the following RxLR-dEER and C-terminal W/Y/L motifs identified from the sequences themselves, nearly no functional domains have been found. Recently, protein structures of several RxLRs were revealed to comprise alpha-helical bundle repeats. However, approximately half of all RxLRs lack obvious W/Y/L motifs, which are associated with helical structures. In this study, secondary structure prediction of the putative RxLR proteins was performed. We found that the C-terminus of the majority of these RxLR proteins, irrespective of the presence of W/Y/L motifs, contains abundant short alpha-helices. Since a large-scale experimental determination of protein structures has been difficult to date, results of the current study extend our understanding on the oomycete RxLR effectors in protein secondary structures from individual members to the entire family. Moreover, we identified less alpha-helix-rich proteins from secretomes of several oomycete and fungal organisms in which RxLRs have not been identified, providing additional evidence that these organisms are unlikely to harbor RxLR-like proteins. Therefore, these results provide additional information that will aid further studies on the evolution and functional mechanisms of RxLR effectors. PMID:26252511

  8. Disordered nucleiome: Abundance of intrinsic disorder in the DNA- and RNA-binding proteins in 1121 species from Eukaryota, Bacteria and Archaea.

    PubMed

    Wang, Chen; Uversky, Vladimir N; Kurgan, Lukasz

    2016-05-01

    Intrinsically disordered proteins (IDPs) are abundant in various proteomes, where they play numerous important roles and complement biological activities of ordered proteins. Among functions assigned to IDPs are interactions with nucleic acids. However, often, such assignments are made based on the guilty-by-association principle. The validity of the extension of these correlations to all nucleic acid binding proteins has never been analyzed on a large scale across all domains of life. To fill this gap, we perform a comprehensive computational analysis of the abundance of intrinsic disorder and intrinsically disordered domains in nucleiomes (∼548 000 nucleic acid binding proteins) of 1121 species from Archaea, Bacteria and Eukaryota. Nucleiome is a whole complement of proteins involved in interactions with nucleic acids. We show that relative to other proteins in the corresponding proteomes, the DNA-binding proteins have significantly increased disorder content and are significantly enriched in disordered domains in Eukaryotes but not in Archaea and Bacteria. The RNA-binding proteins are significantly enriched in the disordered domains in Bacteria, Archaea and Eukaryota, while the overall abundance of disorder in these proteins is significantly increased in Bacteria, Archaea, animals and fungi. The high abundance of disorder in nucleiomes supports the notion that the nucleic acid binding proteins often require intrinsic disorder for their functions and regulation.

  9. Disordered nucleiome: Abundance of intrinsic disorder in the DNA- and RNA-binding proteins in 1121 species from Eukaryota, Bacteria and Archaea.

    PubMed

    Wang, Chen; Uversky, Vladimir N; Kurgan, Lukasz

    2016-05-01

    Intrinsically disordered proteins (IDPs) are abundant in various proteomes, where they play numerous important roles and complement biological activities of ordered proteins. Among functions assigned to IDPs are interactions with nucleic acids. However, often, such assignments are made based on the guilty-by-association principle. The validity of the extension of these correlations to all nucleic acid binding proteins has never been analyzed on a large scale across all domains of life. To fill this gap, we perform a comprehensive computational analysis of the abundance of intrinsic disorder and intrinsically disordered domains in nucleiomes (∼548 000 nucleic acid binding proteins) of 1121 species from Archaea, Bacteria and Eukaryota. Nucleiome is a whole complement of proteins involved in interactions with nucleic acids. We show that relative to other proteins in the corresponding proteomes, the DNA-binding proteins have significantly increased disorder content and are significantly enriched in disordered domains in Eukaryotes but not in Archaea and Bacteria. The RNA-binding proteins are significantly enriched in the disordered domains in Bacteria, Archaea and Eukaryota, while the overall abundance of disorder in these proteins is significantly increased in Bacteria, Archaea, animals and fungi. The high abundance of disorder in nucleiomes supports the notion that the nucleic acid binding proteins often require intrinsic disorder for their functions and regulation. PMID:27037624

  10. Abundant and broad expression of transcription-induced chimeras and protein products in mammalian genomes.

    PubMed

    Lu, Guanting; Wu, Jin; Zhao, Gangbin; Wang, Zhiqiang; Chen, Weihua; Mu, Shijie

    2016-02-12

    The expression of transcription-induced chimeras (TICs) was underestimated due to strategic and logical reasons. In order to thoroughly examine TICs, systematic survey of TIC events was conducted in mammalian genomes using ESTs, followed by experimental validation using RT-PCR and real-time quantitative PCR (qPCR). The expression of ∼98% TIC events in at least one tissue or cell line from both mouse and human was verified. Besides, ∼40% TICs were broadly expressed, and ∼33% of TICs showed expression levels comparable to or higher than their upstream parental genes. We further identified putative chimeric proteins in public databases and validated two using Western blotting. GO analysis revealed that proteins resided in one multi-protein complex or functioning in metabolic or signaling pathway tended to produce fused products. Taken together, we have shown substantial evidence for the underestimated TIC events; and TICs could be a novel regulated way to further increases the proteome complexity in mammalian genomes. Possible regulation mechanisms and evolution of TICs were also discussed. PMID:26718406

  11. LEA polypeptide profiling of recalcitrant and orthodox legume seeds reveals ABI3-regulated LEA protein abundance linked to desiccation tolerance.

    PubMed

    Delahaie, Julien; Hundertmark, Michaela; Bove, Jérôme; Leprince, Olivier; Rogniaux, Hélène; Buitink, Julia

    2013-11-01

    In contrast to orthodox seeds that acquire desiccation tolerance during maturation, recalcitrant seeds are unable to survive drying. These desiccation-sensitive seeds constitute an interesting model for comparative analysis with phylogenetically close species that are desiccation tolerant. Considering the importance of LEA (late embryogenesis abundant) proteins as protective molecules both in drought and in desiccation tolerance, the heat-stable proteome was characterized in cotyledons of the legume Castanospermum australe and it was compared with that of the orthodox model legume Medicago truncatula. RNA sequencing identified transcripts of 16 homologues out of 17 LEA genes for which polypeptides are detected in M. truncatula seeds. It is shown that for 12 LEA genes, polypeptides were either absent or strongly reduced in C. australe cotyledons compared with M. truncatula seeds. Instead, osmotically responsive, non-seed-specific dehydrins accumulated to high levels in the recalcitrant cotyledons compared with orthodox seeds. Next, M. truncatula mutants of the abscisic acid insensitive3 (ABI3) gene were characterized. Mature Mtabi3 seeds were found to be desiccation sensitive when dried below a critical water content of 0.4 g H2O g DW(-1). Characterization of the LEA proteome of the Mtabi3 seeds revealed a subset of LEA proteins with severely reduced abundance that were also found to be reduced or absent in C. australe cotyledons. Transcripts of these genes were indeed shown to be ABI3 responsive. The results highlight those LEA proteins that are critical to desiccation tolerance and suggest that comparable regulatory pathways responsible for their accumulation are missing in both desiccation-sensitive genotypes, revealing new insights into the mechanistic basis of the recalcitrant trait in seeds.

  12. Rotavirus disrupts cytoplasmic P bodies during infection.

    PubMed

    Bhowmick, Rahul; Mukherjee, Arpita; Patra, Upayan; Chawla-Sarkar, Mamta

    2015-12-01

    Cytoplasmic Processing bodies (P bodies), the RNA-protein aggregation foci of translationally stalled and potentially decaying mRNA, have been reported to be differentially modulated by viruses. Rotavirus, the causative agent of acute infantile gastroenteritis is a double stranded RNA virus which completes its entire life-cycle exclusively in host cell cytoplasm. In this study, the fate of P bodies was investigated upon rotavirus infection. It was found that P bodies get disrupted during rotavirus infection. The disruption occurred by more than one different mechanism where deadenylating P body component Pan3 was degraded by rotavirus NSP1 and exonuclease XRN1 along with the decapping enzyme hDCP1a were relocalized from cytoplasm to nucleus. Overall the study highlights decay and subcellular relocalization of P body components as novel mechanisms by which rotavirus subverts cellular antiviral responses.

  13. Translation of tobacco mosaic virus RNA In Acetabularia cell cytoplasm.

    PubMed

    Cairns, E; Sarkar, S; Schweiger, H G

    1978-11-01

    Isolated Acetabularia nuclei were microinjected with Tobacco Mosaic Virus RNA and then implanted into an anucleate posterior fragment of an Acetabularia cell. Injected RNA was translated in the Acetabularia cytoplasm from the first to twelfth day after implantation of the nuclei. The production of specific virus proteins was detected and localized in the Acetabularia cytoplasm by an immunofluorescence precipitation technique.

  14. Norvaline and Norleucine May Have Been More Abundant Protein Components during Early Stages of Cell Evolution

    NASA Astrophysics Data System (ADS)

    Alvarez-Carreño, Claudia; Becerra, Arturo; Lazcano, Antonio

    2013-10-01

    The absence of the hydrophobic norvaline and norleucine in the inventory of protein amino acids is readdressed. The well-documented intracellular accumulation of these two amino acids results from the low-substrate specificity of the branched-chain amino acid biosynthetic enzymes that act over a number of related α-ketoacids. The lack of absolute substrate specificity of leucyl-tRNA synthase leads to a mischarged norvalyl-tRNALeu that evades the translational proofreading activites and produces norvaline-containing proteins, (cf. Apostol et al. J Biol Chem 272:28980-28988, 1997). A similar situation explains the presence of minute but detectable amounts of norleucine in place of methionine. Since with few exceptions both leucine and methionine are rarely found in the catalytic sites of most enzymes, their substitution by norvaline and norleucine, respectively, would have not been strongly hindered in small structurally simple catalytic polypeptides during the early stages of biological evolution. The report that down-shifts of free oxygen lead to high levels of intracellular accumulation of pyruvate and the subsequent biosynthesis of norvaline (Soini et al. Microb Cell Factories 7:30, 2008) demonstrates the biochemical and metabolic consequences of the development of a highly oxidizing environment. The results discussed here also suggest that a broader definition of biomarkers in the search for extraterrestrial life may be required.

  15. Norvaline and norleucine may have been more abundant protein components during early stages of cell evolution.

    PubMed

    Alvarez-Carreño, Claudia; Becerra, Arturo; Lazcano, Antonio

    2013-10-01

    The absence of the hydrophobic norvaline and norleucine in the inventory of protein amino acids is readdressed. The well-documented intracellular accumulation of these two amino acids results from the low-substrate specificity of the branched-chain amino acid biosynthetic enzymes that act over a number of related α-ketoacids. The lack of absolute substrate specificity of leucyl-tRNA synthase leads to a mischarged norvalyl-tRNA(Leu) that evades the translational proofreading activities and produces norvaline-containing proteins, (cf. Apostol et al. J Biol Chem 272:28980-28988, 1997). A similar situation explains the presence of minute but detectable amounts of norleucine in place of methionine. Since with few exceptions both leucine and methionine are rarely found in the catalytic sites of most enzymes, their substitution by norvaline and norleucine, respectively, would have not been strongly hindered in small structurally simple catalytic polypeptides during the early stages of biological evolution. The report that down-shifts of free oxygen lead to high levels of intracellular accumulation of pyruvate and the subsequent biosynthesis of norvaline (Soini et al. Microb Cell Factories 7:30, 2008) demonstrates the biochemical and metabolic consequences of the development of a highly oxidizing environment. The results discussed here also suggest that a broader definition of biomarkers in the search for extraterrestrial life may be required. PMID:24013929

  16. Molecular evolution of myelin basic protein, an abundant structural myelin component.

    PubMed

    Nawaz, Schanila; Schweitzer, Jörn; Jahn, Olaf; Werner, Hauke B

    2013-08-01

    Rapid nerve conduction in jawed vertebrates is facilitated by the myelination of axons, which evolved in ancient cartilaginous fish. We aim to understand the coevolution of myelin and the major myelin proteins. We found that myelin basic protein (MBP) derived from living cartilaginous fish (sharks and rays) associated with the plasma membrane of glial cells similar to the phosphatidylinositol (4,5)-bisphosphate (PIP₂)-binding marker PH-PLCδ1, and that ionomycin-induced PIP₂-hydrolysis led to its cellular redistribution. We identified two paralogous mbp genes in multiple teleost species, consistent with a genome duplication at the root of the teleost clade. Zebrafish mbpb is organized in a complex transcription unit together with the unrelated gene-of-the-oligodendrocyte-lineage (golli) while mbpa does not encode GOLLI. Moreover, the embryonic expression of mbpa and mbpb differed, indicating functional specialization after duplication. However, both mbpa and mbpb-mRNAs were detected in mature oligodendrocytes and Schwann cells, MBPa and MBPb were mass spectrometrically identified in zebrafish myelin, both associated with the plasma membrane via PIP₂, and the ratio of nonsynonymous to synonymous nucleotide-substitution rates (Ka/Ks) was low. Together, this indicates selective pressure to conserve many aspects of the cellular expression and function of MBP across vertebrate species. We propose that the PIP₂-binding function of MBP is evolutionarily old and that its emergence in ancient gnathostomata provided glial cells with the competence to myelinate. PMID:24040667

  17. Molecular evolution of myelin basic protein, an abundant structural myelin component.

    PubMed

    Nawaz, Schanila; Schweitzer, Jörn; Jahn, Olaf; Werner, Hauke B

    2013-08-01

    Rapid nerve conduction in jawed vertebrates is facilitated by the myelination of axons, which evolved in ancient cartilaginous fish. We aim to understand the coevolution of myelin and the major myelin proteins. We found that myelin basic protein (MBP) derived from living cartilaginous fish (sharks and rays) associated with the plasma membrane of glial cells similar to the phosphatidylinositol (4,5)-bisphosphate (PIP₂)-binding marker PH-PLCδ1, and that ionomycin-induced PIP₂-hydrolysis led to its cellular redistribution. We identified two paralogous mbp genes in multiple teleost species, consistent with a genome duplication at the root of the teleost clade. Zebrafish mbpb is organized in a complex transcription unit together with the unrelated gene-of-the-oligodendrocyte-lineage (golli) while mbpa does not encode GOLLI. Moreover, the embryonic expression of mbpa and mbpb differed, indicating functional specialization after duplication. However, both mbpa and mbpb-mRNAs were detected in mature oligodendrocytes and Schwann cells, MBPa and MBPb were mass spectrometrically identified in zebrafish myelin, both associated with the plasma membrane via PIP₂, and the ratio of nonsynonymous to synonymous nucleotide-substitution rates (Ka/Ks) was low. Together, this indicates selective pressure to conserve many aspects of the cellular expression and function of MBP across vertebrate species. We propose that the PIP₂-binding function of MBP is evolutionarily old and that its emergence in ancient gnathostomata provided glial cells with the competence to myelinate.

  18. Reorganization of cytoplasmic structures during cell fusion.

    PubMed

    Zheng, Q A; Chang, D C

    1991-11-01

    In order to provide a better understanding of the dynamic process of cell fusion, we studied the reorganization of cytoplasmic structures in electro-fused CV-1 cells. Using fluorescence microscopy and double staining methods, we examined correlations between the structural patterns of the major cytoskeletal proteins (microtubules, actin and vimentin intermediate filaments) and the distribution of various organelles (endoplasmic reticulum, mitochondria and nuclei) at different stages of cell fusion. Our results suggest that microtubules appear to play a primary role in the process of cytoplasmic reorganization. At the early stage of cell fusion, microtubules were observed to infiltrate rapidly into the newly formed cytoplasmic bridges and establish a connection between the cytoskeletal networks of fusing cells. The reorganization of microtubules was found to be correlated with the redistribution of endoplasmic reticulum (ER), vimentin intermediate filaments, mitochondria, and the aggregation of nuclei. The F-actin system, on the other hand, appeared to be independent of the reorganization of the other cytoplasmic structures. The principal function of F-actin during cell fusion is probably to widen the cytoplasmic bridges by lamellipodial extension.

  19. The X11L/X11{beta}/MINT2 and X11L2/X11{gamma}/MINT3 scaffold proteins shuttle between the nucleus and cytoplasm

    SciTech Connect

    Sumioka, Akio; Saito, Yuhki; Sakuma, Megumi; Araki, Yoichi; Yamamoto, Tohru; Suzuki, Toshiharu

    2008-03-10

    The X11/MINT family proteins are adaptor scaffolding proteins involved in formation of multiprotein complexes, and trafficking and metabolism of membrane proteins such as the beta-amyloid precursor protein. We found that a significant portion of X11L and X11L2 are recovered in nuclear fraction of mouse brain homogenates. EGFP-X11s were not detected in the nucleus of N2a neuroblastoma cells; however, administration of leptomycin B (LMB) induced substantial nuclear accumulation of EGFP-X11L and EGFP-X11L2, while EGFP-X11 showed little accumulation. Fluorescence loss in photobleaching (FLIP) analysis indicated that EGFP-X11L2 and EGFP-X11L are shuttled between the cytoplasm and nucleus, the former more effectively than the latter. We identified a nuclear export signal (NES) in the N-terminus of X11L2, mutation of which induces nuclear accumulation of EGFP-X11L2 in the absence of LMB. X11L2 fused to the Gal4 DNA binding domain (DBD) showed transcriptional activity, suggesting that X11L2 could function as a transcriptional activator if tethered near a promoter. Interestingly, attenuation of the nucleo-cytoplasmic shuttling of GAL4-DBD-X11L2 by mutating the NES or attaching the SV40 nuclear localization signal significantly decreased the apparent transcriptional activity. Our observations suggest that X11L2 functions in the nucleus by a mechanism distinct from conventional transactivators.

  20. The abundant and essential HU proteins in Deinococcus deserti and Deinococcus radiodurans are translated from leaderless mRNA.

    PubMed

    Bouthier de la Tour, Claire; Blanchard, Laurence; Dulermo, Rémi; Ludanyi, Monika; Devigne, Alice; Armengaud, Jean; Sommer, Suzanne; de Groot, Arjan

    2015-12-01

    HU proteins have an important architectural role in nucleoid organization in bacteria. Compared with HU of many bacteria, HU proteins from Deinococcus species possess an N-terminal lysine-rich extension similar to the eukaryotic histone H1 C-terminal domain involved in DNA compaction. The single HU gene in Deinococcus radiodurans, encoding DrHU, is required for nucleoid compaction and cell viability. Deinococcus deserti contains three expressed HU genes, encoding DdHU1, DdHU2 and DdHU3. Here, we show that either DdHU1 or DdHU2 is essential in D. deserti. DdHU1 and DdHU2, but not DdHU3, can substitute for DrHU in D. radiodurans, indicating that DdHU3 may have a non-essential function different from DdHU1, DdHU2 and DrHU. Interestingly, the highly abundant DrHU and DdHU1 proteins, and also the less expressed DdHU2, are translated in Deinococcus from leaderless mRNAs, which lack a 5'-untranslated region and, hence, the Shine-Dalgarno sequence. Unexpectedly, cloning the DrHU or DdHU1 gene under control of a strong promoter in an expression plasmid, which results in leadered transcripts, strongly reduced the DrHU and DdHU1 protein level in D. radiodurans compared with that obtained from the natural leaderless gene. We also show that the start codon position for DrHU and DdHU1 should be reannotated, resulting in proteins that are 15 and 4 aa residues shorter than initially reported. The expression level and start codon correction were crucial for functional characterization of HU in Deinococcus.

  1. A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties*

    PubMed Central

    Rivera-Najera, Lucero Y.; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O.; García-Hernández, Enrique; Solórzano, Rosa M.; Reyes, José L.; Covarrubias, Alejandra A.

    2014-01-01

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association. PMID:25271167

  2. A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties.

    PubMed

    Rivera-Najera, Lucero Y; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O; García-Hernández, Enrique; Solórzano, Rosa M; Reyes, José L; Covarrubias, Alejandra A

    2014-11-14

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association. PMID:25271167

  3. Proteome profiling of the growth phases of Leishmania pifanoi promastigotes in axenic culture reveals differential abundance of immunostimulatory proteins.

    PubMed

    Alcolea, Pedro J; Alonso, Ana; García-Tabares, Francisco; Mena, María del Carmen; Ciordia, Sergio; Larraga, Vicente

    2016-06-01

    Leishmaniasis is a term that encompasses a compendium of neglected tropical diseases caused by dimorphic and digenetic protozoan parasites from the genus Leishmania (Kinetoplastida: Trypanosomatidae). The clinical manifestations of neotropical cutaneous leishmaniasis (NCL) caused by Leishmania pifanoi and other species of the "Leishmania mexicana complex" mainly correspond to anergic diffuse cutaneous leishmaniasis (ADCL), which is the origin of considerable morbidity. Despite the outstanding advances in the characterization of the trypanosomatid genomes and proteomes, the biology of this species has been scarcely explored. However, the close relation of L. pifanoi to the sequenced species L. mexicana and others included in the "L. mexicana complex" allowed us to perform a two-dimension electrophoresis (2DE) approach to the promastigote proteome at the differential expression level. Protein identifications were performed by matrix-assisted laser desorption-ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). This insight has revealed similarities and differences between L. pifanoi and other species responsible for cutaneous and visceral leishmaniasis. Interestingly, certain proteins that were previously described as immunostimulatory (elongation factor 1β, trypanothione peroxidase, heat shock protein 70, enolase, GDP-forming succinyl-CoA and aldehyde dehydrogenase) are more abundant in the final growth stages of promastigotes (late-logarithmic and/or stationary phase) in the case of L. pifanoi. PMID:26992294

  4. Stabilization and Degradation Mechanisms of Cytoplasmic Ataxin-1

    PubMed Central

    Kohiyama, Mayumi F.; Lagalwar, Sarita

    2015-01-01

    Aggregation-prone proteins in neurodegenerative disease disrupt cellular protein stabilization and degradation pathways. The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is caused by a coding polyglutamine expansion in the Ataxin-1 gene (ATXN1), which gives rise to the aggregation-prone mutant form of ATXN1 protein. Cerebellar Purkinje neurons, preferentially vulnerable in SCA1, produce ATXN1 protein in both cytoplasmic and nuclear compartments. Cytoplasmic stabilization of ATXN1 by phosphorylation and 14-3-3-mediated mechanisms ultimately drive translocation of the protein to the nucleus where aggregation may occur. However, experimental inhibition of phosphorylation and 14-3-3 binding results in rapid degradation of ATXN1, thus preventing nuclear translocation and cellular toxicity. The exact mechanism of cytoplasmic ATXN1 degradation is currently unknown; further investigation of degradation may provide future therapeutic targets. This review examines the present understanding of cytoplasmic ATXN1 stabilization and potential degradation mechanisms during normal and pathogenic states. PMID:27168726

  5. Increased Abundance of Proteins Involved in Phytosiderophore Production in Boron-Tolerant Barley1[C][W

    PubMed Central

    Patterson, John; Ford, Kris; Cassin, Andrew; Natera, Siria; Bacic, Antony

    2007-01-01

    Boron (B) phytotoxicity affects cereal-growing regions worldwide. Although B-tolerant barley (Hordeum vulgare) germplasm is available, molecules responsible for this tolerance mechanism have not been defined. We describe and use a new comparative proteomic technique, iTRAQ peptide tagging (iTRAQ), to compare the abundances of proteins from B-tolerant and -intolerant barley plants from a ‘Clipper’ × ‘Sahara’ doubled-haploid population selected on the basis of a presence or absence of two B-tolerance quantitative trait loci. iTRAQ was used to identify three enzymes involved in siderophore production (Iron Deficiency Sensitive2 [IDS2], IDS3, and a methylthio-ribose kinase) as being elevated in abundance in the B-tolerant plants. Following from this result, we report a potential link between iron, B, and the siderophore hydroxymugineic acid. We believe that this study highlights the potency of the iTRAQ approach to better understand mechanisms of abiotic stress tolerance in cereals, particularly when applied in conjunction with bulked segregant analysis. PMID:17478636

  6. The Drosophila CLOCK protein undergoes daily rhythms in abundance, phosphorylation, and interactions with the PER-TIM complex.

    PubMed

    Lee, C; Bae, K; Edery, I

    1998-10-01

    We report the in vivo characterization of the Drosophila CLOCK protein (dCLOCK), a transcription factor that is required for the expression of the circadian clock genes period (per) and timeless (tim). dCLOCK undergoes circadian fluctuations in abundance, is phosphorylated throughout a daily cycle, and interacts with PER, TIM, and/or the PER-TIM complex during the night but not during most of the day. Our results suggest that PER and TIM participate in transcriptional autoinhibition by physically interacting with dCLOCK or a dCLOCK-containing complex. Nevertheless, in the absence of PER or TIM, the levels of dCLOCK are constitutively low, indicating that PER and TIM also act as positive elements in the feedback loop by stimulating the production of dCLOCK.

  7. Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

    PubMed Central

    Zhao, Jianguo; Xu, Weijie; Ross, Jason W.; Walters, Eric M.; Butler, Stephen P.; Whyte, Jeff J.; Kelso, Lindsey; Fatemi, Mostafa; Vanderslice, Nicholas C.; Giroux, Keith; Spate, Lee D.; Samuel, Melissa S.; Murphy, Cliff N.; Wells, Kevin D.; Masiello, Nick C.; Prather, Randall S.; Velander, William H.

    2015-01-01

    Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world’s population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock. PMID:26387706

  8. Characterization of an 18-kilodalton Brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis.

    PubMed

    Goldbaum, F A; Leoni, J; Wallach, J C; Fossati, C A

    1993-08-01

    Some anticytoplasmic protein monoclonal antibodies (MAbs) from mice immunized by infection with Brucella ovis cells have been obtained. One of these MAbs, BI24, was used to purify by immunoaffinity a protein with a pI of 5.6 and a molecular mass of 18 kDa. This protein was present in all of the rough and smooth Brucella species studied, but it could not be detected in Yersinia enterocolitica 09. Three internal peptides of this protein were partially sequenced; no homology with other bacterial proteins was found. The immunogenicity of the 18-kDa protein was studied with both human and bovine sera by a capture enzyme-linked immunosorbent assay system with MAb BI24.

  9. Characterization of an 18-kilodalton Brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis.

    PubMed Central

    Goldbaum, F A; Leoni, J; Wallach, J C; Fossati, C A

    1993-01-01

    Some anticytoplasmic protein monoclonal antibodies (MAbs) from mice immunized by infection with Brucella ovis cells have been obtained. One of these MAbs, BI24, was used to purify by immunoaffinity a protein with a pI of 5.6 and a molecular mass of 18 kDa. This protein was present in all of the rough and smooth Brucella species studied, but it could not be detected in Yersinia enterocolitica 09. Three internal peptides of this protein were partially sequenced; no homology with other bacterial proteins was found. The immunogenicity of the 18-kDa protein was studied with both human and bovine sera by a capture enzyme-linked immunosorbent assay system with MAb BI24. Images PMID:8370742

  10. Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli.

    PubMed Central

    Yenugu, Suresh; Hamil, Katherine G; Birse, Charles E; Ruben, Steven M; French, Frank S; Hall, Susan H

    2003-01-01

    During passage through the epididymis, sperm interact with secreted epididymal proteins that promote maturation, including the acquisition of motility and fertilization competence. Viewed previously as distinct from sperm maturation, host defence capabilities are now recognized functions of the human epididymis 2 (HE2) family of sperm-binding proteins. We analysed the potent dose and time-dependent bactericidal activity of recombinant HE2alpha, HE2beta1 and HE2beta2 and found that the full-length proteins (10 microg/ml or approximately 1 microM) caused more than a 50% decrease in Escherichia coli colony forming units within 15 min. By contrast, human beta-defensin-1, at a similar concentration, required more than 90 min to exhibit similar antibacterial activity. The epididymis-specific lipocalin, LCN6, failed to kill bacteria. Higher concentrations (25-100 microg/ml) of HE2 proteins and a longer duration of treatment resulted in near total inhibition of bacterial growth. The C-terminal peptides of HE2alpha, HEbeta1 and HEbeta2 proteins exhibited antibacterial activity similar to their full-length counterparts, indicating that the antibacterial activity of HE2 proteins resides in these C-terminal regions. Antibacterial activities of HE2 proteins and peptides were slightly inhibited by NaCl concentrations of up to 150 mM, while human beta-defensin-1 activity was nearly eliminated. Reduction and alkylation of disulphide bonds in HE2 proteins and their C-terminal peptides abolished their antibacterial activity. Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic E. coli membranes. A much longer exposure time was required for human beta-defensin-1-mediated permeabilization of membranes, suggesting a possible difference in mode of action compared with the HE2 antibacterial peptides. PMID:12628001

  11. Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS

    PubMed Central

    Xie, Xitao; Yi, Zhengping; Bowen, Benjamin; Wolf, Cassandra; Flynn, Charles R.; Sinha, Sandeep; Mandarino, Lawrence J.; Meyer, Christian

    2010-01-01

    Abnormalities in adipocytes play an important role in various conditions, including the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, but little is known about alterations at the protein level. We therefore sought to 1) comprehensively characterize the human adipocyte proteome for the first time, and 2) demonstrate feasibility of measuring adipocyte protein abundances by one-dimensional SDS-PAGE and High Performance Liquid Chromatography -Electron Spray Ionization - tandem Mass Spectrometry (HPLC-ESI-MS/MS). In adipocytes isolated from ~0.5 g subcutaneous abdominal adipose tissue of three healthy, lean subjects we identified a total of 1493 proteins. Triplicate analysis indicated a 22.5% coefficient of variation of protein abundances. Proteins ranged from 5.8 to 629 kDa and included a large number of proteins involved in lipid metabolism, such as fatty acid transport, fatty acid oxidation, lipid storage, lipolysis and lipid droplet maintenance. Furthermore, we found most glycolysis enzymes and numerous proteins associated with oxidative stress, protein synthesis and degradation as well as some adipokines. 22% of all proteins were of mitochondrial origin. These results provide the first detailed characterization of the human adipocyte proteome, suggest an important role of adipocyte mitochondria, and demonstrate feasibility of this approach to examine alterations of adipocyte protein abundances in human diseases. PMID:20812759

  12. The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.

    PubMed Central

    Zhang, Z; Tanaka, Y; Nonaka, S; Aizawa, H; Kawasaki, H; Nakata, T; Hirokawa, N

    1993-01-01

    Overlapping cDNA clones encoding the heavy chain of rat brain cytoplasmic dynein have been isolated. The isolated cDNA clones contain an open reading frame of 13,932 bp encoding 4644 aa (M(r), 532,213). The deduced protein sequence of the heavy chain of rat brain dynein shows significant similarity to sea urchin flagellar beta-dynein (27.0% identical) and to Dictyostelium cytoplasmic dynein (53.5% identical) throughout the entire sequence. The heavy chain of rat brain (cytoplasmic) dynein contains four putative nucleotide-binding consensus sequences [GX4GK(T/S)] in the central one-third region that are highly similar to those of sea urchin and Dictyostelium dyneins. The N-terminal one-third of the heavy chain of rat brain (cytoplasmic) dynein shows high similarity (43.8% identical) to that of Dictyostelium cytoplasmic dynein but poor similarity (19.4% identical) to that of sea urchin flagellar dynein. These results suggested that the C-terminal two-thirds of the dynein molecule is conserved and plays an essential role in microtubule-dependent motility activity, whereas the N-terminal regions are different between cytoplasmic and flagellar dyneins. Images Fig. 1 PMID:7690137

  13. Expression in yeast of the T-urf13 protein from Texas male-sterile maize mitochondria confers sensitivity to methomyl and to Texas-cytoplasm-specific fungal toxins.

    PubMed

    Huang, J; Lee, S H; Lin, C; Medici, R; Hack, E; Myers, A M

    1990-02-01

    The mitochondrial gene T-urf13 from maize (Zea mays L.) with Texas male-sterile (T) cytoplasm codes for a unique 13 kd polypeptide, T-URF13, which is implicated in cytoplasmic male sterility and sensitivity to the insecticide methomyl and to host-specific fungal toxins produced by Helminthosporium maydis race T (HmT toxin) and Phyllosticta maydis (Pm toxin). A chimeric gene coding for T-URF13 fused to the mitochondrial targeting peptide from the Neurospora crassa ATP synthase subunit 9 precursor was constructed. Expression of this gene in the yeast Saccharomyces cerevisiae yielded a polypeptide that was translocated into the membrane fraction of mitochondria and processed to give a protein the same size as maize T-URF13. Methomyl, HmT toxin and Pm toxin inhibited growth of yeast cells expressing the gene fusion on medium containing glycerol as sole carbon source and stimulated respiration with NADH as substrate by isolated mitochondria from these cells. These effects were not observed in yeast cells expressing T-URF13 without a targeting peptide. The results show that T-URF13 is sufficient to confer sensitivity to methomyl and the fungal toxins in a heterologous eukaryotic system, and suggest that mitochondrial localization of T-URF13 is critical for these functions.

  14. Photoreception and signal transduction in corals: proteomic and behavioral evidence for cytoplasmic calcium as a mediator of light responsivity.

    PubMed

    Hilton, J Daniel; Brady, Aisling K; Spaho, Skender A; Vize, Peter D

    2012-12-01

    Little is known about how corals sense and respond to light. In this report the proteome of coral is explored using 2D protein electrophoresis in two species, Montastraea cavernosa and Acropora millepora. Multiple protein species have major shifts in abundance in both species when sampled in daylight compared to corals sampled late in the night. These changes were observed both in larvae lacking zooxanthellae and in adult tissue containing zooxanthellae, including both Pacific and Caribbean corals. When larvae kept in the dark were treated with either thapsigargin or ionomycin, compounds that raise the level of cytoplasmic calcium, the night pattern of proteins shifted to the day pattern. This implies that photoreceptors responding to light elevate calcium levels and that calcium acts as the second messenger relaying light responses in corals. Corals spawn at night, and spawning can be delayed by exposure to light or pushed forward by early artificial sunsets. In a series of behavioral experiments, treatment of corals with ionomycin or thapsigargin was found to delay broadcast spawning in M. franksi, demonstrating that pharmacologically altering cytoplasmic calcium levels generates the same response as light exposure. Together these results show that the photo-responsive cells of corals detect and respond to light by altering cytoplasmic calcium levels, similarly to the transduction pathways in complex invertebrate eyes. The primacy of cytoplasmic calcium levels in light responsivity has broad implications for coral reproduction, including predicting how different species spawn at different times after sunset and how reproductive isolation is achieved during coral speciation.

  15. Abundance of some skeletal muscle mitochondrial proteins is associated with increased blood serum insulin in bovine fetuses.

    PubMed

    Pajak, Beata; Pawlikowska, Patrycja; Cassar-Malek, Isabelle; Picard, Brigitte; Hocquette, Jean-François; Orzechowski, Arkadiusz

    2010-12-01

    The aim of this study was to investigate the evolution of the abundance of cytochrome oxidase c subunit IV (NCOIV) and beta subunit of ATP synthase (β-ATP) during the last third of gestation in bovine skeletal muscles. Semitendinosus, longissimus thoracis and rectus abdominis muscles were chosen for the immunoblotting of the respective protein levels. Muscle and blood samples from bovine fetuses of randomly selected breeds were collected at 180, 210, and 260 days post-conception (dpc). The muscle tissue expressions of NCOIV, β-ATP were compared to blood glucose and insulin. At 260 dpc, protein levels of NCOIV raised in skeletal muscles. Additionally, β-ATP in semitendinosus and longissimus thoracis were elevated and paralleled by higher concentrations of blood serum insulin. It corroborates our previous observations indicating that accelerated metabolic differentiation of bovine skeletal muscles is associated with elevated blood insulin and occurs during the last trimester of gestation. Our observations point to the connection between insulin-sensitivity and the molecular mechanisms of mitochondrial contribution to ontogenesis of skeletal muscles.

  16. Integrated analysis of transcriptomic and proteomic data of Desulfovibrio vulgaris: Zero-Inflated Poisson regression models to predict abundance of undetected proteins

    SciTech Connect

    Nie, Lei; Wu, Gang; Brockman, Fred J.; Zhang, Weiwen

    2006-05-04

    Abstract Advances in DNA microarray and proteomics technologies have enabled high-throughput measurement of mRNA expression and protein abundance. Parallel profiling of mRNA and protein on a global scale and integrative analysis of these two data types could provide additional insight into the metabolic mechanisms underlying complex biological systems. However, because protein abundance and mRNA expression are affected by many cellular and physical processes, there have been conflicting results on the correlation of these two measurements. In addition, as current proteomic methods can detect only a small fraction of proteins present in cells, no correlation study of these two data types has been done thus far at the whole-genome level. In this study, we describe a novel data-driven statistical model to integrate whole-genome microarray and proteomic data collected from Desulfovibrio vulgaris grown under three different conditions. Based on the Poisson distribution pattern of proteomic data and the fact that a large number of proteins were undetected (excess zeros), Zero-inflated Poisson models were used to define the correlation pattern of mRNA and protein abundance. The models assumed that there is a probability mass at zero representing some of the undetected proteins because of technical limitations. The models thus use abundance measurements of transcripts and proteins experimentally detected as input to generate predictions of protein abundances as output for all genes in the genome. We demonstrated the statistical models by comparatively analyzing D. vulgaris grown on lactate-based versus formate-based media. The increased expressions of Ech hydrogenase and alcohol dehydrogenase (Adh)-periplasmic Fe-only hydrogenase (Hyd) pathway for ATP synthesis were predicted for D. vulgaris grown on formate.

  17. Cycling of intracellular pH during cell division of Xenopus embryos is a cytoplasmic activity depending on protein synthesis and phosphorylation

    PubMed Central

    1990-01-01

    In Xenopus embryos, the successive and rapid cell divisions that follow fertilization are accompanied by periodic oscillations of intracellular pH (pHi). Cycling of pHi occurs in phase with several other oscillatory activities, namely nuclear divisions, M phase-promoting factor (MPF) activity, and surface contraction waves (SCWs). We report that treatments that abolish cycling of MPF activity and the SCWs also suppress the pHi oscillations, whereas those that block cell division without affecting neither MPF activity nor the SCWs do not suppress the pHi oscillations. Experiments on enucleated oocytes, matured in vitro and activated, demonstrated that the activity governing the rhythmicity of the pHi oscillations resided in the cytoplasm of the oocyte. In this respect, the activity responsible for the pHi oscillations was different from that which drives the SCWs, which necessitated the presence of the oocyte germinal vesicle (Ohsumi et al., 1986), but more closely resembled MPF activity that did not require the presence of the oocyte germinal vesicle (Dabauvalle et al., 1988). In mature eggs enucleated at the time of egg activation, the pHi oscillations were similar to those in control nucleated eggs, whereas the period between two peaks of SCWs was 35-60 min vs. 20-35 min in nucleated control eggs. Previous studies had shown that the periodicity of SCWs was larger in anucleate egg fragments than in their nucleate counterparts (Sakai and Kubota, 1981), the difference being on the order of 6-15 min (Shinagawa, 1983). However, in these previous studies, enucleation was performed 30-50 min after fertilization. Our results clearly demonstrate that the periodicity of the SCWs is lengthened when the interval between egg activation and enucleation is shortened, thereby providing an easier way to assess the nuclear dependency of the SCWs. Finally, the various possibilities concerning the role of pHi cycling during cell division are discussed. PMID:2116420

  18. Internal Sense of Direction: Sensing and Signaling from Cytoplasmic Chemoreceptors

    PubMed Central

    Collins, Kieran D.; Lacal, Jesus

    2014-01-01

    SUMMARY Chemoreceptors sense environmental signals and drive chemotactic responses in Bacteria and Archaea. There are two main classes of chemoreceptors: integral inner membrane and soluble cytoplasmic proteins. The latter were identified more recently than integral membrane chemoreceptors and have been studied much less thoroughly. These cytoplasmic chemoreceptors are the subject of this review. Our analysis determined that 14% of bacterial and 43% of archaeal chemoreceptors are cytoplasmic, based on currently sequenced genomes. Cytoplasmic chemoreceptors appear to share the same key structural features as integral membrane chemoreceptors, including the formations of homodimers, trimers of dimers, and 12-nm hexagonal arrays within the cell. Cytoplasmic chemoreceptors exhibit varied subcellular locations, with some localizing to the poles and others appearing both cytoplasmic and polar. Some cytoplasmic chemoreceptors adopt more exotic locations, including the formations of exclusively internal clusters or moving dynamic clusters that coalesce at points of contact with other cells. Cytoplasmic chemoreceptors presumably sense signals within the cytoplasm and bear diverse signal input domains that are mostly N terminal to the domain that defines chemoreceptors, the so-called MA domain. Similar to the case for transmembrane receptors, our analysis suggests that the most common signal input domain is the PAS (Per-Arnt-Sim) domain, but a variety of other N-terminal domains exist. It is also common, however, for cytoplasmic chemoreceptors to have C-terminal domains that may function for signal input. The most common of these is the recently identified chemoreceptor zinc binding (CZB) domain, found in 8% of all cytoplasmic chemoreceptors. The widespread nature and diverse signal input domains suggest that these chemoreceptors can monitor a variety of cytoplasmically based signals, most of which remain to be determined. PMID:25428939

  19. Looking Deep Inside: Detection of Low-Abundance Proteins in Leaf Extracts of Arabidopsis and Phloem Exudates of Pumpkin1[W

    PubMed Central

    Fröhlich, Andreas; Gaupels, Frank; Sarioglu, Hakan; Holzmeister, Christian; Spannagl, Manuel; Durner, Jörg; Lindermayr, Christian

    2012-01-01

    The field of proteomics suffers from the immense complexity of even small proteomes and the enormous dynamic range of protein concentrations within a given sample. Most protein samples contain a few major proteins, which hamper in-depth proteomic analysis. In the human field, combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) have been used for reduction of the dynamic range of protein concentrations; however, this technique is not established in plant research. In this work, we present the application of CPLL to Arabidopsis (Arabidopsis thaliana) leaf proteins. One- and two-dimensional gel electrophoresis showed a decrease in high-abundance proteins and an enrichment of less abundant proteins in CPLL-treated samples. After optimization of the CPLL protocol, mass spectrometric analyses of leaf extracts led to the identification of 1,192 proteins in control samples and an additional 512 proteins after the application of CPLL. Upon leaf infection with virulent Pseudomonas syringae DC3000, CPLL beads were also used for investigating the bacterial infectome. In total, 312 bacterial proteins could be identified in infected Arabidopsis leaves. Furthermore, phloem exudates of pumpkin (Cucurbita maxima) were analyzed. CPLL prefractionation caused depletion of the major phloem proteins 1 and 2 and improved phloem proteomics, because 67 of 320 identified proteins were detectable only after CPLL treatment. In sum, our results demonstrate that CPLL beads are a time- and cost-effective tool for reducing major proteins, which often interfere with downstream analyses. The concomitant enrichment of less abundant proteins may facilitate a deeper insight into the plant proteome. PMID:22555880

  20. Heterogeneity of Maize Cytoplasmic Genomes among Male-Sterile Cytoplasms

    PubMed Central

    Pring, D. R.; Levings, C. S.

    1978-01-01

    Maize mitochondrial and chloroplast DNA's were prepared from normal (fertile) lines or single crosses and from members of the T, C, and S groups of male-sterile cytoplasms. Restriction endonucleases HindIII, BamI, EcoRI, and SalI were used to restrict the DNA, and the resultant fragments were electrophoresed in agarose gels. The results show that the N (fertile), T, C, and S cytoplasms each contained distinct mitochondrial DNA (mtDNA). These distinctive patterns were unaffected by nuclear genotype. No evidence of paternal inheritance of mtDNA was observed. Chloroplast DNA (ctDNA) from the N, C, and T cytoplasms was indistinguishable by HindIII, SalI, or EcoRI endonuclease digestion. The S cytoplasm ctDNA, however, was slightly different from that of other cytoplasms, as indicated by a slight displacement of one band in HindIII digests. The molecular weight of maize ctDNA was estimated to be as high as 88 x 106. Estimates of the minimum molecular weight of maize mtDNA ranged from 116–131 x 106, but the patterns were to complex for an unambiguous determination. Based on HindIII data, a comparison of the molecular weight of mtDNA bands common to the N, T. C, and S cytoplasms suggests that C cytoplasm most closely resembles N cytoplasm. The T and S sources are more divergent from the C and N cytoplasms. These results indicate a possible gradation of relatedness among male-sterile cytoplasms. The marked variation in mtDNA, with apparently less variation in ctDNA, represents circumstantial, but compelling, evidence that mtDNA may be involved in the male sterility and disease susceptibility traits in maize. PMID:17248823

  1. Competition between Heterochromatic Loci Allows the Abundance of the Silencing Protein, Sir4, to Regulate de novo Assembly of Heterochromatin

    PubMed Central

    Larin, Michelle L.; Harding, Katherine; Williams, Elizabeth C.; Lianga, Noel; Doré, Carole; Pilon, Sophie; Langis, Éric; Yanofsky, Corey; Rudner, Adam D.

    2015-01-01

    Changes in the locations and boundaries of heterochromatin are critical during development, and de novo assembly of silent chromatin in budding yeast is a well-studied model for how new sites of heterochromatin assemble. De novo assembly cannot occur in the G1 phase of the cell cycle and one to two divisions are needed for complete silent chromatin assembly and transcriptional repression. Mutation of DOT1, the histone H3 lysine 79 (K79) methyltransferase, and SET1, the histone H3 lysine 4 (K4) methyltransferase, speed de novo assembly. These observations have led to the model that regulated demethylation of histones may be a mechanism for how cells control the establishment of heterochromatin. We find that the abundance of Sir4, a protein required for the assembly of silent chromatin, decreases dramatically during a G1 arrest and therefore tested if changing the levels of Sir4 would also alter the speed of de novo establishment. Halving the level of Sir4 slows heterochromatin establishment, while increasing Sir4 speeds establishment. yku70Δ and ubp10Δ cells also speed de novo assembly, and like dot1Δ cells have defects in subtelomeric silencing, suggesting that these mutants may indirectly speed de novo establishment by liberating Sir4 from telomeres. Deleting RIF1 and RIF2, which suppresses the subtelomeric silencing defects in these mutants, rescues the advanced de novo establishment in yku70Δ and ubp10Δ cells, but not in dot1Δ cells, suggesting that YKU70 and UBP10 regulate Sir4 availability by modulating subtelomeric silencing, while DOT1 functions directly to regulate establishment. Our data support a model whereby the demethylation of histone H3 K79 and changes in Sir4 abundance and availability define two rate-limiting steps that regulate de novo assembly of heterochromatin. PMID:26587833

  2. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    PubMed

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  3. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    PubMed

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  4. Preferential binding of the neutrophil cytoplasmic granule-derived bactericidal/permeability increasing protein to target bacteria. Implications and use as a means of purification.

    PubMed

    Mannion, B A; Kalatzis, E S; Weiss, J; Elsbach, P

    1989-04-15

    The specificity of the basic bactericidal/permeability increasing protein (BPI) of polymorphonuclear leukocytes (PMN) for gram-negative bacteria is attributable to its strong attraction for the negatively charged envelope LPS. The antibacterial activity of PMN homogenates or extracts toward Escherichia coli corresponds to their BPI content and is blocked by anti-BPI IgG, suggesting that BPI action is unaffected by the presence of other PMN proteins. To test if BPI is preferentially bound to E. coli when other antibacterial proteins are present, we have measured binding in buffered (pH 7.5) balanced salts solution of [125I] human BPI to E. coli J5 in the presence and absence of other human PMN granule proteins. BPI binding is saturable with an apparent K = 23 nM and 2.2 million binding sites/cell. While binding of [125I] human BPI is competitively inhibited by human or rabbit BPI, it is only weakly inhibited by myeloperoxidase, lysozyme, or cathepsin G. In contrast, myeloperoxidase binding to E. coli is strongly inhibited by BPI. Moreover, incubation of E. coli with crude extracts of PMN or CML spleen results in near quantitative binding of BPI, identified by silver staining and immunoblotting after SDS-PAGE of the washed E. coli pellet, without recognizable binding of other leukocyte proteins (greater than 98% of added total protein is recovered in supernatant). After addition of 200 mM MgCl2, approximately 80% of bound BPI is released as fully active and pure protein (as judged by SDS-PAGE and HPLC). Thus the selective and reversible binding of BPI in crude PMN extracts to target bacteria provides a one-step "affinity" purification procedure.

  5. Compartmentalization of membrane trafficking, glucose transport, glycolysis, actin, tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm.

    PubMed

    Au, Catherine E; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Kearney, Robert E; Smith, Charles E; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Simon, Paul H G; Mandato, Cr