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Sample records for 12s-lipoxygenase protein associates

  1. 12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells

    SciTech Connect

    Weisinger, Gary . E-mail: gary_w@tasmc.health.gov.il; Limor, Rona; Marcus-Perlman, Yonit; Knoll, Esther; Kohen, Fortune; Schinder, Vera; Firer, Michael; Stern, Naftali

    2007-05-11

    The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to {alpha}-actin, a component of the cytoplasmic myofilaments. 12-LO/{alpha}-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to {alpha}-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein {alpha}-actin.

  2. Microtubules, Tubulins and Associated Proteins.

    ERIC Educational Resources Information Center

    Raxworthy, Michael J.

    1988-01-01

    Reviews much of what is known about microtubules, which are biopolymers consisting predominantly of subunits of the globular protein, tubulin. Describes the functions of microtubules, their structure and assembly, microtube associated proteins, and microtubule-disrupting agents. (TW)

  3. Microtubules, Tubulins and Associated Proteins.

    ERIC Educational Resources Information Center

    Raxworthy, Michael J.

    1988-01-01

    Reviews much of what is known about microtubules, which are biopolymers consisting predominantly of subunits of the globular protein, tubulin. Describes the functions of microtubules, their structure and assembly, microtube associated proteins, and microtubule-disrupting agents. (TW)

  4. Epistructural Tension Promotes Protein Associations

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel

    2012-05-01

    Epistructural tension is the reversible work per unit area required to span the aqueous interface of a soluble protein structure. The parameter accounts for the free-energy cost of imperfect hydration, involving water molecules with a shortage of hydrogen-bonding partnerships relative to bulk levels. The binding hot spots along protein-protein interfaces are identified with residues that contribute significantly to the epistructural tension in the free subunits. Upon association, such residues either displace or become deprived of low-coordination vicinal water molecules.

  5. Bayesian Estimator of Protein-Protein Association Probabilities

    SciTech Connect

    2008-05-28

    The Bayesian Estimator of Protein-Protein Association Probabilities (BEPro3) is a software tool for estimating probabilities of protein-protein association between bait and prey protein pairs using data from multiple-bait, multiple-replicate, protein LC-MS/MS affinity isolation experiments. BEPro3 is public domain software, has been tested on Windows XP and version 10.4 or newer of the Mac OS 10.4, and is freely available. A user guide, example dataset with analysis and additional documentation are included with the BEPro3 download.

  6. Evolution of organelle-associated protein profiling.

    PubMed

    Yan, Wei; Aebersold, Ruedi; Raines, Elaine W

    2009-02-15

    Identification of the protein constituents of cell organelles forms the basis for studies to define the roles of specific proteins in organelle structure and functions. Over the past decade, the use of mass spectrometry-based proteomics has dissected various organelles and allowed the association of many novel proteins with particular organelles. This review chronicles the evolution of organelle proteomics technology, and discusses how many limitations, such as organelle heterogeneity and purity, can be avoided with recently developed quantitative profiling approaches. Although many challenges remain, quantitative profiling of organelles holds the promise to begin to address the complex and dynamic shuttling of proteins among organelles that will be critical for application of this advanced technology to disease-based changes in organelle function.

  7. Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

    PubMed Central

    Mezzina, Mariela P.

    2016-01-01

    Phasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies and in silico predictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant α-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have both in vivo and in vitro chaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins. PMID:27287326

  8. Tracking membrane protein association in model membranes.

    PubMed

    Reffay, Myriam; Gambin, Yann; Benabdelhak, Houssain; Phan, Gilles; Taulier, Nicolas; Ducruix, Arnaud; Hodges, Robert S; Urbach, Wladimir

    2009-01-01

    Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue.We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well.After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 A, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a

  9. Multifunctional Microtubule-Associated Proteins in Plants

    PubMed Central

    Krtková, Jana; Benáková, Martina; Schwarzerová, Kateřina

    2016-01-01

    Microtubules (MTs) are involved in key processes in plant cells, including cell division, growth and development. MT-interacting proteins modulate MT dynamics and organization, mediating functional and structural interaction of MTs with other cell structures. In addition to conventional microtubule-associated proteins (MAPs) in plants, there are many other MT-binding proteins whose primary function is not related to the regulation of MTs. This review focuses on enzymes, chaperones, or proteins primarily involved in other processes that also bind to MTs. The MT-binding activity of these multifunctional MAPs is often performed only under specific environmental or physiological conditions, or they bind to MTs only as components of a larger MT-binding protein complex. The involvement of multifunctional MAPs in these interactions may underlie physiological and morphogenetic events, e.g., under specific environmental or developmental conditions. Uncovering MT-binding activity of these proteins, although challenging, may contribute to understanding of the novel functions of the MT cytoskeleton in plant biological processes. PMID:27148302

  10. Hydrophobic folding units at protein-protein interfaces: implications to protein folding and to protein-protein association.

    PubMed Central

    Tsai, C. J.; Nussinov, R.

    1997-01-01

    A hydrophobic folding unit cutting algorithm, originally developed for dissecting single-chain proteins, has been applied to a dataset of dissimilar two-chain protein-protein interfaces. Rather than consider each individual chain separately, the two-chain complex has been treated as a single chain. The two-chain parsing results presented in this work show hydrophobicity to be a critical attribute of two-state versus three-state protein-protein complexes. The hydrophobic folding units at the interfaces of two-state complexes suggest that the cooperative nature of the two-chain protein folding is the outcome of the hydrophobic effect, similar to its being the driving force in a single-chain folding. In analogy to the protein-folding process, the two-chain, two-state model complex may correspond to the formation of compact, hydrophobic nuclei. On the other hand, the three-state model complex involves binding of already folded monomers, similar to the association of the hydrophobic folding units within a single chain. The similarity between folding entities in protein cores and in two-state protein-protein interfaces, despite the absence of some chain connectivities in the latter, indicates that chain linkage does not necessarily affect the native conformation. This further substantiates the notion that tertiary, non-local interactions play a critical role in protein folding. These compact, hydrophobic, two-chain folding units, derived from structurally dissimilar protein-protein interfaces, provide a rich set of data useful in investigations of the role played by chain connectivity and by tertiary interactions in studies of binding and of folding. Since they are composed of non-contiguous pieces of protein backbones, they may also aid in defining folding nuclei. PMID:9232644

  11. Sneddon syndrome associated with Protein S deficiency.

    PubMed

    Sayin, Refah; Bilgili, Serap Gunes; Karadag, Ayse Serap; Tombul, Temel

    2012-01-01

    Sneddon syndrome (SS) is rare, arterio-occlusive disorder characterized by generalized livedo racemosa of the skin and various central nervous symptoms due to occlusion of medium-sized arteries of unknown. Seizure, cognitive impairment, hypertension, and history of repetitive miscarriages are the other symptoms seen in this disease. Livedo racemosa involves persisting irreversible skin lesions red or blue in color with irregular margins. Usually, SS occurs in women of childbearing age. Protein S deficiency is an inherited or acquired disorder associated with an increased risk of thrombosis. We present a 33-year-old woman with SS with diffuse livedo racemosa, recurrent cerebrovascular diseases, migraine-type headache, sinus vein thrombosis, and protein S deficiency. Protein S deficiency and with Sneddon syndrome rarely encountered in the literature.

  12. Ribosome-associated protein quality control

    PubMed Central

    Brandman, Onn; Hegde, Ramanujan S

    2016-01-01

    Protein synthesis by the ribosome can fail for numerous reasons including faulty mRNA, insufficient availability of charged tRNAs and genetic errors. All organisms have evolved mechanisms to recognize stalled ribosomes and initiate pathways for recycling, quality control and stress signaling. Here we review the discovery and molecular dissection of the eukaryotic ribosome-associated quality-control pathway for degradation of nascent polypeptides arising from interrupted translation. PMID:26733220

  13. Microtubule-associated proteins from Antarctic fishes.

    PubMed

    Detrich, H W; Neighbors, B W; Sloboda, R D; Williams, R C

    1990-01-01

    Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.

  14. Protein-Associated Lipid of Bacillus stearothermophilus

    PubMed Central

    Card, George L.; Szuba, Joan C.; Shimizu, Marilyn

    1979-01-01

    The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 ± 0.02 μmol of lipid phosphorus per mg of protein, compared with 0.54 ± 0.05 μmol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 ± 4% cardiolipin, 7 ± 1% phosphatidylglycerol, and 15 ± 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F1 component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with 32P and [2-3H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex. PMID:159285

  15. Characterization of microtubule-associated protein 1-associated protein kinases from rat brain.

    PubMed

    Fujii, T; Watanabe, M; Nakamura, A

    1996-01-01

    The microtubule-associated protein (MAP) 1 preparation, MAP1A and 1B, obtained from rat brain microtubules was associated with protein kinases that were insensitive to cAMP, cGMP, calcium, calcium/calmodulin and calcium/phosphatidylserine. The fractionation of highly purified MAP1 by phosphocellulose chromatography revealed that protein kinase activity to phosphorylate phosvitin was separated into three major peaks (MAP1 kinases A, B and C). MAP1 was recovered in the MAP1 kinase A fraction and phosphorylated by the contained kinase. MAP1 kinase A is a novel protein kinase that is remarkably activated by poly-L-lysine and poly-L-arginine, but very insensitive to heparin among the kinases. Photoaffinity labeling using [alpha-32P]8-azido ATP indicated that the 65 kDa polypeptide is identified as an ATP-binding protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the highly purified MAP1 and MAP1 kinase A fractions. MAP1 kinases B and C may be identified as casein kinase I- and II-like kinases. The present results show that MAP1 is associated with at least three kinases and provide an insight for understanding thoroughly the MAP1-mediated microtubule functions.

  16. Structure prediction of magnetosome-associated proteins

    PubMed Central

    Nudelman, Hila; Zarivach, Raz

    2014-01-01

    Magnetotactic bacteria (MTB) are Gram-negative bacteria that can navigate along geomagnetic fields. This ability is a result of a unique intracellular organelle, the magnetosome. These organelles are composed of membrane-enclosed magnetite (Fe3O4) or greigite (Fe3S4) crystals ordered into chains along the cell. Magnetosome formation, assembly, and magnetic nano-crystal biomineralization are controlled by magnetosome-associated proteins (MAPs). Most MAP-encoding genes are located in a conserved genomic region – the magnetosome island (MAI). The MAI appears to be conserved in all MTB that were analyzed so far, although the MAI size and organization differs between species. It was shown that MAI deletion leads to a non-magnetic phenotype, further highlighting its important role in magnetosome formation. Today, about 28 proteins are known to be involved in magnetosome formation, but the structures and functions of most MAPs are unknown. To reveal the structure–function relationship of MAPs we used bioinformatics tools in order to build homology models as a way to understand their possible role in magnetosome formation. Here we present a predicted 3D structural models’ overview for all known Magnetospirillum gryphiswaldense strain MSR-1 MAPs. PMID:24523717

  17. Associations between individual cow factors and milk-protein production.

    PubMed

    Sargeant, J M; Martin, S W; Lissemore, K D; Leslie, K E; Gibson, J P; Scott, H M; Kelton, D F

    1998-02-06

    Associations between stage of lactation, cow characteristics, and protein production were evaluated using data from a 2-year period on 75 Ontario, 5 Alberta, and 3 Nova Scotia dairy farms. Individual-cow protein production was defined by 305-day protein yield and by the estimated breeding value for protein yield. Lactation curves for average daily protein yield were computed by parity, breed, and season of calving. Mean protein yield was highest in early lactation. However, there was no pronounced peak in daily protein yield. Parity was positively associated with 305-day protein yield and negatively associated with the estimated breeding values for protein yield. First-calf heifers had lower protein yields in early lactation and a slower rate of decline in protein yield in late lactation, as compared to later parity cows. Holstein cows had higher unadjusted protein yields and lower protein yields after adjusting for milk yield than other breeds. Holstein cows had significantly higher protein yields early in lactation compared to other breeds, but the rate of decline in protein production in late lactation was also greater. Season was associated with 305-day protein yield; the highest protein yields occurred in cows calving in the fall and winter months, but these cows had the greatest rate of decline in protein production in late lactation.

  18. Golgi linked protein glycosylation and associated diseases.

    PubMed

    Ungar, Daniel

    2009-09-01

    One of the Golgi's main functions is the glycosylation of secreted proteins. A large variety of glycan chains can be synthesized in the Golgi, and it is increasingly clear that these are critical in basic cellular functions as well as the development of multicellular organisms. The structurally best-documented glycans are N-glycans, yet these are also the most enigmatic in their function. In contrast, O-glycan function is far better understood, but here the structures and biosynthetic pathways are very incomplete. The critical importance of glycans is highlighted by the broad spectrum of diseases they are associated with, such as a number of inherited diseases, but also cancers or diabetes. The molecular clues to these, however, are only just being elucidated. Although some glycan structures are known to be involved in signaling or adhesion to the extracellular matrix, for most the functions are not yet known. This review aims at summarizing current knowledge as much as to point out critical areas key for future progress.

  19. TTRAP is a novel PML nuclear bodies-associated protein

    SciTech Connect

    Xu Guanlan; Pan Yukun; Wang Bingyin; Huang Lu; Tian Ling; Xue Jinglun; Chen Jinzhong Jia, William

    2008-10-24

    PML nuclear body (PML NB) is an important macromolecular nuclear structure that is involved in many essential aspects of cellular function. Tens of proteins have been found in PML NBs, and promyelocytic leukemia protein (PML) has been proven to be essential for the formation of this structure. Here, we showed that TRAF and TNF receptor-associated protein (TTRAP) was a novel PML NBs-associated protein. TTRAP colocalized with three important PML NBs-associated proteins, PML, DAXX and Sp100 in the typical fashion of PML NBs. By yeast mating assay, TTRAP was identified to interact with these PML NBs-associated proteins. The transcription and expression of TTRAP could be induced by IFN-{gamma}, representing another common feature of PML NBs-associated proteins. These results would not only be important for understanding PML NBs but also be helpful in studying the TTRAP function in the future.

  20. HIV-associated thromboembolic phenomenon due to protein C deficiency.

    PubMed

    Goyal, Anmol; Shah, Ira

    2014-01-01

    HIV-infected individuals are at a high risk of developing arterial and venous thromboembolism. Opportunistic infections, protease inhibitors, low CD4 count, antiphospholipid antibodies, protein S, and protein C deficiencies are some important risk factors associated with it. However, thromboembolic phenomenon due to protein C deficiency has been rarely reported. We report a case of a 12-year-old girl with facial palsy due to middle cerebral artery infarct because of HIV infection and associated protein C deficiency.

  1. Determination of reversible protein equilibrium association coefficients using light scattering

    NASA Astrophysics Data System (ADS)

    Larkin, Michael

    2009-03-01

    The characterization in solution of reversible protein associations as well as associations between proteins and small molecules is essential in many areas of science. Understanding cellular function or developing and formulating pharmaceuticals or other biologically active materials often requires quantitation of such associations. Most pharmaceuticals have functionality due solely to association with molecules within the body, and the discovery and accurate characterization of these associations is a key element for pharmaceutical development. Unfortunately, most methods used to measure associations of proteins require either immobilizing the protein on a surface (e.g. surface plasmon resonance), which potentially alters the protein characteristics, or require considerable time and effort and large quantities of sample (e.g. analytical ultracentrifugation, isothermal titration calorimetry). Light scattering based measurements of reversible association coefficients require much less sample and may be performed much more rapidly than other free solution techniques. In this talk I describe how static and dynamic light scattering may each independently be used to measure equilibrium association coefficients between proteins in free solution, and may also be used to observe and quantitate the association of small molecules with them. I present background theory for both static and dynamic light scattering measurements of equilibrium associations, and examples of measurements made of both model systems and of systems with commercial relevance in the pharmaceutical industry.

  2. Dynein light chain association sequences can facilitate nuclear protein import.

    PubMed

    Moseley, Gregory W; Roth, Daniela Martino; DeJesus, Michelle A; Leyton, Denisse L; Filmer, Richard P; Pouton, Colin W; Jans, David A

    2007-08-01

    Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures. We also find that P-protein DLC-AS, as well as analogous sequences from other proteins, acts as an independent module that can confer enhancement of nuclear accumulation to proteins carrying the P-protein NLS, as well as several heterologous NLSs. Photobleaching experiments in live cells demonstrate that the MT-dependent enhancement of NLS-mediated nuclear accumulation by the P-protein DLC-AS involves an increased rate of nuclear import. This is the first report of DLC-AS enhancement of NLS function, identifying a novel mechanism regulating nuclear transport with relevance to viral and cellular protein biology. Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application.

  3. Neurodegenerative diseases and widespread aggregation are associated with supersaturated proteins

    PubMed Central

    Ciryam, Prajwal; Tartaglia, Gian Gaetano; Morimoto, Richard I.; Dobson, Christopher M.; Vendruscolo, Michele

    2013-01-01

    Summary The maintenance of protein solubility is a fundamental aspect of protein homeostasis, as aggregation is associated with cytotoxicity and a variety of human diseases. Numerous proteins unrelated in sequence and structure, however, can misfold and aggregate, and widespread aggregation can occur in living systems under stress or ageing. A crucial question in this context is why only certain proteins aggregate in vivo while others do not. We identify here the proteins most vulnerable to aggregation as those whose cellular concentrations are high relative to their solubilities. These supersaturated proteins represent a metastable sub-proteome involved in pathological aggregation during stress and ageing, and are overrepresented in biochemical processes associated with neurodegenerative disorders. Consequently, such cellular processes become dysfunctional when the ability to keep intrinsically supersaturated proteins soluble is compromised. Thus, the simultaneous analysis of abundance and solubility can rationalize the diverse cellular pathologies linked to neurodegenerative diseases and aging. PMID:24183671

  4. Reverse Nearest Neighbor Search on a Protein-Protein Interaction Network to Infer Protein-Disease Associations

    PubMed Central

    Suratanee, Apichat; Plaimas, Kitiporn

    2017-01-01

    The associations between proteins and diseases are crucial information for investigating pathological mechanisms. However, the number of known and reliable protein-disease associations is quite small. In this study, an analysis framework to infer associations between proteins and diseases was developed based on a large data set of a human protein-protein interaction network integrating an effective network search, namely, the reverse k-nearest neighbor (RkNN) search. The RkNN search was used to identify an impact of a protein on other proteins. Then, associations between proteins and diseases were inferred statistically. The method using the RkNN search yielded a much higher precision than a random selection, standard nearest neighbor search, or when applying the method to a random protein-protein interaction network. All protein-disease pair candidates were verified by a literature search. Supporting evidence for 596 pairs was identified. In addition, cluster analysis of these candidates revealed 10 promising groups of diseases to be further investigated experimentally. This method can be used to identify novel associations to better understand complex relationships between proteins and diseases. PMID:28757797

  5. Reverse Nearest Neighbor Search on a Protein-Protein Interaction Network to Infer Protein-Disease Associations.

    PubMed

    Suratanee, Apichat; Plaimas, Kitiporn

    2017-01-01

    The associations between proteins and diseases are crucial information for investigating pathological mechanisms. However, the number of known and reliable protein-disease associations is quite small. In this study, an analysis framework to infer associations between proteins and diseases was developed based on a large data set of a human protein-protein interaction network integrating an effective network search, namely, the reverse k-nearest neighbor (RkNN) search. The RkNN search was used to identify an impact of a protein on other proteins. Then, associations between proteins and diseases were inferred statistically. The method using the RkNN search yielded a much higher precision than a random selection, standard nearest neighbor search, or when applying the method to a random protein-protein interaction network. All protein-disease pair candidates were verified by a literature search. Supporting evidence for 596 pairs was identified. In addition, cluster analysis of these candidates revealed 10 promising groups of diseases to be further investigated experimentally. This method can be used to identify novel associations to better understand complex relationships between proteins and diseases.

  6. Integrated protein function prediction by mining function associations, sequences, and protein-protein and gene-gene interaction networks.

    PubMed

    Cao, Renzhi; Cheng, Jianlin

    2016-01-15

    Protein function prediction is an important and challenging problem in bioinformatics and computational biology. Functionally relevant biological information such as protein sequences, gene expression, and protein-protein interactions has been used mostly separately for protein function prediction. One of the major challenges is how to effectively integrate multiple sources of both traditional and new information such as spatial gene-gene interaction networks generated from chromosomal conformation data together to improve protein function prediction. In this work, we developed three different probabilistic scores (MIS, SEQ, and NET score) to combine protein sequence, function associations, and protein-protein interaction and spatial gene-gene interaction networks for protein function prediction. The MIS score is mainly generated from homologous proteins found by PSI-BLAST search, and also association rules between Gene Ontology terms, which are learned by mining the Swiss-Prot database. The SEQ score is generated from protein sequences. The NET score is generated from protein-protein interaction and spatial gene-gene interaction networks. These three scores were combined in a new Statistical Multiple Integrative Scoring System (SMISS) to predict protein function. We tested SMISS on the data set of 2011 Critical Assessment of Function Annotation (CAFA). The method performed substantially better than three base-line methods and an advanced method based on protein profile-sequence comparison, profile-profile comparison, and domain co-occurrence networks according to the maximum F-measure. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Predicting protein function by frequent functional association pattern mining in protein interaction networks.

    PubMed

    Cho, Young-Rae; Zhang, Aidong

    2010-01-01

    Predicting protein function from protein interaction networks has been challenging because of the complexity of functional relationships among proteins. Most previous function prediction methods depend on the neighborhood of or the connected paths to known proteins. However, their accuracy has been limited due to the functional inconsistency of interacting proteins. In this paper, we propose a novel approach for function prediction by identifying frequent patterns of functional associations in a protein interaction network. A set of functions that a protein performs is assigned into the corresponding node as a label. A functional association pattern is then represented as a labeled subgraph. Our frequent labeled subgraph mining algorithm efficiently searches the functional association patterns that occur frequently in the network. It iteratively increases the size of frequent patterns by one node at a time by selective joining, and simplifies the network by a priori pruning. Using the yeast protein interaction network, our algorithm found more than 1400 frequent functional association patterns. The function prediction is performed by matching the subgraph, including the unknown protein, with the frequent patterns analogous to it. By leave-one-out cross validation, we show that our approach has better performance than previous link-based methods in terms of prediction accuracy. The frequent functional association patterns generated in this study might become the foundations of advanced analysis for functional behaviors of proteins in a system level.

  8. Predicting Protein-protein Association Rates using Coarse-grained Simulation and Machine Learning

    NASA Astrophysics Data System (ADS)

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

    2017-04-01

    Protein-protein interactions dominate all major biological processes in living cells. We have developed a new Monte Carlo-based simulation algorithm to study the kinetic process of protein association. We tested our method on a previously used large benchmark set of 49 protein complexes. The predicted rate was overestimated in the benchmark test compared to the experimental results for a group of protein complexes. We hypothesized that this resulted from molecular flexibility at the interface regions of the interacting proteins. After applying a machine learning algorithm with input variables that accounted for both the conformational flexibility and the energetic factor of binding, we successfully identified most of the protein complexes with overestimated association rates and improved our final prediction by using a cross-validation test. This method was then applied to a new independent test set and resulted in a similar prediction accuracy to that obtained using the training set. It has been thought that diffusion-limited protein association is dominated by long-range interactions. Our results provide strong evidence that the conformational flexibility also plays an important role in regulating protein association. Our studies provide new insights into the mechanism of protein association and offer a computationally efficient tool for predicting its rate.

  9. CB1 Cannabinoid Receptors and their Associated Proteins

    PubMed Central

    Howlett, Allyn C.; Blume, Lawrence C.; Dalton, George D.

    2011-01-01

    CB1 receptors are G-protein coupled receptors (GPCRs) abundant in neurons, in which they modulate neurotransmission. The CB1 receptor influence on memory and learning is well recognized, and disease states associated with CB1 receptors are observed in addiction disorders, motor dysfunction, schizophrenia, and in bipolar, depression, and anxiety disorders. Beyond the brain, CB1 receptors also function in liver and adipose tissues, vascular as well as cardiac tissue, reproductive tissues and bone. Signal transduction by CB1 receptors occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPK), inhibit voltage-gated Ca2+ channels, activate K+ currents (Kir), and influence Nitric Oxide (NO) signaling. CB1 receptors are observed in internal organelles as well as plasma membrane. β-Arrestins, adaptor protein AP-3, and G-protein receptor-associated sorting protein 1 (GASP1) modulate cellular trafficking. Cannabinoid Receptor Interacting Protein 1a (CRIP1a) is an accessory protein whose function has not been delineated. Factor Associated with Neutral sphingomyelinase (FAN) regulates ceramide signaling. Such diversity in cellular signaling and modulation by interacting proteins suggests that agonists and allosteric modulators could be developed to specifically regulate unique, cell type-specific responses. PMID:20166926

  10. Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

    PubMed

    Chen, L; Haider, K; Ponda, M; Cariappa, A; Rowitch, D; Pillai, S

    2001-06-15

    A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

  11. Identification of proteins associated with amyloidosis by polarity index method.

    PubMed

    Polanco, Carlos; Samaniego, José Lino; Uversky, Vladimir N; Castañón-González, Jorge Alberto; Buhse, Thomas; Leopold-Sordo, Marili; Madero-Arteaga, Alejandro; Morales-Reyes, Alicia; Tavera-Sierra, Lourdes; González-Bernal, Jesus A; Arias-Estrada, Miguel

    2015-01-01

    There is a natural protein form, insoluble and resistant to proteolysis, adopted by many proteins independently of their amino acid sequences via specific misfolding-aggregation process. This dynamic process occurs in parallel with or as an alternative to physiologic folding, generating toxic protein aggregates that are deposited and accumulated in various organs and tissues. These proteinaceous deposits typically represent bundles of β-sheet-enriched fibrillar species known as the amyloid fibrils that are responsible for serious pathological conditions, including but not limited to neurodegenerative diseases, grouped under the term amyloidoses. The proteins that might adopt this fibrillar conformation are some globular proteins and natively unfolded (or intrinsically disordered) proteins. Our work shows that intrinsically disordered and intrinsically ordered proteins can be reliably identified, discriminated, and differentiated by analyzing their polarity profiles generated using a computational tool known as the polarity index method (Polanco & Samaniego, 2009; Polanco et al., 2012; 2013; 2013a; 2014; 2014a; 2014b; 2014c; 2014d). We also show that proteins expressed in neurons can be differentiated from proteins in these two groups based on their polarity profiles, and also that this computational tool can be used to identify proteins associated with amyloidoses. The efficiency of the proposed method is high (i.e. 70%) as evidenced by the analysis of peptides and proteins in the APD2 database (2012), AVPpred database (2013), and CPPsite database (2013), the set of selective antibacterial peptides from del Rio et al. (2001), the sets of natively unfolded and natively folded proteins from Oldfield et al. (2005), the set of human revised proteins expressed in neurons, and non-human revised proteins expressed in neurons, from the Uniprot database (2014), and also the set of amyloidogenic proteins from the AmyPDB database (2014).

  12. Protein function prediction using guilty by association from interaction networks.

    PubMed

    Piovesan, Damiano; Giollo, Manuel; Ferrari, Carlo; Tosatto, Silvio C E

    2015-12-01

    Protein function prediction from sequence using the Gene Ontology (GO) classification is useful in many biological problems. It has recently attracted increasing interest, thanks in part to the Critical Assessment of Function Annotation (CAFA) challenge. In this paper, we introduce Guilty by Association on STRING (GAS), a tool to predict protein function exploiting protein-protein interaction networks without sequence similarity. The assumption is that whenever a protein interacts with other proteins, it is part of the same biological process and located in the same cellular compartment. GAS retrieves interaction partners of a query protein from the STRING database and measures enrichment of the associated functional annotations to generate a sorted list of putative functions. A performance evaluation based on CAFA metrics and a fair comparison with optimized BLAST similarity searches is provided. The consensus of GAS and BLAST is shown to improve overall performance. The PPI approach is shown to outperform similarity searches for biological process and cellular compartment GO predictions. Moreover, an analysis of the best practices to exploit protein-protein interaction networks is also provided.

  13. RAIN: RNA–protein Association and Interaction Networks

    PubMed Central

    Junge, Alexander; Refsgaard, Jan C.; Garde, Christian; Pan, Xiaoyong; Santos, Alberto; Alkan, Ferhat; Anthon, Christian; von Mering, Christian; Workman, Christopher T.; Jensen, Lars Juhl; Gorodkin, Jan

    2017-01-01

    Protein association networks can be inferred from a range of resources including experimental data, literature mining and computational predictions. These types of evidence are emerging for non-coding RNAs (ncRNAs) as well. However, integration of ncRNAs into protein association networks is challenging due to data heterogeneity. Here, we present a database of ncRNA–RNA and ncRNA–protein interactions and its integration with the STRING database of protein–protein interactions. These ncRNA associations cover four organisms and have been established from curated examples, experimental data, interaction predictions and automatic literature mining. RAIN uses an integrative scoring scheme to assign a confidence score to each interaction. We demonstrate that RAIN outperforms the underlying microRNA-target predictions in inferring ncRNA interactions. RAIN can be operated through an easily accessible web interface and all interaction data can be downloaded. Database URL: http://rth.dk/resources/rain PMID:28077569

  14. The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

    PubMed Central

    Solomon, K R; Rudd, C E; Finberg, R W

    1996-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8650218

  15. A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association

    PubMed Central

    Cabantous, Stéphanie; Nguyen, Hau B.; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A.; Favre, Gilles; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2013-01-01

    Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence. PMID:24092409

  16. A new protein-protein interaction sensor based on tripartite split-GFP association.

    PubMed

    Cabantous, Stéphanie; Nguyen, Hau B; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A; Favre, Gilles; Terwilliger, Thomas C; Waldo, Geoffrey S

    2013-10-04

    Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

  17. Engineering nanoparticle-protein associations for protein crystal nucleation and nanoparticle arrangement

    NASA Astrophysics Data System (ADS)

    Benoit, Denise N.

    Engineering the nanoparticle - protein association offers a new way to form protein crystals as well as new approaches for arrangement of nanoparticles. Central to this control is the nanoparticle surface. By conjugating polymers on the surface with controlled molecular weights many properties of the nanoparticle can be changed including its size, stability in buffers and the association of proteins with its surface. Large molecular weight poly(ethylene glycol) (PEG) coatings allow for weak associations between proteins and nanoparticles. These interactions can lead to changes in how proteins crystallize. In particular, they decrease the time to nucleation and expand the range of conditions over which protein crystals form. Interestingly, when PEG chain lengths are too short then protein association is minimized and these effects are not observed. One important feature of protein crystals nucleated with nanoparticles is that the nanoparticles are incorporated into the crystals. What results are nanoparticles placed at well-defined distances in composite protein-nanoparticle crystals. Crystals on the size scale of 10 - 100 micrometers exhibit optical absorbance, fluorescence and super paramagnetic behavior derivative from the incorporated nanomaterials. The arrangement of nanoparticles into three dimensional arrays also gives rise to new and interesting physical and chemical properties, such as fluorescence enhancement and varied magnetic response. In addition, anisotropic nanomaterials aligned throughout the composite crystal have polarization dependent optical properties.

  18. Prioritizing disease candidate proteins in cardiomyopathy-specific protein-protein interaction networks based on "guilt by association" analysis.

    PubMed

    Li, Wan; Chen, Lina; He, Weiming; Li, Weiguo; Qu, Xiaoli; Liang, Binhua; Gao, Qianping; Feng, Chenchen; Jia, Xu; Lv, Yana; Zhang, Siya; Li, Xia

    2013-01-01

    The cardiomyopathies are a group of heart muscle diseases which can be inherited (familial). Identifying potential disease-related proteins is important to understand mechanisms of cardiomyopathies. Experimental identification of cardiomyophthies is costly and labour-intensive. In contrast, bioinformatics approach has a competitive advantage over experimental method. Based on "guilt by association" analysis, we prioritized candidate proteins involving in human cardiomyopathies. We first built weighted human cardiomyopathy-specific protein-protein interaction networks for three subtypes of cardiomyopathies using the known disease proteins from Online Mendelian Inheritance in Man as seeds. We then developed a method in prioritizing disease candidate proteins to rank candidate proteins in the network based on "guilt by association" analysis. It was found that most candidate proteins with high scores shared disease-related pathways with disease seed proteins. These top ranked candidate proteins were related with the corresponding disease subtypes, and were potential disease-related proteins. Cross-validation and comparison with other methods indicated that our approach could be used for the identification of potentially novel disease proteins, which may provide insights into cardiomyopathy-related mechanisms in a more comprehensive and integrated way.

  19. Identification of nuclear structural protein alterations associated with seminomas.

    PubMed

    Leman, Eddy S; Magheli, Ahmed; Yong, Koh Meng Aw; Netto, George; Hinz, Stefan; Getzenberg, Robert H

    2009-12-15

    Currently, there are no specific markers available for the early detection and for monitoring testicular cancer. Based upon an approach that targets nuclear structure, we have identified a set of proteins that are specific for seminomas, which may then have clinical utility for the disease. Utilizing samples obtained from men with no evidence of testicular cancer (n = 5) as well as those with seminomas (n = 6), nuclear matrix proteins were extracted and separated using a high-resolution two-dimensional electrophoresis gel system. The proteins were identified by mass spectrometry analysis. These analyses revealed seven nuclear matrix proteins associated with the normal testes, which did not appear in the seminomas. In the seminomas, four nuclear matrix proteins were identified to be associated with the disease that were absent in the normal testes. Mass spectrometric and immunoblot analyses of these proteins revealed that one of the proteins identified in the normal testes appears to be StAR-related lipid transfer protein 7 (StARD7). In the non-seminoma tissues, one of the identified proteins appears to be cell division protein kinase 10 (CDK10). Both StarD7 and CDK10 could potentially be involved in cell differentiation and growth, and thus may serve as potential targets for therapy of prognostication of seminomas. This is the first study to examine the role of nuclear structural proteins as potential biomarkers in testicular cancer. We are currently examining the roles of some of the identified proteins as potential biomarkers for the disease. (c) 2009 Wiley-Liss, Inc.

  20. Nanoparticles-cell association predicted by protein corona fingerprints

    NASA Astrophysics Data System (ADS)

    Palchetti, S.; Digiacomo, L.; Pozzi, D.; Peruzzi, G.; Micarelli, E.; Mahmoudi, M.; Caracciolo, G.

    2016-06-01

    In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface

  1. Few serum proteins mediate APOE’s association with dementia

    PubMed Central

    2017-01-01

    The latent variable “δ” (for “dementia”) appears to be uniquely responsible for the dementing aspects of cognitive impairment. Age, depression, gender and the apolipoprotein E (APOE) e4 allele are independently associated with δ. In this analysis, we explore serum proteins as potential mediators of APOE’s specific association with δ in a large, ethnically diverse longitudinal cohort, the Texas Alzheimer’s Research and Care Consortium (TARCC). APOE was associated only with C-Reactive Protein (CRP), Adiponectin (APN) and Amphiregulin (AREG), although the latter two’s associations did not survive Bonferroni correction for multiple comparisons. All three proteins were associated with δ and had weak potential mediation effects on APOE’s association with that construct. Our findings suggest that APOE’s association with cognitive performance is specific to δ and partially mediated by serum inflammatory proteins. The majority of APOE’s significant unadjusted effect on δ is unexplained. It may instead arise from direct central nervous system effects, possibly on native intelligence. If so, then APOE may exert a life-long influence over δ and therefore all-cause dementia risk. PMID:28291794

  2. Quantitative Protein Localization Signatures Reveal an Association between Spatial and Functional Divergences of Proteins

    PubMed Central

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-01-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  3. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    PubMed

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-03-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  4. Screening serum hepatocellular carcinoma-associated proteins by SELDI-based protein spectrum analysis

    PubMed Central

    Cui, Jie-Feng; Liu, Yin-Kun; Zhou, Hai-Jun; Kang, Xiao-Nan; Huang, Cheng; He, Yi-Feng; Tang, Zhao-You; Uemura, Toshimasa

    2008-01-01

    AIM: To find out potential serum hepatocellular carcinoma (HCC)-associated proteins with low molecular weight and low abundance by SELDI-based serum protein spectra analysis, that will have much application in the diagnosis or differentiated diagnosis of HCC, as well as giving a better understanding of the mechanism of hepato-carcinogenesis. METHODS: Total serum samples were collected with informed consent from 81 HCC patients with HBV(+)/cirrhosis(+), 36 cirrhosis patients and 43 chronic hepatitis B patients. Serum protein fingerprint profiles were first generated by selected WCX2 protein chip capture integrating with SELDI-TOF-MS, then normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard. Comparative analysis of the intensity of corresponding protein fingerprint peaks in normalized protein spectra, some protein peaks with significant difference between HCC and cirrhosis or chronic hepatitis B were found. RESULTS: One hundred and twenty-eight serum protein peaks between 2000 and 30 000Da were identified under the condition of signal-to-noise > 5 and minimum threshold for cluster > 20%. Eighty-seven of these proteins were showed significant differences in intensity between HCC and cirrhosis (P < 0.05). Of the above differential proteins, 45 proteins had changes greater than two-fold, including 15 upregulated proteins and 30 downregulated proteins in HCC serum. Between HCC and chronic hepatitis B, 9 of 52 differential proteins (P < 0.05) had intensities of more than two-fold, including 2 upregulated proteins and 7 downregulated proteins in HCC serum. Between cirrhosis and chronic hepatitis B, 28 of 79 significant differential proteins (P < 0.05) changes greater than two-fold in intensity, including 17 upregulated proteins and 11 downregulated proteins in cirrhosis serum. For the analysis of these leading differential proteins in subtraction difference mode among three diseases, the five common downregulated proteins in HCC serum (M/Z 2870

  5. Screening serum hepatocellular carcinoma-associated proteins by SELDI-based protein spectrum analysis.

    PubMed

    Cui, Jie-Feng; Liu, Yin-Kun; Zhou, Hai-Jun; Kang, Xiao-Nan; Huang, Cheng; He, Yi-Feng; Tang, Zhao-You; Uemura, Toshimasa

    2008-02-28

    To find out potential serum hepatocellular carcinoma (HCC)-associated proteins with low molecular weight and low abundance by SELDI-based serum protein spectra analysis, that will have much application in the diagnosis or differentiated diagnosis of HCC, as well as giving a better understanding of the mechanism of hepato-carcinogenesis. Total serum samples were collected with informed consent from 81 HCC patients with HBV(+)/cirrhosis(+), 36 cirrhosis patients and 43 chronic hepatitis B patients. Serum protein fingerprint profiles were first generated by selected WCX2 protein chip capture integrating with SELDI-TOF-MS, then normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard. Comparative analysis of the intensity of corresponding protein fingerprint peaks in normalized protein spectra, some protein peaks with significant difference between HCC and cirrhosis or chronic hepatitis B were found. One hundred and twenty-eight serum protein peaks between 2000 and 30000 Da were identified under the condition of signal-to-noise > 5 and minimum threshold for cluster > 20%. Eighty-seven of these proteins were showed significant differences in intensity between HCC and cirrhosis (P < 0.05). Of the above differential proteins, 45 proteins had changes greater than two-fold, including 15 upregulated proteins and 30 downregulated proteins in HCC serum. Between HCC and chronic hepatitis B, 9 of 52 differential proteins (P < 0.05) had intensities of more than two-fold, including 2 upregulated proteins and 7 downregulated proteins in HCC serum. Between cirrhosis and chronic hepatitis B, 28 of 79 significant differential proteins (P < 0.05) changes greater than two-fold in intensity, including 17 upregulated proteins and 11 downregulated proteins in cirrhosis serum. For the analysis of these leading differential proteins in subtraction difference mode among three diseases, the five common downregulated proteins in HCC serum (M/Z 2870, 3941, 2688, 3165, 5483

  6. Association of Ebola Virus Matrix Protein VP40 with Microtubules

    DTIC Science & Technology

    2005-04-01

    dynein has been reported for African swine fever virus protein 54 (1) as well as VP26 of herpes simplex virus (12), and binding to members of the plus...associated motor pro- teins for movement of viral particles to the site of budding has been proposed for African swine fever virus and vaccinia virus (22...Fernandez-Zapatero, L. Soto, C. Canto, I. Rodriguez-Crespo, L. Dixon, and J. M. Escribano. 2001. African swine fever virus protein p54 interacts with

  7. A cellular protein that associates with the transforming protein of Rous sarcoma virus is also a heat-shock protein.

    PubMed Central

    Oppermann, H; Levinson, W; Bishop, J M

    1981-01-01

    A single viral protein (pp60src) mediates neoplastic transformation of cells infected with Rous sarcoma virus. Immunoprecipitation of pp60src has revealed two cellular proteins (Mr 50,000 and 89,000) that appear to associate with pp60src in a specific manner. Neither of the cellular proteins has been well characterized, but it is thought that both may participate in the function of pp60src. Treatment of avian cells with unphysiological temperature or certain chemical agents amplifies the production of several proteins in the manner of the "heat shock" response earlier described for Drosophila. We report here that one of these proteins, with a molecular weight of 89,000 is identical to the 89-kilodalton protein found associated with pp60src. The 89-kilodalton protein is a major constituent of both uninfected and infected cells, even in the absence of inducing agents, but only a small fraction of this protein appears to associate with pp60src in cells transformed by Rous sarcoma virus. The complex containing pp60src and the 89-kilodalton protein can be precipitated by an immune reaction involving pp60src alone. The complexed form of the 89-kilodalton protein did not react directly with antibodies but regained its reactivity subsequent to release from the complex. We conclude that the 89-kilodalton protein is bound to pp60src in a relatively stable complex. We suggest that the 89-kilodalton protein may have overlapping roles in viral oncogenesis and the heat shock response, and that evidence on the function of the protein in either setting may illuminate its function in the other. In addition, it may prove profitable to search for other overlaps between the cellular response to heat shock and the neoplastic transformation of cells by pp60src. Images PMID:6262754

  8. Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

    PubMed Central

    Miczak, A; Kaberdin, V R; Wei, C L; Lin-Chao, S

    1996-01-01

    The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. Images Fig. 1 Fig. 2 PMID:8632981

  9. Surfactant-associated proteins: structure, function and clinical implications.

    PubMed

    Ketko, Anastasia K; Donn, Steven M

    2014-01-01

    Surfactant replacement therapy is now the standard of care for infants with respiratory distress syndrome. As the understanding of surfactant structure and function has evolved, surfactant-associated proteins are now understood to be essential components of pulmonary surfactant. Their structural and functional diversity detail the complexity of their contributions to normal pulmonary physiology, and deficiency states result in significant pathology. Engineering synthetic surfactant protein constructs has been a major research focus for replacement therapies. This review highlights what is known about surfactant proteins and how this knowledge is pivotal for future advancements in treating respiratory distress syndrome as well as other pulmonary diseases characterized by surfactant deficiency or inactivation.

  10. Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Perez, Adam M.; Finnigan, Gregory C.; Roelants, Françoise M.; Thorner, Jeremy

    2016-01-01

    Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast Saccharomyces cerevisiae is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle. In particular, septin-associated protein kinases couple cell cycle progression with cellular morphogenesis. Thus, septin-containing structures serve as signaling platforms that integrate a multitude of signals and coordinate key downstream networks required for cell cycle passage. This review summarizes what we currently understand about how the action of septin-associated protein kinases and their substrates control information flow to drive the cell cycle into and out of mitosis, to regulate bud growth, and especially to direct timely and efficient execution of cytokinesis and cell abscission. Thus, septin structures represent a regulatory node at the intersection of many signaling pathways. In addition, and importantly, the activities of certain septin-associated protein kinases also regulate the state of organization of the septins themselves, creating a complex feedback loop. PMID:27847804

  11. Protein-Based Three-Dimensional Memories and Associative Processors

    NASA Astrophysics Data System (ADS)

    Birge, Robert

    2008-03-01

    The field of bioelectronics has benefited from the fact that nature has often solved problems of a similar nature to those which must be solved to create molecular electronic or photonic devices that operate with efficiency and reliability. Retinal proteins show great promise in bioelectronic devices because they operate with high efficiency (˜0.65%), high cyclicity (>10^7), operate over an extended wavelength range (360 -- 630 nm) and can convert light into changes in voltage, pH, absorption or refractive index. This talk will focus on a retinal protein called bacteriorhodopsin, the proton pump of the organism Halobacterium salinarum. Two memories based on this protein will be described. The first is an optical three-dimensional memory. This memory stores information using volume elements (voxels), and provides as much as a thousand-fold improvement in effective capacity over current technology. A unique branching reaction of a variant of bacteriorhodopsin is used to turn each protein into an optically addressed latched AND gate. Although three working prototypes have been developed, a number of cost/performance and architectural issues must be resolved prior to commercialization. The major issue is that the native protein provides a very inefficient branching reaction. Genetic engineering has improved performance by nearly 500-fold, but a further order of magnitude improvement is needed. Protein-based holographic associative memories will also be discussed. The human brain stores and retrieves information via association, and human intelligence is intimately connected to the nature and enormous capacity of this associative search and retrieval process. To a first order approximation, creativity can be viewed as the association of two seemingly disparate concepts to form a totally new construct. Thus, artificial intelligence requires large scale associative memories. Current computer hardware does not provide an optimal environment for creating artificial

  12. Protein complexes associated with the Kaposi's sarcoma-associated herpesvirus-encoded LANA

    SciTech Connect

    Kaul, Rajeev; Verma, Subhash C.; Robertson, Erle S. . E-mail: erle@mail.med.upenn.edu

    2007-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposi's sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA is known to modulate viral and cellular gene expression. We report here on some initial proteomic studies to identify cellular proteins associated with the amino and carboxy-terminal domains of LANA. The results of these studies show an association of known cellular proteins which support LANA functions and have identified additional LANA-associated proteins. These results provide new evidence for complexes involving LANA with a number of previously unreported functional classes of proteins including DNA polymerase, RNA helicase and cell cycle control proteins. The results also indicate that the amino terminus of LANA can interact with its carboxy-terminal domain. This interaction is potentially important for facilitating associations with other cell cycle regulatory proteins which include CENP-F identified in association with both the amino and carboxy-termini. These novel associations add to the diversity of LANA functions in relation to the maintenance of latency and subsequent transformation of KSHV infected cells.

  13. Mechanisms of Protein-Ligand Association and Its Modulation by Protein Mutations

    PubMed Central

    Held, Martin; Metzner, Philipp; Prinz, Jan-Hendrik; Noé, Frank

    2011-01-01

    Protein-ligand interactions are essential for nearly all biological processes, and yet the biophysical mechanism that enables potential binding partners to associate before specific binding occurs remains poorly understood. Fundamental questions include which factors influence the formation of protein-ligand encounter complexes, and whether designated association pathways exist. To address these questions, we developed a computational approach to systematically analyze the complete ensemble of association pathways. Here, we use this approach to study the binding of a phosphate ion to the Escherichia coli phosphate-binding protein. Various mutants of the protein are considered, and their effects on binding free-energy profiles, association rates, and association pathway distributions are quantified. The results reveal the existence of two anion attractors, i.e., regions that initially attract negatively charged particles and allow them to be efficiently screened for phosphate, which is subsequently specifically bound. Point mutations that affect the charge on these attractors modulate their attraction strength and speed up association to a factor of 10 of the diffusion limit, and thus change the association pathways of the phosphate ligand. It is demonstrated that a phosphate that prebinds to such an attractor neutralizes its attraction effect to the environment, making the simultaneous association of a second phosphate ion unlikely. This study suggests ways in which structural properties can be used to tune molecular association kinetics so as to optimize the efficiency of binding, and highlights the importance of kinetic properties. PMID:21281585

  14. Phospholipid transfer protein in human plasma associates with proteins linked to immunity and inflammation.

    PubMed

    Cheung, Marian C; Vaisar, Tomás; Han, Xianlin; Heinecke, Jay W; Albers, John J

    2010-08-31

    Phospholipid transfer protein (PLTP), which associates with apolipoprotein A-I (the major HDL protein), plays a key role in lipoprotein remodeling. Because its level in plasma increases during acute inflammation, it may also play previously unsuspected roles in the innate immune system. To gain further insight into its potential physiological functions, we isolated complexes containing PLTP from plasma by immunoaffinity chromatography and determined their composition. Shotgun proteomics revealed that only 6 of the 24 proteins detected in the complexes were apolipoproteins. The most abundant proteins were clusterin (apoJ), PLTP itself, coagulation factors, complement factors, and apoA-I. Remarkably, 20 of the 24 proteins had known protein-protein interactions. Biochemical studies confirmed two previously established interactions and identified five new ones between PLTP and proteins. Moreover, clusterin, apoA-I, and apoE preserved the lipid-transfer activity of recombinant PLTP in the absence of lipid, indicating that these interactions may have functional significance. Unexpectedly, lipids accounted for only 3% of the mass of the PLTP complexes. Collectively, our observations indicate that PLTP in human plasma resides on lipid-poor complexes dominated by clusterin and proteins implicated in host defense and inflammation. They further suggest that protein-protein interactions drive the formation of PLTP complexes in plasma.

  15. Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm.

    PubMed

    Roberts, Kenneth P; Ensrud-Bowlin, Kathy M; Piehl, Laura B; Parent, Karlye R; Bernhardt, Miranda L; Hamilton, David W

    2008-12-01

    Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.

  16. Immunogenicity of self tumor associated proteins is enhanced through protein truncation

    PubMed Central

    Kottke, Tim; Shim, Kevin G; Alonso-Camino, Vanesa; Zaidi, Shane; Maria Diaz, Rosa; Pulido, Jose; Thompson, Jill; Rajani, Karishma R; Evgin, Laura; Ilett, Elizabeth; Pandha, Hardev; Harrington, Kevin; Selby, Peter; Melcher, Alan; Vile, Richard

    2016-01-01

    We showed previously that therapy with Vesicular Stomatitis Virus (VSV) expressing tumor-associated proteins eradicates established tumors. We show here that when cellular cDNA were cloned into VSV which retained their own poly-A signal, viral species emerged in culture which had deleted the cellular poly-A signal and also contained a truncated form of the protein coding sequence. Typically, the truncation occurred such that a Tyrosine-encoding codon was converted into a STOP codon. We believe that the truncation of tumor-associated proteins expressed from VSV in this way occurred to preserve the ability of the virus to replicate efficiently. Truncated cDNA expressed from VSV were significantly more effective than full length cDNA in treating established tumors. Moreover, tumor therapy with truncated cDNA was completely abolished by depletion of CD4+ T cells, whereas therapy with full length cDNA was CD8+ T cell dependent. These data show that the type/potency of antitumor immune responses against self-tumor-associated proteins can be manipulated in vivo through the nature of the self protein (full length or truncated). Therefore, in addition to generation of neoantigens through sequence mutation, immunological tolerance against self-tumor-associated proteins can be broken through manipulation of protein integrity, allowing for rational design of better self-immunogens for cancer immunotherapy. PMID:27933315

  17. Cancer associated proteins in blood plasma: Determining normal variation.

    PubMed

    Stenemo, Markus; Teleman, Johan; Sjöström, Martin; Grubb, Gabriel; Malmström, Erik; Malmström, Johan; Niméus, Emma

    2016-07-01

    Protein biomarkers have the potential to improve diagnosis, stratification of patients into treatment cohorts, follow disease progression and treatment response. One distinct group of potential biomarkers comprises proteins which have been linked to cancer, known as cancer associated proteins (CAPs). We determined the normal variation of 86 CAPs in 72 individual plasma samples collected from ten individuals using SRM mass spectrometry. Samples were collected weekly during 5 weeks from ten volunteers and over one day at nine fixed time points from three volunteers. We determined the degree of the normal variation depending on interpersonal variation, variation due to time of day, and variation over weeks and observed that the variation dependent on the time of day appeared to be the most important. Subdivision of the proteins resulted in two predominant protein groups containing 21 proteins with relatively high variation in all three factors (day, week and individual), and 22 proteins with relatively low variation in all factors. We present a strategy for prioritizing biomarker candidates for future studies based on stratification over their normal variation and have made all data publicly available. Our findings can be used to improve selection of biomarker candidates in future studies and to determine which proteins are most suitable depending on study design.

  18. A multidimensional proteomic approach to identify hypertrophy-associated proteins.

    PubMed

    Lindsey, Merry L; Goshorn, Danielle K; Comte-Walters, Susana; Hendrick, Jennifer W; Hapke, Elizabeth; Zile, Michael R; Schey, Kevin

    2006-04-01

    Left ventricular hypertrophy (LVH) is a leading cause of congestive heart failure. The exact mechanisms that control cardiac growth and regulate the transition to failure are not fully understood, in part due to the lack of a complete inventory of proteins associated with LVH. We investigated the proteomic basis of LVH using the transverse aortic constriction model of pressure overload in mice coupled with a multidimensional approach to identify known and novel proteins that may be relevant to the development and maintenance of LVH. We identified 123 proteins that were differentially expressed during LVH, including LIM proteins, thioredoxin, myoglobin, fatty acid binding protein 3, the abnormal spindle-like microcephaly protein (ASPM), and cytoskeletal proteins such as actin and myosin. In addition, proteins with unknown functions were identified, providing new directions for future research in this area. We also discuss common pitfalls and strategies to overcome the limitations of current proteomic technologies. Together, the multidimensional approach provides insight into the proteomic changes that occur in the LV during hypertrophy.

  19. Identification of urinary proteins potentially associated with diabetic kidney disease

    PubMed Central

    Marikanty, R. K.; Gupta, M. K.; Cherukuvada, S. V. B.; Kompella, S. S. S; Prayaga, A. K.; Konda, S.; Polisetty, R. V.; Idris, M. M.; Rao, P. V.; Chandak, G. R.; Dakshinamurty, K. V.

    2016-01-01

    Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. Although several parameters are used to evaluate renal damage, in many instances, there is no pathological change until damage is already advanced. Mass spectrometry-based proteomics is a novel tool to identify newer diagnostic markers. To identify urinary proteins associated with renal complications in diabetes, we collected urine samples from 10 type 2 diabetes patients each with normoalbuminuria, micro- and macro-albuminuria and compared their urinary proteome with that of 10 healthy individuals. Urinary proteins were concentrated, depleted of albumin and five other abundant plasma proteins and in-gel trypsin digested after prefractionation on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The peptides were analyzed using a nanoflow reverse phase liquid chromatography system coupled to linear trap quadrupole-Orbitrap mass spectrometer. We identified large number of proteins in each group, of which many were exclusively present in individual patient groups. A total of 53 proteins were common in all patients but were absent in the controls. The majority of the proteins were functionally binding, biologically involved in metabolic processes, and showed enrichment of alternative complement and blood coagulation pathways. In addition to identifying reported proteins such as α2-HS-glycoprotein and Vitamin D binding protein, we detected novel proteins such as CD59, extracellular matrix protein 1 (ECM1), factor H, and myoglobin in the urine of macroalbuminuria patients. ECM1 and factor H are known to influence mesangial cell proliferation, and CD59 causes microvascular damage by influencing membrane attack complex deposition, suggestive their biological relevance to DN. Thus, we have developed a proteome database where various proteins exclusively present in the patients may be further investigated for their role as stage-specific markers and possible therapeutic targets. PMID

  20. Identification of urinary proteins potentially associated with diabetic kidney disease.

    PubMed

    Marikanty, R K; Gupta, M K; Cherukuvada, S V B; Kompella, S S S; Prayaga, A K; Konda, S; Polisetty, R V; Idris, M M; Rao, P V; Chandak, G R; Dakshinamurty, K V

    2016-01-01

    Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. Although several parameters are used to evaluate renal damage, in many instances, there is no pathological change until damage is already advanced. Mass spectrometry-based proteomics is a novel tool to identify newer diagnostic markers. To identify urinary proteins associated with renal complications in diabetes, we collected urine samples from 10 type 2 diabetes patients each with normoalbuminuria, micro- and macro-albuminuria and compared their urinary proteome with that of 10 healthy individuals. Urinary proteins were concentrated, depleted of albumin and five other abundant plasma proteins and in-gel trypsin digested after prefractionation on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The peptides were analyzed using a nanoflow reverse phase liquid chromatography system coupled to linear trap quadrupole-Orbitrap mass spectrometer. We identified large number of proteins in each group, of which many were exclusively present in individual patient groups. A total of 53 proteins were common in all patients but were absent in the controls. The majority of the proteins were functionally binding, biologically involved in metabolic processes, and showed enrichment of alternative complement and blood coagulation pathways. In addition to identifying reported proteins such as α2-HS-glycoprotein and Vitamin D binding protein, we detected novel proteins such as CD59, extracellular matrix protein 1 (ECM1), factor H, and myoglobin in the urine of macroalbuminuria patients. ECM1 and factor H are known to influence mesangial cell proliferation, and CD59 causes microvascular damage by influencing membrane attack complex deposition, suggestive their biological relevance to DN. Thus, we have developed a proteome database where various proteins exclusively present in the patients may be further investigated for their role as stage-specific markers and possible therapeutic targets.

  1. Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages.

    PubMed

    Montgomery, Stephanie A; Berglund, Peter; Beard, Clayton W; Johnston, Robert E

    2006-08-01

    Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

  2. Bacterial protein signals are associated with Crohn’s disease

    PubMed Central

    Juste, Catherine; Kreil, David P; Beauvallet, Christian; Guillot, Alain; Vaca, Sebastian; Carapito, Christine; Mondot, Stanislas; Sykacek, Peter; Sokol, Harry; Blon, Florence; Lepercq, Pascale; Levenez, Florence; Valot, Benoît; Carré, Wilfrid; Loux, Valentin; Pons, Nicolas; David, Olivier; Schaeffer, Brigitte; Lepage, Patricia; Martin, Patrice; Monnet, Véronique; Seksik, Philippe; Beaugerie, Laurent; Ehrlich, S Dusko; Gibrat, Jean-François; Van Dorsselaer, Alain; Doré, Joël

    2014-01-01

    Objective No Crohn’s disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. Design We first developed and validated a workflow—including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS—to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. Results Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. Conclusions This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis. PMID:24436141

  3. Bacterial protein signals are associated with Crohn's disease.

    PubMed

    Juste, Catherine; Kreil, David P; Beauvallet, Christian; Guillot, Alain; Vaca, Sebastian; Carapito, Christine; Mondot, Stanislas; Sykacek, Peter; Sokol, Harry; Blon, Florence; Lepercq, Pascale; Levenez, Florence; Valot, Benoît; Carré, Wilfrid; Loux, Valentin; Pons, Nicolas; David, Olivier; Schaeffer, Brigitte; Lepage, Patricia; Martin, Patrice; Monnet, Véronique; Seksik, Philippe; Beaugerie, Laurent; Ehrlich, S Dusko; Gibrat, Jean-François; Van Dorsselaer, Alain; Doré, Joël

    2014-10-01

    No Crohn's disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. We first developed and validated a workflow-including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS-to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  4. Protein-driven inference of miRNA–disease associations

    PubMed Central

    Mørk, Søren; Pletscher-Frankild, Sune; Palleja Caro, Albert; Gorodkin, Jan; Jensen, Lars Juhl

    2014-01-01

    Motivation: MicroRNAs (miRNAs) are a highly abundant class of non-coding RNA genes involved in cellular regulation and thus also diseases. Despite miRNAs being important disease factors, miRNA–disease associations remain low in number and of variable reliability. Furthermore, existing databases and prediction methods do not explicitly facilitate forming hypotheses about the possible molecular causes of the association, thereby making the path to experimental follow-up longer. Results: Here we present miRPD in which miRNA–Protein–Disease associations are explicitly inferred. Besides linking miRNAs to diseases, it directly suggests the underlying proteins involved, which can be used to form hypotheses that can be experimentally tested. The inference of miRNAs and diseases is made by coupling known and predicted miRNA–protein associations with protein–disease associations text mined from the literature. We present scoring schemes that allow us to rank miRNA–disease associations inferred from both curated and predicted miRNA targets by reliability and thereby to create high- and medium-confidence sets of associations. Analyzing these, we find statistically significant enrichment for proteins involved in pathways related to cancer and type I diabetes mellitus, suggesting either a literature bias or a genuine biological trend. We show by example how the associations can be used to extract proteins for disease hypothesis. Availability and implementation: All datasets, software and a searchable Web site are available at http://mirpd.jensenlab.org. Contact: lars.juhl.jensen@cpr.ku.dk or gorodkin@rth.dk PMID:24273243

  5. Analysis of the proteins associated with platelet detergent resistant membranes.

    PubMed

    Szklanna, Paulina B; Foy, Martina; Wynne, Kieran; Byrne, Dwayne; Maguire, Patricia B

    2016-09-01

    Proteomic studies have facilitated the identification of proteins associated with the detergent-resistant membrane (DRM) fraction in a variety of cell types. Here, we have undertaken label-free quantitative (LFQ) proteomic profiling of the proteins associated with detergent-resistant plasma and internal membranes from resting and activated platelets. One hundred forty-one proteins were identified and raw data is available via ProteomeXchange with identifier PXD002554. The proteins identified include a myriad of important platelet signaling and trafficking proteins including Rap1b, Src, SNAP-23, syntaxin-11, and members of the previously unattributed Ragulator complex. Mean LFQ intensities calculated across three technical replicates for the three biological donors revealed that several important platelet signaling proteins altered their detergent solubility upon activation, including GPIbα, GPIbβ, Src, and 14-3-3ζ. Altered detergent solubility for GPIbα, following activation using a variety of platelet agonists, was confirmed by immunoblotting and further coimmunoprecipitation experiments revealed that GPIbα forms a complex with 14-3-3ζ that shifts into DRMs following activation. Taken together, proteomic profiling of platelet DRMs allowed greater insight in the complex biology of both DRMs and platelets and will be a useful subproteome to study platelet-related disease. All MS data have been deposited in the ProteomeXchange with identifier PXD002554 (http://proteomecentral.proteomexchange.org/dataset/PXD002554). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Jupiter, a new Drosophila protein associated with microtubules.

    PubMed

    Karpova, Nina; Bobinnec, Yves; Fouix, Sylvaine; Huitorel, Philippe; Debec, Alain

    2006-05-01

    In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.

  7. Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Shang, E S; Foley, D M; Blanco, D R; Champion, C I; Mirzabekov, T; Sokolov, Y; Kagan, B L; Miller, J N; Lovett, M A

    1995-01-01

    We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease. Images PMID:7593626

  8. Thermodynamics of folding and association of lattice-model proteins

    NASA Astrophysics Data System (ADS)

    Cellmer, Troy; Bratko, Dusan; Prausnitz, John M.; Blanch, Harvey

    2005-05-01

    Closely related to the "protein folding problem" is the issue of protein misfolding and aggregation. Protein aggregation has been associated with the pathologies of nearly 20 human diseases and presents serious difficulties during the manufacture of pharmaceutical proteins. Computational studies of multiprotein systems have recently emerged as a powerful complement to experimental efforts aimed at understanding the mechanisms of protein aggregation. We describe the thermodynamics of systems containing two lattice-model 64-mers. A parallel tempering algorithm abates problems associated with glassy systems and the weighted histogram analysis method improves statistical quality. The presence of a second chain has a substantial effect on single-chain conformational preferences. The melting temperature is substantially reduced, and the increase in the population of unfolded states is correlated with an increase in interactions between chains. The transition from two native chains to a non-native aggregate is entropically favorable. Non-native aggregates receive ˜25% of their stabilizing energy from intraprotein contacts not found in the lowest-energy structure. Contact maps show that for non-native dimers, nearly 50% of the most probable interprotein contacts involve pairs of residues that form native contacts, suggesting that a domain-swapping mechanism is involved in self-association.

  9. Encounter complexes and dimensionality reduction in protein–protein association

    PubMed Central

    Kozakov, Dima; Li, Keyong; Hall, David R; Beglov, Dmitri; Zheng, Jiefu; Vakili, Pirooz; Schueler-Furman, Ora; Paschalidis, Ioannis Ch; Clore, G Marius; Vajda, Sandor

    2014-01-01

    An outstanding challenge has been to understand the mechanism whereby proteins associate. We report here the results of exhaustively sampling the conformational space in protein–protein association using a physics-based energy function. The agreement between experimental intermolecular paramagnetic relaxation enhancement (PRE) data and the PRE profiles calculated from the docked structures shows that the method captures both specific and non-specific encounter complexes. To explore the energy landscape in the vicinity of the native structure, the nonlinear manifold describing the relative orientation of two solid bodies is projected onto a Euclidean space in which the shape of low energy regions is studied by principal component analysis. Results show that the energy surface is canyon-like, with a smooth funnel within a two dimensional subspace capturing over 75% of the total motion. Thus, proteins tend to associate along preferred pathways, similar to sliding of a protein along DNA in the process of protein-DNA recognition. DOI: http://dx.doi.org/10.7554/eLife.01370.001 PMID:24714491

  10. Preeclampsia Is Associated with Low Concentrations of Protein Z

    PubMed Central

    Erez, Offer; Hoppensteadt, Debra; Romero, Roberto; Espinoza, Jimmy; Goncalves, Luis; Nien, Jyh Kae; Kusanovic, Juan Pedro; Fareed, Jawed; Gotsch, Francesca; Pineles, Beth; Chaiworapongsa, Tinnakorn

    2008-01-01

    Objective Protein Z, a vitamin K-dependent plasma protein, has an important role in the regulation of the coagulation cascade. Protein Z deficiency has been associated with unexplained pregnancy loss and adverse pregnancy outcome in patients with thrombophilia. This study was conducted to determine if preeclampsia (PE), small-for-gestational age (SGA) and fetal demise are associated with changes in maternal plasma concentrations of protein Z. Study Design This cross-sectional study included normal pregnant women (N=71), patients with PE (N=130), patients who delivered a SGA neonate (N=58), and patients with fetal demise (N=58). Maternal plasma protein Z concentrations were measured by a sensitive and specific immunoassay. Protein Z deficiency was defined as maternal plasma concentrations ≤5th percentile of the normal pregnancy group (≤1.59μg/mL). Non-parametric statistics were used for analysis. Results 1) Patients with preeclampsia had a lower median plasma concentration of protein Z than normal pregnant women (PE: median: 1.6 μg/mL, range: 0.2-3.3 μg/mLl vs. normal pregnancy: median: 2.4 μg/mL, range: 1.1-3.4 μg/mLl; p<0.01); 2) Patients with SGA (median: 2.3 μg/mL, range: 0.2-3.8 μg/mL) and fetal demise (median: 2.6 μg/mL, range: 0.2-4.3 μg/mL) did not have significant different median protein Z concentrations from normal pregnant women (p>0.05); and 3) Women in the PE and fetal demise groups had significantly higher rate of protein Z deficiency than those with normal pregnancy outcome. Conclusion 1) PE, but neither SGA nor fetal demise, is associated with significantly lower maternal median plasma concentration of protein Z concentrations than normal pregnancy; and 2) a high rate of protein Z deficiency was observed in patients with PE and fetal demise. PMID:17701666

  11. An updated nomenclature for keratin-associated proteins (KAPs).

    PubMed

    Gong, Hua; Zhou, Huitong; McKenzie, Grant W; Yu, Zhidong; Clerens, Stefan; Dyer, Jolon M; Plowman, Jeffrey E; Wright, Mathew W; Arora, Reena; Bawden, C Simon; Chen, Yulin; Li, Jinquan; Hickford, Jonathan G H

    2012-01-01

    Most protein in hair and wool is of two broad types: keratin intermediate filament-forming proteins (commonly known as keratins) and keratin-associated proteins (KAPs). Keratin nomenclature was reviewed in 2006, but the KAP nomenclature has not been revised since 1993. Recently there has been an increase in the number of KAP genes (KRTAPs) identified in humans and other species, and increasingly reports of variation in these genes. We therefore propose that an updated naming system is needed to accommodate the complexity of the KAPs. It is proposed that the system is founded in the previous nomenclature, but with the abbreviation sp-KAPm-nL*x for KAP proteins and sp-KRTAPm-n(p/L)*x for KAP genes. In this system "sp" is a unique letter-based code for different species as described by the protein knowledge-based UniProt. "m" is a number identifying the gene or protein family, "n" is a constituent member of that family, "p" signifies a pseudogene if present, "L" if present signifies "like" and refers to a temporary "place-holder" until the family is confirmed and "x" signifies a genetic variant or allele. We support the use of non-italicised text for the proteins and italicised text for the genes. This nomenclature is not that different to the existing system, but it includes species information and also describes genetic variation if identified, and hence is more informative. For example, GenBank sequence JN091630 would historically have been named KRTAP7-1 for the gene and KAP7-1 for the protein, but with the proposed nomenclature would be SHEEP-KRTAP7-1*A and SHEEP-KAP7-1*A for the gene and protein respectively. This nomenclature will facilitate more efficient storage and retrieval of data and define a common language for the KAP proteins and genes from all mammalian species.

  12. Loss of protein association causes cardiolipin degradation in Barth syndrome

    PubMed Central

    Xu, Yang; Phoon, Colin K.L.; Berno, Bob; D’Souza, Kenneth; Hoedt, Esthelle; Zhang, Guoan; Neubert, Thomas A.; Epand, Richard M.; Ren, Mindong; Schlame, Michael

    2016-01-01

    Cardiolipin is a specific mitochondrial phospholipid that has a high affinity for proteins and that stabilizes the assembly of supercomplexes involved in oxidative phosphorylation. We found that sequestration of cardiolipin in protein complexes is critical to protect it from degradation. The turnover of cardiolipin is slower by almost an order of magnitude than the turnover of other phospholipids. However, in Barth syndrome, cardiolipin is rapidly degraded via the intermediate monolyso-cardiolipin. Treatments that induce supercomplex assembly decrease the turnover of cardiolipin and the concentration of monolyso-cardiolipin whereas dissociation of supercomplexes has the opposite effect. Our data suggest that cardiolipin is uniquely protected from normal lipid turnover by its association with proteins, but in Barth syndrome, where this association is compromised, cardiolipin becomes unstable, which causes the accumulation of monolyso-cardiolipin. PMID:27348092

  13. Crystallization of cyclase-associated protein from Dictyostelium discoideum.

    PubMed

    Hofmann, Andreas; Hess, Sonja; Noegel, Angelika A; Schleicher, Michael; Wlodawer, Alexander

    2002-10-01

    Cyclase-associated protein (CAP) is a conserved two-domain protein that helps to activate the catalytic activity of adenylyl cyclase in the cyclase-bound state through interaction with Ras, which binds to the cyclase in a different region. With its other domain, CAP can bind monomeric actin and therefore also carries a cytoskeletal function. The protein is thus involved in Ras/cAMP-dependent signal transduction and most likely serves as an adapter protein translocating the adenylyl cyclase complex to the actin cytoskeleton. Crystals belonging to the orthorhombic space group C222, with unit-cell parameters a = 71.2, b = 75.1, c = 162.9 A, have been obtained from Dictyostelium discoideum CAP carrying a C-terminal His tag. A complete native data set extending to 2.2 A resolution was collected from a single crystal using an in-house X-ray system. The asymmetric unit contains one molecule of CAP.

  14. Enzymes Associated with Protein Bodies Isolated from Ungerminated Barley Seeds

    PubMed Central

    Ory, Robert L.; Henningsen, Knud W.

    1969-01-01

    Protein bodies were isolated intact from dormant barley seeds, Hordeum vulgare, var. Kenia, by a combination of buffer extractions and centrifugations over a sucrose gradient. Examination of the protein bodies pellet in the electron microscope shows 2 types of protein bodies in a wide variation of sizes. The majority of them stain evenly with osmium, are contained within a single membrane, and have no other structural components. The other type, mostly the larger particles, has a fine structure of orderly dark and light-stained layers attached to the protein bodies. Two acid hydrolases are associated with these particles: acid phosphatase activity, specific for sodium phytate but inactive on β-glycerol phosphate, glucose 1-phosphate, fructose 1,6-diphosphate and adenosine triphosphate; and acid protease activity. Images PMID:5397495

  15. Enzymes associated with protein bodies isolated from ungerminated barley seeds.

    PubMed

    Ory, R L; Henningsen, K W

    1969-11-01

    Protein bodies were isolated intact from dormant barley seeds, Hordeum vulgare, var. Kenia, by a combination of buffer extractions and centrifugations over a sucrose gradient. Examination of the protein bodies pellet in the electron microscope shows 2 types of protein bodies in a wide variation of sizes. The majority of them stain evenly with osmium, are contained within a single membrane, and have no other structural components. The other type, mostly the larger particles, has a fine structure of orderly dark and light-stained layers attached to the protein bodies. Two acid hydrolases are associated with these particles: acid phosphatase activity, specific for sodium phytate but inactive on beta-glycerol phosphate, glucose 1-phosphate, fructose 1,6-diphosphate and adenosine triphosphate; and acid protease activity.

  16. RNA immunoprecipitation for determining RNA-protein associations in vivo.

    PubMed

    Gilbert, Chris; Svejstrup, Jesper Q

    2006-08-01

    Similar to chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) can be used to detect the association of individual proteins with specific nucleic acid regions, in this case on RNA. Live cells are treated with formaldehyde to generate protein-RNA cross-links between molecules that are in close proximity in vivo. RNA sequences that cross-link with a given protein are isolated by immunoprecipitation of the protein, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated RNA by reverse transcription PCR. The basics of RIP are very similar to those of ChIP, but with some important caveats. This unit describes the RIP procedure for Saccharomyces cerevisiae. Although the corresponding steps for metazoan cells have not yet been worked out, it is likely that the yeast procedure can easily be adapted for use in other organisms.

  17. Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

    PubMed Central

    Molnár, János; Szakács, Gergely; Tusnády, Gábor E.

    2016-01-01

    Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations. PMID:26986070

  18. Matrix Gla Protein polymorphisms are associated with coronary artery calcification

    USDA-ARS?s Scientific Manuscript database

    Matrix Gla Protein (MGP) is a key regulator of vascular calcification. Genetic variation at the MGP locus could modulate the development of coronary artery calcification (CAC). We examined the cross-sectional association between MGP SNPs [rs1800802 (T-138C), rs1800801 (G-7A),and rs4236 (Ala102Thr)...

  19. ODV-associated proteins of the Pieris rapae granulovirus.

    PubMed

    Wang, Xiao-Feng; Zhang, Bao-Qin; Xu, Hai-Jun; Cui, Ying-Jun; Xu, Yi-Peng; Zhang, Min-Juan; Han, Yeon Soo; Lee, Yong Seok; Bao, Yan-Yuan; Zhang, Chuan-Xi

    2011-06-03

    Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses, NPV) and Betabaculovirus (granuloviruses, GV) are two main genera of the family Baculoviridae. The virion proteomes of Alphabaculovirus have been well studied; however, the Betabaculovirus virion compositions remain unclear. Pieris rapae granulovirus (PrGV) can kill larvae of P. rapae, a worldwide and important pest of mustard family crops. In this study, the occlusion-derived virus (ODV)-associated proteins of PrGV were identified using three mass spectrometry (MS) approaches. The MS analyses demonstrated that 47 proteins were present in PrGV-ODV. Of the 47 PrGV-ODV proteins, 33 have homologues identified previously in other baculovirus ODV/BVs, whereas 14 (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were newly identified ODV proteins. Seven of the 14 newly identified ODV proteins are specific to Betabaculovirus, including Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114. Furthermore, the data derived from these MS approaches were validated by immunoblotting analysis using antisera prepared from 11 randomly selected recombinant PrGV-ODV proteins (including 5 Betabaculovirus-unique proteins). Comparison analyses revealed the similar and different compositions between Betabaculovirus and Alphabaculovirus virions, which deepen our understanding of the baculovirus virion structure and provide helpful information on Betabaculovirus--host interaction studies.

  20. Effects of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions.

    PubMed Central

    Ahmed, K; Wilson, M J; Goueli, S A; Williams-Ashman, H G

    1978-01-01

    Studies are presented on the influence of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions involving both exogenous and endogenous substrates. The activities toward the model acidic protein substrate, dephosphophosvitin, were maximal at 160--200mM-NaCl (or -KCl or -NH4Cl). Under these conditions, spermidine and spermine added in concentrations up to 2mM were essentially without effect. However, without addition of NaCl to the medium, marked stimulation of these reactions was elicited by these polyamines at 1--2mM concentrations. The stimulatory effects were not due to non-specific changes in the ionic strength or to substitution of spermine for Mg2+, as maximal stimulation by 1 mM-spermine was observed only at optimal (2--4mM) Mg2+ concentrations. Qualitatively similar effects of polyamines were observed with enzyme preparations from the prostates of castrated rats, and with chromatin and non-histone-protein preparations from other tissues besides ventral prostate. When phosphorylation of endogenous non-histone proteins of the chromatin was measured, spermine stimulated both the initial rates and the final extent of transphosphorylation, even in the presence of optimal concentration of NaCl. By contrast, spermine or spermidine had no effect on the chromatin- and non-histone-protein-associated protein kinase reactions determined with lysine-rich histones as substrates. Chemically NN-dimethylated dephosphophosvitin was a less active substrate for the chromatin-associated protein kinase, but its phosphorylation was more markedly stimulated by spermine in comparison with unmodified dephosphophosvitin. These observations hint that the polyamine stimulations of the various protein kinase reactions may be due to effects on the conformations of the non-histone protein substrates rather than on the kinases themselves. PMID:747650

  1. Lipid nanotechnologies for structural studies of membrane-associated proteins.

    PubMed

    Stoilova-McPhie, Svetla; Grushin, Kirill; Dalm, Daniela; Miller, Jaimy

    2014-11-01

    We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo-electron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ∼20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ∼10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment. © 2014 Wiley Periodicals, Inc.

  2. Comparative effects of cryosolvents on tubulin association, thermal stability, and binding of microtubule-associated proteins.

    PubMed

    Pajot-Augy, E

    1993-06-01

    Organic cryosolvents essential for cryopreservation of living cells have a colligative effect on water properties, but also affect cellular structures such as the membrane, actin, or tubulin cytoskeleton. The effects of cryosolvents on actin and its binding proteins are starting to be well investigated. In parallel, tubulin assembly characteristics were investigated comparatively, with 0-30% 1,2-propanediol, dimethyl sulfoxide, or glycerol, and with or without microtubule-associated proteins, at 37 or 4 degrees C. Tubulin association was monitored by spectrometry and sedimentation, providing the concentration in free protein, cold-depolymerizable microtubules, and cold-resistant associations. At 37 degrees C, 1,2-propanediol and dimethyl sulfoxide induce a similar association level and cold stability of the assemblies. Glycerol yields a lower level of tubulin association. Cold stability of the assemblies requires the presence of solvent, the amount of which is modulated by microtubule-associated proteins (MAPs): 15% 1,2-propanediol or dimethyl sulfoxide, decreasing down to 10% with MAPs, or 10% glycerol with MAPs only. At 4 degrees C, some cold-stable association is promoted by 1,2-propanediol or dimethyl sulfoxide above 10-15%, in the presence or absence of MAPs, but not with glycerol. In addition, protein content of the various fractions obtained with MAPs and 30% solvent was examined by densitometry of electrophoresis gels. Cold-labile associations obtained at 37 degrees C with 1,2-propanediol or dimethyl sulfoxide are lacking in tubulin and enriched in tau proteins relative to control or glycerol. Associations formed at 37 degrees C and stable to subsequent cold treatment, or at 4 degrees C, regardless of the solvent, present a large tubulin content, as well as few tau proteins and high-molecular-weight MAPs.

  3. Chicken Egg Shell Membrane Associated Proteins and Peptides.

    PubMed

    Makkar, Sarbjeet; Liyanage, Rohana; Kannan, Lakshmi; Packialakshmi, Balamurugan; Lay, Jack O; Rath, Narayan C

    2015-11-11

    Egg shells are poultry industry byproducts with potential for use in various biological and agricultural applications. We have been interested in the membranes underlying the calcareous shell as a feed supplement, which showed potential to improve immunity and performance of post hatch poultry. Therefore, to determine their protein and peptide profiles, we extracted the egg shell membranes (ESM) from fresh unfertilized eggs with methanol and guanidine hydrochloride (GdHCl) to obtain soluble proteins for analysis by mass spectrometry. The methanol extract was subjected to matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), high-performance reverse phase liquid chromatographic separation (HPLC), and tandem mass spectrometry (MS/MS) to determine its peptide and protein profiles. The GdHCl extract was subjected to ESI-HPLC-MS/MS following trypsin digestion of reduced/alkylated proteins. Nine proteins from the methanol extract and >275 proteins from the GdHCl extract were tentatively identified. The results suggested the presence of several abundant proteins from egg whites, such as ovoalbumin, ovotransferrin, and lysozyme as well as many others associated with antimicrobial, biomechanical, cytoskeletal organizational, cell signaling, and enzyme activities. Collagens, keratin, agrin, and laminin were some of the structural proteins present in the ESM. The methanol-soluble fraction contained several clusterin peptides and defensins, particularly, two isoforms of gallin. The ratios of the two isoforms of gallin differed between the membranes obtained from brown and white eggs. The high abundance of several antimicrobial, immunomodulatory, and other bioactive proteins in the ESM along with its potential to entrap various microbes and antigens may make it a suitable vehicle for oral immunization of post hatch poultry and improve their disease resistance.

  4. Balanced Protein–Water Interactions Improve Properties of Disordered Proteins and Non-Specific Protein Association

    PubMed Central

    2015-01-01

    Some frequently encountered deficiencies in all-atom molecular simulations, such as nonspecific protein–protein interactions being too strong, and unfolded or disordered states being too collapsed, suggest that proteins are insufficiently well solvated in simulations using current state-of-the-art force fields. To address these issues, we make the simplest possible change, by modifying the short-range protein–water pair interactions, and leaving all the water–water and protein–protein parameters unchanged. We find that a modest strengthening of protein–water interactions is sufficient to recover the correct dimensions of intrinsically disordered or unfolded proteins, as determined by direct comparison with small-angle X-ray scattering (SAXS) and Förster resonance energy transfer (FRET) data. The modification also results in more realistic protein-protein affinities, and average solvation free energies of model compounds which are more consistent with experiment. Most importantly, we show that this scaling is small enough not to affect adversely the stability of the folded state, with only a modest effect on the stability of model peptides forming α-helix and β-sheet structures. The proposed adjustment opens the way to more accurate atomistic simulations of proteins, particularly for intrinsically disordered proteins, protein–protein association, and crowded cellular environments. PMID:25400522

  5. Gene Ontology Function prediction in Mollicutes using Protein-Protein Association Networks

    PubMed Central

    2011-01-01

    Background Many complex systems can be represented and analysed as networks. The recent availability of large-scale datasets, has made it possible to elucidate some of the organisational principles and rules that govern their function, robustness and evolution. However, one of the main limitations in using protein-protein interactions for function prediction is the availability of interaction data, especially for Mollicutes. If we could harness predicted interactions, such as those from a Protein-Protein Association Networks (PPAN), combining several protein-protein network function-inference methods with semantic similarity calculations, the use of protein-protein interactions for functional inference in this species would become more potentially useful. Results In this work we show that using PPAN data combined with other approximations, such as functional module detection, orthology exploitation methods and Gene Ontology (GO)-based information measures helps to predict protein function in Mycoplasma genitalium. Conclusions To our knowledge, the proposed method is the first that combines functional module detection among species, exploiting an orthology procedure and using information theory-based GO semantic similarity in PPAN of the Mycoplasma species. The results of an evaluation show a higher recall than previously reported methods that focused on only one organism network. PMID:21486441

  6. Purification of recombinant nacre-associated mineralization protein AP7 fused with maltose-binding protein.

    PubMed

    Huang, Yu-Chieh; Chang, Hsun-Hui; Mou, Yun; Chi, Peter; Chan, Jerry Chun Chung; Luo, Shih-Chi

    2014-08-01

    Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7 kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we report the first successful expression and purification of recombinant AP7 using the maltose-binding protein (MBP) fusion approach. We obtain a high-yield production of recombinant MBP-AP7 protein inE. coli (∼60 mg/L). We also establish an efficient protocol to remove the MBP fusion protein by Factor Xa, followed by purification using size-exclusion chromatography. Characterization of the recombinant AP7 protein has been carried out using MALDI-TOF, peptide mass fingerprinting, and circular dichroism (CD). The mass data confirm that the purified recombinant protein is AP7. The CD data suggest that the recombinant AP7 protein exists as partially disordered structure at neutral pH. The calcium carbonate precipitation assay shows that both MBP-AP7 and AP7 exhibit morphological modification on calcite crystallites. The co-precipitation of MBP-tagged AP7 derivatives and calcium carbonate generate different types of AP7 composite calcite and vaterite crystals. This system should be helpful to establish a model for understanding the structure/function relationship between the protein and inorganic mineral interaction.

  7. MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

    PubMed

    Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

    2017-03-01

    Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins.

  8. Root carbon and protein metabolism associated with heat tolerance.

    PubMed

    Huang, Bingru; Rachmilevitch, Shimon; Xu, Jichen

    2012-05-01

    Extensive past efforts have been taken toward understanding heat tolerance mechanisms of the aboveground organs. Root systems play critical roles in whole-plant adaptation to heat stress, but are less studied. This review discusses recent research results revealing some critical physiological and metabolic factors underlying root thermotolerance, with a focus on temperate perennial grass species. Comparative analysis of differential root responses to supraoptimal temperatures by a heat-adapted temperate C3 species, Agrostis scabra, which can survive high soil temperatures up to 45 °C in geothermal areas in Yellow Stone National Park, and a heat-sensitive cogeneric species, Agrostis stolonifera, suggested that efficient carbon and protein metabolism is critical for root thermotolerance. Superior root thermotolerance in a perennial grass was associated with a greater capacity to control respiratory costs through respiratory acclimation, lowering carbon investment in maintenance for protein turnover, and efficiently partitioning carbon into different metabolic pools and alternative respiration pathways. Proteomic analysis demonstrated that root thermotolerance was associated with an increased maintenance of stability and less degradation of proteins, particularly those important for metabolism and energy production. In addition, thermotolerant roots are better able to maintain growth and activity during heat stress by activating stress defence proteins such as those participating in antioxidant defence (i.e. superoxide dismutase, peroxidase, glutathione S-transferase) and chaperoning protection (i.e. heat shock protein).

  9. HLA-H and associated proteins in patients with hemochromatosis.

    PubMed Central

    Beutler, E.; West, C.; Gelbart, T.

    1997-01-01

    BACKGROUND: The 845A(C282Y) mutation in the HLA-H gene accounts for most cases of hereditary hemochromatosis in patients who are of European origin. Some lack this mutation, however, and it is not present in Asian patients. Thus, other mutations either in HLA-H or associated proteins may be present in such patients. HLA-H associates with beta-2-microglobulin. Calreticulin associates with class 1 HLA proteins and appears to be identical with mobilferrin, a putative iron transport protein. These two proteins are therefore candidates for mutations in patients with hemochromatosis. MATERIALS AND METHODS: We have sequenced the coding region and parts of introns of the HLA-H gene, the beta-2-microglobulin gene, and the calreticulin (mobilferrin) gene of 10, 7, and 5 hemochromatosis patients, respectively, selecting those who were not homozygous for the 845A(C282Y) mutation. The number of chromosomes at risk studied were 18 for HLA-H, 14 for beta-2-microglobulin and 10 for calreticulin. RESULTS: We detected 3 new intronic polymorphisms in the HLA-H gene, each a point mutation. Some differences from published sequences of beta-2-microglobulin and calreticulin were documented, but these were uniformly present in all samples. CONCLUSIONS: The lack of additional mutations in the HLA-H gene is remarkable, and we speculate that the C282Y mutation may be a gain-of-function change. PMID:9234244

  10. Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

    PubMed

    Shima, F; Yamawaki-Kataoka, Y; Yanagihara, C; Tamada, M; Okada, T; Kariya, K; Kataoka, T

    1997-03-01

    Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

  11. Transcriptional robustness and protein interactions are associated in yeast.

    PubMed

    Bekaert, Michaël; Conant, Gavin C

    2011-05-05

    Robustness to insults, both external and internal, is a characteristic feature of life. One level of biological organization for which noise and robustness have been extensively studied is gene expression. Cells have a variety of mechanisms for buffering noise in gene expression, but it is not completely clear what rules govern whether or not a given gene uses such tools to maintain appropriate expression. Here, we show a general association between the degree to which yeast cells have evolved mechanisms to buffer changes in gene expression and whether they possess protein-protein interactions. We argue that this effect bears an affinity to epistasis, because yeast appears to have evolved regulatory mechanisms such that distant changes in gene copy number for a protein-protein interaction partner gene can alter a gene's expression. This association is not unexpected given recent work linking epistasis and the deleterious effects of changes in gene dosage (i.e., the dosage balance hypothesis). Using gene expression data from artificial aneuploid strains of bakers' yeast, we found that genes coding for proteins that physically interact with other proteins show less expression variation in response to aneuploidy than do other genes. This effect is even more pronounced for genes whose products interact with proteins encoded on aneuploid chromosomes. We further found that genes targeted by transcription factors encoded on aneuploid chromosomes were more likely to change in expression after aneuploidy. We suggest that these observations can be best understood as resulting from the higher fitness cost of misexpression in epistatic genes and a commensurate greater regulatory control of them.

  12. Attractive protein-polymer interactions markedly alter the effect of macromolecular crowding on protein association equilibria.

    PubMed

    Jiao, Ming; Li, Hong-Tao; Chen, Jie; Minton, Allen P; Liang, Yi

    2010-08-04

    The dependence of the fluorescence of catalase upon the concentration of added superoxide dismutase (SOD) indicates that SOD binds to saturable sites on catalase. The affinity of SOD for these sites varies with temperature, and with the concentration of each of three nominally inert polymeric additives--dextran 70, Ficoll 70, and polyethylene glycol 2000. At room temperature (25.0 degrees C) and higher, the addition of high concentrations of polymer is found to significantly enhance the affinity of SOD for catalase, but with decreasing temperature the enhancing effect of polymer addition diminishes, and at 8.0 degrees C, addition of polymer has little or no effect on the affinity of SOD for catalase. The results presented here provide the first experimental evidence for the existence of competition between a repulsive excluded volume interaction between protein and polymer, which tends to enhance association of dilute protein, and an attractive interaction between protein and polymer, which tends to inhibit protein association. The net effect of high concentrations of polymer upon protein associations depends upon the relative strength of these two types of interactions at the temperature of measurement, and may vary significantly between different proteins and/or polymers.

  13. The functional domain grouping of microtubule associated proteins

    PubMed Central

    Deane, Charlotte M; Wakefield, James G

    2008-01-01

    Microtubules (MTs), which play crucial roles in normal cell function, are regulated by MT associated proteins (MAPs). Using a combinatorial approach that includes biochemistry, proteomics and bioinformatics, we have recently identified 270 putative MAPs from Drosophila embryos and characterized some of those required for correct progression through mitosis. Here we identify functional groups of these MAPs using a reciprocal hits sequence alignment technique and assign InterPro functional domains to 28 previously uncharacterized proteins. This approach gives insight into the potential functions of MAPs and how their roles may affect MTs. PMID:19704789

  14. Heat Shock Proteins in Association with Heat Tolerance in Grasses

    PubMed Central

    Xu, Yan; Zhan, Chenyang; Huang, Bingru

    2011-01-01

    The grass family Poaceae includes annual species cultivated as major grain crops and perennial species cultivated as forage or turf grasses. Heat stress is a primary factor limiting growth and productivity of cool-season grass species and is becoming a more significant problem in the context of global warming. Plants have developed various mechanisms in heat-stress adaptation, including changes in protein metabolism such as the induction of heat shock proteins (HSPs). This paper summarizes the structure and function of major HSPs, recent research progress on the association of HSPs with grass tolerance to heat stress, and incorporation of HSPs in heat-tolerant grass breeding. PMID:22084689

  15. p95vav associates with the nuclear protein Ku-70.

    PubMed Central

    Romero, F; Dargemont, C; Pozo, F; Reeves, W H; Camonis, J; Gisselbrecht, S; Fischer, S

    1996-01-01

    The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70. The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav. A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif. Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction. The potential role of Vav/Ku-70 complexes is discussed. PMID:8524317

  16. A novel family of plant nuclear envelope-associated proteins.

    PubMed

    Pawar, Vidya; Poulet, Axel; Détourné, Gwénaëlle; Tatout, Christophe; Vanrobays, Emmanuel; Evans, David E; Graumann, Katja

    2016-10-01

    This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.

  17. Characterization of associated proteins and phospholipids in natural rubber latex.

    PubMed

    Sansatsadeekul, Jitlada; Sakdapipanich, Jitladda; Rojruthai, Porntip

    2011-06-01

    Non-rubber components present in natural rubber (NR) latex, such as proteins and phospholipids, are presumed to be distributed in the serum fraction as well as surrounding the rubber particle surface. The phospholipid-protein layers covering the rubber particle surface are especially interesting due to their ability to enhance the colloidal stability of NR latex. In this study, we have characterized the components surrounding the NR particle surface and investigated their role in the colloidal stability of NR particles. Proteins from the cream fraction were proteolytically removed from the NR latex and compare to those from the serum fractions using SDS-polyacrylamide gel electrophoresis revealing that both fractions contained similar proteins in certain molecular weights such as 14.5, 25 and 27 kDa. Phospholipids removed from latex by treatment with NaOH were analyzed using (1)H-NMR spectroscopy and several major signals were assignable to -(CH(2))(n)-, -CH(2)OP, -CH(2)OC═O and -OCH(2)CH(2)NH-. These signals are important evidence that indicates phospholipids associate with the rubber chain. The colloidal behavior of rubber lattices before and after removal of protein-lipid membrane was evaluated by zeta potential analysis and scanning electron microscope (SEM). The lowest zeta potential value of NR particles was observed at pH 10, consequently leading to the highest stability of rubber particles. Additionally, SEM micrographs clearly displayed a gray ring near the particle surface corresponding to the protein-lipid membrane layer.

  18. Herp enhances ER-associated protein degradation by recruiting ubiquilins

    SciTech Connect

    Kim, Tae-Yeon; Kim, Eunmin; Yoon, Sungjoo Kim; Yoon, Jong-Bok

    2008-05-02

    ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3{delta}, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3{delta} was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates.

  19. Protein requirements for sister telomere association in human cells

    PubMed Central

    Canudas, Silvia; Houghtaling, Benjamin R; Kim, Ju Youn; Dynek, Jasmin N; Chang, William G; Smith, Susan

    2007-01-01

    Previous studies in human cells indicate that sister telomeres have distinct requirements for their separation at mitosis. In cells depleted for tankyrase 1, a telomeric poly(ADP-ribose) polymerase, sister chromatid arms and centromeres separate normally, but telomeres remain associated and cells arrest in mitosis. Here, we use biochemical and genetic approaches to identify proteins that might mediate the persistent association at sister telomeres. We use immunoprecipitation analysis to show that the telomeric proteins, TRF1 (an acceptor of PARsylation by tankyrase 1) and TIN2 (a TRF1 binding partner) each bind to the SA1 ortholog of the cohesin Scc3 subunit. Sucrose gradient sedimentation shows that TRF1 cosediments with the SA1–cohesin complex. Depletion of the SA1 cohesin subunit or the telomeric proteins (TRF1 and TIN2) restores the normal resolution of sister telomeres in mitosis in tankyrase 1-depleted cells. Moreover, depletion of TRF1 and TIN2 or SA1 abrogates the requirement for tankyrase 1 in mitotic progression. Our studies indicate that sister telomere association in human cells is mediated by a novel association between a cohesin subunit and components of telomeric chromatin. PMID:17962804

  20. Repeat-containing protein effectors of plant-associated organisms

    PubMed Central

    Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

    2015-01-01

    Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

  1. Adeno-associated virus rep protein synthesis during productive infection

    SciTech Connect

    Redemann, B.E.; Mendelson, E.; Carter, B.J.

    1989-02-01

    Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.

  2. Differences in protein-protein association networks for lung adenocarcinoma: A retrospective study

    PubMed Central

    Datta, Anisha; Sikdar, Sinjini; Gill, Ryan

    2014-01-01

    Various methods to determine the connectivity scores between groups of proteins associated with lung adenocarcinoma are examined. Proteins act together to perform a wide range of functions within biological processes. Hence, identification of key proteins and their interactions within protein networks can provide invaluable information on disease mechanisms. Differential network analysis provides a means of identifying differences in the interactions among proteins between two networks. We use connectivity scores based on the method of partial least squares to quantify the strength of the interactions between each pair of proteins. These scores are then used to perform permutation-based statistical tests. This examines if there are significant differences between the network connectivity scores for individual proteins or classes of proteins. The expression data from a study on lung adenocarcinoma is used in this study. Connectivity scores are computed for a group of 109 subjects who were in the complete remission and as well as for a group of 51 subjects whose cancer had progressed. The distributions of the connectivity scores are similar for the two networks yet subtle but statistically significant differences have been identified and their impact discussed. PMID:25489174

  3. Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles.

    PubMed

    Adessi, C; Chapel, A; Vinçon, M; Rabilloud, T; Klein, G; Satre, M; Garin, J

    1995-10-01

    Magnetic isolation of endocytic vesicles from Dictyostelium discoideum was accomplished after feeding the amoebae with iron oxide particles. Proteins associated with the endocytic vesicles were resolved by SDS-PAGE and digested 'in-gel' with endoproteinase Lys-C or Asp-N to generate peptides for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles: namely, the A, B, D, E and 110 kDa subunits of a vacuolar type H(+)-ATPase, actin, a Rab 7-like GTPase, a p34 protein corresponding to a new cysteine proteinase and the 25 kDa product of a recently sequenced D. discoideum open reading frame.

  4. Identification of protein-protein interactions of the occlusion-derived virus-associated proteins of Helicoverpa armigera nucleopolyhedrovirus.

    PubMed

    Peng, Ke; Wu, Minzhi; Deng, Fei; Song, Jingjiao; Dong, Chunsheng; Wang, Hualin; Hu, Zhihong

    2010-03-01

    The purpose of this study was to identify protein-protein interactions among the components of the occlusion-derived virus (ODV) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV), a group II alphabaculovirus in the family Baculoviridae. To achieve this, 39 selected genes of potential ODV structural proteins were cloned and expressed in the Gal4 yeast two-hybrid (Y2H) system. The direct-cross Y2H assays identified 22 interactions comprising 13 binary interactions [HA9-ODV-EC43, ODV-E56-38K, ODV-E56-PIF3, LEF3-helicase, LEF3-alkaline nuclease (AN), GP41-38K, GP41-HA90, 38K-PIF3, 38K-PIF2, VP80-HA100, ODV-E66-PIF3, ODV-E66-PIF2 and PIF3-PIF2] and nine self-associations (IE1, HA44, LEF3, HA66, GP41, CG30, 38K, PIF3 and P24). Five of these interactions - LEF3-helicase and LEF3-AN, and the self-associations of IE1, LEF3 and 38K - have been reported previously in Autographa californica multiple nucleopolyhedrovirus. As HA44 and HA100 were two newly identified ODV proteins of group II viruses, their interactions were further confirmed. The self-association of HA44 was verified with a His pull-down assay and the interaction of VP80-HA100 was confirmed by a co-immunoprecipitation assay. A summary of the protein-protein interactions of baculoviruses reported so far, comprising 68 interactions with 45 viral proteins and five host proteins, is presented, which will facilitate our understanding of the molecular mechanisms of baculovirus infection.

  5. Ric-8 Proteins Are Molecular Chaperones That Direct Nascent G Protein α Subunit Membrane Association

    PubMed Central

    Gabay, Meital; Pinter, Mary E.; Wright, Forrest A.; Chan, PuiYee; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Tall, Gregory G.

    2013-01-01

    Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide–binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A– or Ric-8B–deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A−/− ES cells, which resulted from reduced steady-state amounts of Gαi, Gαq, and Gα13 proteins to <5% of those of wild-type cells. The amounts of Gαs and total Gβ protein were partially reduced in Ric-8A−/− cells compared to those in wild-type cells, and only the amount of Gαs was reduced substantially in Ric-8B−/− cells. The abundances of mRNAs encoding the G protein α subunits were largely unchanged by loss of Ric-8A or Ric-8B. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous Gαi and Gαq were efficiently translated in Ric-8A−/− cells but integrated into endomembranes poorly; however, the reduced amounts of G protein α subunits that reached the membrane still bound to nascent Gβγ. Finally, Gαi, Gαq, and Gβ1 proteins exhibited accelerated rates of degradation in Ric-8A−/− cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent Gα subunits with cellular membranes. PMID:22114146

  6. Why are proteins with glutamine- and asparagine-rich regions associated with protein misfolding diseases?

    NASA Astrophysics Data System (ADS)

    Cruzeiro, Leonor

    2005-12-01

    The possibility that vibrational excited states (VESs) are the drivers of protein folding and function (the VES hypothesis) is explored to explain the reason why Gln- and Asn-rich proteins are associated with degenerative diseases. The Davydov/Scott model is extended to describe energy transfer from the water solution to the protein and vice versa. Computer simulations show that, on average, Gln and Asn residues lead to an initial larger absorption of energy from the environment to the protein, something that can explain the greater structural instability of prions. The sporadic, inherited and infectious character of prion diseases is discussed in the light of the VES hypothesis. An alternative treatment for prion diseases is suggested.

  7. BIMOLECULAR FLUORESCENCE COMPLEMENTATION ANALYSIS OF INDUCIBLE PROTEIN INTERACTIONS: EFFECTS OF FACTORS AFFECTING PROTEIN FOLDING ON FLUORESCENT PROTEIN FRAGMENT ASSOCIATION

    PubMed Central

    Robida, Aaron M; Kerppola, Tom K

    2009-01-01

    Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. We investigated the temporal resolution and the quantitative accuracy of BiFC analysis using fragments of different fluorescent proteins. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of YFP fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 minutes after rapamycin addition and a ten-fold increase in the mean fluorescence intensity in 8 hours. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and YFP produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment of the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized prior to rapamycin addition formed BiFC complexes with the same efficiency as newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggest that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular

  8. Modeling associated protein-DNA pattern discovery with unified scores.

    PubMed

    Chan, Tak-Ming; Lo, Leung-Yau; Sze-To, Ho-Yin; Leung, Kwong-Sak; Xiao, Xinshu; Wong, Man-Hon

    2013-01-01

    Understanding protein-DNA interactions, specifically transcription factor (TF) and transcription factor binding site (TFBS) bindings, is crucial in deciphering gene regulation. The recent associated TF-TFBS pattern discovery combines one-sided motif discovery on both the TF and the TFBS sides. Using sequences only, it identifies the short protein-DNA binding cores available only in high-resolution 3D structures. The discovered patterns lead to promising subtype and disease analysis applications. While the related studies use either association rule mining or existing TFBS annotations, none has proposed any formal unified (both-sided) model to prioritize the top verifiable associated patterns. We propose the unified scores and develop an effective pipeline for associated TF-TFBS pattern discovery. Our stringent instance-level evaluations show that the patterns with the top unified scores match with the binding cores in 3D structures considerably better than the previous works, where up to 90 percent of the top 20 scored patterns are verified. We also introduce extended verification from literature surveys, where the high unified scores correspond to even higher verification percentage. The top scored patterns are confirmed to match the known WRKY binding cores with no available 3D structures and agree well with the top binding affinities of in vivo experiments.

  9. A novel nucleoid-associated protein specific to the actinobacteria

    PubMed Central

    Swiercz, Julia P.; Nanji, Tamiza; Gloyd, Melanie; Guarné, Alba; Elliot, Marie A.

    2013-01-01

    Effective chromosome organization is central to the functioning of any cell. In bacteria, this organization is achieved through the concerted activity of multiple nucleoid-associated proteins. These proteins are not, however, universally conserved, and different groups of bacteria have distinct subsets that contribute to chromosome architecture. Here, we describe the characterization of a novel actinobacterial-specific protein in Streptomyces coelicolor. We show that sIHF (SCO1480) associates with the nucleoid and makes important contributions to chromosome condensation and chromosome segregation during Streptomyces sporulation. It also affects antibiotic production, suggesting an additional role in gene regulation. In vitro, sIHF binds DNA in a length-dependent but sequence-independent manner, without any obvious structural preferences. It does, however, impact the activity of topoisomerase, significantly altering DNA topology. The sIHF–DNA co-crystal structure reveals sIHF to be composed of two domains: a long N-terminal helix and a C-terminal helix-two turns-helix domain with two separate DNA interaction sites, suggesting a potential role in bridging DNA molecules. PMID:23427309

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

    SciTech Connect

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

    2005-05-15

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

  11. Structural and functional characterization of synapse-associated protein-97

    NASA Astrophysics Data System (ADS)

    Wang, Lei

    Synapse-associated protein-97 (SAP97) as a scaffold protein plays an important role in regulating neural signal transmission in the central nervous system by coupling with activated membrane receptors, ion channels, and downstream signaling proteins. SAP97 consists of six functional domains: L27, PDZ1, PDZ2, PDZ3, SH3, and GK. Each of these domains mediates the interactions of SAP97 with other proteins. Understanding the molecular mechanism of these interactions in neural signal transmission is a goal of this study. Here high-resolution nuclear magnetic resonance spectroscopy and fluorescence anisotropy are employed towards the goal of the structural and functional characterization of SAP97; specifically, we (a) characterize the binding of the PDZ domains of SAP97 with the C-terminus of NR2B, and determine the structure of the PDZ1-NR2B; (b) characterize the binding of the PDZ domains with the C-terminus of stargazin and multiple mutants, and identify the perturbed amino acids in PDZ2 upon the binding of stargazin; (c) characterize the binding specificity carried by the beta2/beta3 loop of the PDZ3 domain. These results provide insight into the molecular mechanism for the binding specificities of the PDZ domains of SAP97, thereby furthering the development of drugs that target these domains to treat neurological diseases.

  12. Hydroxocobalamin association during cell culture results in pink therapeutic proteins

    PubMed Central

    Prentice, Kenneth M; Gillespie, Ronald; Lewis, Nathan; Fujimori, Kiyoshi; McCoy, Rebecca; Bach, Julia; Connell-Crowley, Lisa; Eakin, Catherine M

    2013-01-01

    Process control of protein therapeutic manufacturing is central to ensuring the product is both safe and efficacious for patients. In this work, we investigate the cause of pink color variability in development lots of monoclonal antibody (mAb) and Fc-fusion proteins. Results show pink-colored product generated during manufacturing is due to association of hydroxocobalamin (OH-Cbl), a form of vitamin B12. OH-Cbl is not part of the product manufacturing process; however we found cyanocobalamin (CN-Cbl) in cell culture media converts to OH-Cbl in the presence of light. OH-Cbl can be released from mAb and Fc-fusion proteins by conversion with potassium cyanide to CN-Cbl, which does not bind. By exploiting the differential binding of CN-Cbl and OH-Cbl, we developed a rapid and specific assay to accurately measure B12 levels in purified protein. Analysis of multiple products and lots using this technique gives insight into color variability during manufacturing. PMID:23924851

  13. Identification of extensin protein associated with sugar beet pectin.

    PubMed

    Nuñez, Alberto; Fishman, Marshall L; Fortis, Laurie L; Cooke, Peter H; Hotchkiss, Arland T

    2009-11-25

    Several studies have suggested that the emulsification properties associated with pectin obtained from sugar beet (Beta vulgaris) are due to the presence of a protein-pectin complex. Nevertheless, the identity of the protein has remained elusive. Pectin, extracted from sugar beet pulp by microwave-assisted extraction, and a commercial sample were both subjected to protease digestion with trypsin. The resulting peptides were separated from the pectin solution by ultrafiltration using a 3 kDa molecular weight cutoff (MWCO) membrane and analyzed using matrix-assisted laser desorption ionization with tandem time-of-flight mass spectrometry. The partial sequences derived from the mass spectrometry analyses of the resulting tryptic peptides are found to be highly consistent with extensin protein matched from the B. vulgaris Genetic Index database and also correspond to previously reported extensin peptides found in sugar beet cell suspension cultures. Further attempts were made to disassociate the protein from pectin using 1 M NaCl and a 100 kDa MWCO membrane; however, no peptides were observed following trypsin digestion of the permeate solution. This evidence suggests the existence of a complex between the pectin and extensin that is not due to ionic interactions. Trypsin digestion of commercial sugar beet pectin also produced the peptide profile observed with the microwave-assisted extracted pectin sample. Atomic force microscopy established that the number of rod-like elements decreased following protease treatment compared to the untreated sample.

  14. Initiation of protein association in tofu formation by metal ions.

    PubMed

    Arii, Yasuhiro; Takenaka, Yasuyuki

    2014-01-01

    Magnesium and calcium ions are important factors in making tofu. However, the molecular role of these ions remains unclear in tofu formation. We have previously shown that magnesium chloride concentration-dependent produced silken tofu-like (SP) and regular tofu-like (RP) precipitates, but was an inconsequential factor for the retention of tofu. We investigated in this present study, the effect of various metal chlorides on the metal chloride concentration-dependent changes in tofu formation. These changes occurred in a similar manner to that of the magnesium ion, in which SP formation was followed by RP formation. It is interesting that the midpoint concentration for the formation of SP and RP represented a good correlation with the stability constant of EDTA. This correlation demonstrated the possibility that metal ions would interact with the carboxyl groups of soy proteins. We consider from these results that metal ions were the initiators of protein association in tofu formation.

  15. Inferring drug-disease associations based on known protein complexes.

    PubMed

    Yu, Liang; Huang, Jianbin; Ma, Zhixin; Zhang, Jing; Zou, Yapeng; Gao, Lin

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html.

  16. STRING: a database of predicted functional associations between proteins.

    PubMed

    von Mering, Christian; Huynen, Martijn; Jaeggi, Daniel; Schmidt, Steffen; Bork, Peer; Snel, Berend

    2003-01-01

    Functional links between proteins can often be inferred from genomic associations between the genes that encode them: groups of genes that are required for the same function tend to show similar species coverage, are often located in close proximity on the genome (in prokaryotes), and tend to be involved in gene-fusion events. The database STRING is a precomputed global resource for the exploration and analysis of these associations. Since the three types of evidence differ conceptually, and the number of predicted interactions is very large, it is essential to be able to assess and compare the significance of individual predictions. Thus, STRING contains a unique scoring-framework based on benchmarks of the different types of associations against a common reference set, integrated in a single confidence score per prediction. The graphical representation of the network of inferred, weighted protein interactions provides a high-level view of functional linkage, facilitating the analysis of modularity in biological processes. STRING is updated continuously, and currently contains 261 033 orthologs in 89 fully sequenced genomes. The database predicts functional interactions at an expected level of accuracy of at least 80% for more than half of the genes; it is online at http://www.bork.embl-heidelberg.de/STRING/.

  17. Functional aspects of membrane association of reggie/flotillin proteins.

    PubMed

    Banning, Antje; Tomasovic, Ana; Tikkanen, Ritva

    2011-12-01

    Flotillin-2 and flotillin-1, also called reggie-1 and reggie-2, are ubiquitously expressed and highly conserved proteins. Originally, they were described as neuronal regeneration proteins, but they appear to function in a wide variety of cellular processes, such as membrane receptor signaling, endocytosis, phagocytosis and cell adhesion. The molecular details of the function of flotillins in these processes have only been partially clarified. Flotillins are associated with cholesterol and sphingolipid enriched membrane microdomains known as rafts, and some findings even suggest that they define their own kind of a microdomain. The mechanism of the membrane association of flotillins appears to rely mainly on acylation (myristoylation and/or palmitoylation), localizing flotillins onto the cytosolic side of the membranes, whereas no transmembrane domains are present. In addition, flotillins show a strong tendency to form homo- and hetero-oligomers with each other. In this review, we will summarize the recent findings on the function of flotillins and discuss the mechanisms that might regulate their function, such as membrane association, oligomerization and phosphorylation.

  18. Iron-Associated Outer Membrane Proteins of Magnetic Bacteria

    DTIC Science & Technology

    1989-06-16

    AD-A210 088 _ _ _ _ _ _ _ _ _ _ _ _ Form Approved WrMN PAGE0MB No ()704-0188 la RPORTSECQ!TY -AssF.(L; i RES’C it MA %CS ()NA 14 J 1 ILL 2a SECURITY...NUMBERS 800N. uicy t.PROGRAM PROiECT rASK P T’O ~80NQunyS.EiLEVE T NO NO NO jACCES ON NO Arlington, VA 22217-5000 61153N IRR 4106 4413-009 1 1 TITLE...include Security Classification) (u) Iron Associated Outer Membrane Proteins of Magnetic Bacteria 12 PERSONAL AuTHOR(S) Blakemore, Richard Peter 1 3a

  19. Protein aggregates are associated with replicative aging without compromising protein quality control

    PubMed Central

    Saarikangas, Juha; Barral, Yves

    2015-01-01

    Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells. In budding yeast, various aging factors segregate to the aging (mother)-lineage, with poorly understood consequences. In this study, we show that yeast mother cells form a protein aggregate during early replicative aging that is maintained as a single, asymmetrically inherited deposit over the remaining lifespan. Surprisingly, deposit formation was not associated with stress or general decline in proteostasis. Rather, the deposit-containing cells displayed enhanced degradation of cytosolic proteasome substrates and unimpaired clearance of stress-induced protein aggregates. Deposit formation was dependent on Hsp42, which collected non-random client proteins of the Hsp104/Hsp70-refolding machinery, including the prion Sup35. Importantly, loss of Hsp42 resulted in symmetric inheritance of its constituents and prolonged the lifespan of the mother cell. Together, these data suggest that protein aggregation is an early aging-associated differentiation event in yeast, having a two-faceted role in organismal fitness. DOI: http://dx.doi.org/10.7554/eLife.06197.001 PMID:26544680

  20. Fragile X mental retardation protein interactions with the microtubule associated protein 1B RNA.

    PubMed

    Menon, Lakshmi; Mader, Samantha Ann; Mihailescu, Mihaela-Rita

    2008-08-01

    Fragile X mental retardation syndrome, the most common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine (RGG) box to bind to a subset of RNA targets that form a G quadruplex structure. We performed a detailed analysis of the interactions between the FMRP RGG box and the microtubule associated protein 1B (MAP1B) mRNA, a relevant in vivo FMRP target. We show that MAP1B RNA forms an intramolecular G quadruplex structure, which is bound with high affinity and specificity by the FMRP RGG box. We determined that hydrophobic interactions are important in the FMRP RGG box-MAP1B RNA association, with minor contributions from electrostatic interactions. Our findings that at low protein:RNA ratios the RNA G quadruplex structure is slightly stabilized, whereas at high ratios is unfolded, suggest a mechanism by which the FMRP concentration variation in response to a neurotransmitter stimulation event could act as a regulatory switch for the protein function, from translation repressor to translation activator.

  1. Simultaneous time-lamination imaging of protein association using a split fluorescent timer protein.

    PubMed

    Takamura, Ayari; Hattori, Mitsuru; Yoshimura, Hideaki; Ozawa, Takeaki

    2015-03-17

    Studies of temporal behaviors of protein association in living cells are crucially important for elucidating the fundamental roles and the mechanism of interactive coordination for cell activities. We developed a method for investigating the temporal alternation of a particular protein assembly using monomeric fluorescent proteins, fluorescent timers (FTs), of which the fluorescent color changes from blue to red over time. We identified a dissection site of the FTs, which allows complementation of the split FT fragments. The split fragments of each FT variant recovered their fluorescence and maintained inherent rates of the color changes upon the reassembly of the fragments in vitro. We applied this method to visualize the aggregation process of α-synuclein in living cells. The size of the aggregates with the temporal information was analyzed from ratio values of the blue and red fluorescence of the reconstituted FTs, from which the aggregation rates were evaluated. This method using the split FT fragments enables tracing and visualizing temporal alternations of various protein associations by single fluorescence measurements at a given time point.

  2. Computing Protein-Protein Association Affinity with Hybrid Steered Molecular Dynamics.

    PubMed

    Rodriguez, Roberto A; Yu, Lili; Chen, Liao Y

    2015-09-08

    Computing protein-protein association affinities is one of the fundamental challenges in computational biophysics/biochemistry. The overwhelming amount of statistics in the phase space of very high dimensions cannot be sufficiently sampled even with today's high-performance computing power. In this article, we extend a potential of mean force (PMF)-based approach, the hybrid steered molecular dynamics (hSMD) approach we developed for ligand-protein binding, to protein-protein association problems. For a protein complex consisting of two protomers, P1 and P2, we choose m (≥3) segments of P1 whose m centers of mass are to be steered in a chosen direction and n (≥3) segments of P2 whose n centers of mass are to be steered in the opposite direction. The coordinates of these m + n centers constitute a phase space of 3(m + n) dimensions (3(m + n)D). All other degrees of freedom of the proteins, ligands, solvents, and solutes are freely subject to the stochastic dynamics of the all-atom model system. Conducting SMD along a line in this phase space, we obtain the 3(m + n)D PMF difference between two chosen states: one single state in the associated state ensemble and one single state in the dissociated state ensemble. This PMF difference is the first of four contributors to the protein-protein association energy. The second contributor is the 3(m + n - 1)D partial partition in the associated state accounting for the rotations and fluctuations of the (m + n - 1) centers while fixing one of the m + n centers of the P1-P2 complex. The two other contributors are the 3(m - 1)D partial partition of P1 and the 3(n - 1)D partial partition of P2 accounting for the rotations and fluctuations of their m - 1 or n - 1 centers while fixing one of the m/n centers of P1/P2 in the dissociated state. Each of these three partial partitions can be factored exactly into a 6D partial partition in multiplication with a remaining factor accounting for the small fluctuations while fixing three

  3. A matrix protein silences transposons and repeats through interaction with retinoblastoma-associated proteins.

    PubMed

    Xu, Yifeng; Wang, Yizhong; Stroud, Hume; Gu, Xiaofeng; Sun, Bo; Gan, Eng-Seng; Ng, Kian-Hong; Jacobsen, Steven E; He, Yuehui; Ito, Toshiro

    2013-02-18

    Epigenetic regulation helps to maintain genomic integrity by suppressing transposable elements (TEs) and also controls key developmental processes, such as flowering time. To prevent TEs from causing rearrangements and mutations, TE and TE-like repetitive DNA sequences are usually methylated, whereas histones are hypoacetylated and methylated on specific residues (e.g., H3 lysine 9 dimethylation [H3K9me2]). TEs and repeats can also attenuate gene expression. However, how various histone modifiers are recruited to target loci is not well understood. Here we show that knockdown of the nuclear matrix protein with AT-hook DNA binding motifs TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK) in Arabidopsis Landsberg erecta results in robust activation of various TEs, the TE-like repeat-containing floral repressor genes FLOWERING LOCUS C (FLC) and FWA. This derepression is associated with chromatin conformational changes, increased histone acetylation, reduced H3K9me2, and even TE transposition. TEK directly binds to an FLC-repressive regulatory region and the silencing repeats of FWA and associates with Arabidopsis homologs of the Retinoblastoma-associated protein 46/48, FVE and MSI5, which mediate histone deacetylation. We propose that the nuclear matrix protein TEK acts in the maintenance of genome integrity by silencing TE and repeat-containing genes.

  4. The evolution and diversification of plant microtubule-associated proteins.

    PubMed

    Gardiner, John

    2013-07-01

    Plant evolution is marked by major advances in structural characteristics that facilitated the highly successful colonization of dry land. Underlying these advances is the evolution of genes encoding specialized proteins that form novel microtubular arrays of the cytoskeleton. This review investigates the evolution of plant families of microtubule-associated proteins (MAPs) through the recently sequenced genomes of Arabidopsis thaliana, Oryza sativa, Selaginella moellendorffii, Physcomitrella patens, Volvox carteri and Chlamydomonas reinhardtii. The families of MAPs examined are AIR9, CLASP, CRIPT, MAP18, MOR1, TON, EB1, AtMAP70, SPR2, SPR1, WVD2 and MAP65 families (abbreviations are defined in the footnote to Table 1). Conjectures are made regarding the evolution of MAPs in plants in relation to the evolution of multicellularity, oriented cell division and vasculature. Angiosperms in particular have high numbers of proteins that are involved in promotion of helical growth or its suppression, and novel plant microtubular structures may have acted as a catalyst for the development of novel plant MAPs. Comparisons of plant MAP gene families with those of animals show that animals may have more flexibility in the structure of their microtubule cytoskeletons than plants, but with both plants and animals possessing many MAP splice variants. © 2013 The Author The Plant Journal © 2013 John Wiley & Sons Ltd.

  5. Protein-tyrosine phosphatase sigma is associated with ulcerative colitis.

    PubMed

    Muise, Aleixo M; Walters, Thomas; Wine, Eytan; Griffiths, Anne M; Turner, Dan; Duerr, Richard H; Regueiro, Miguel D; Ngan, Bo-Yee; Xu, Wei; Sherman, Philip M; Silverberg, Mark S; Rotin, Daniela

    2007-07-17

    Inflammatory bowel disease (IBD), a relatively common chronic debilitating intestinal illness, is composed of two broadly defined groups, Crohn's disease (CD) and ulcerative colitis (UC). Although several susceptibility genes for CD have been recently described, susceptibility genes exclusive for UC have not been forthcoming. Here, we show that receptor protein-tyrosine phosphatase sigma (PTPRS-encoding PTPsigma) knockout mice spontaneously develop mild colitis that becomes severe when challenged with two known inducers of colitis. We also demonstrate that E-cadherin and beta-catenin, two important adherens junction proteins involved in maintenance of barrier defense in the colon, act as colonic substrates for PTPsigma. Furthermore, we show that three SNPs (rs886936, rs17130, and rs8100586) that flank exon 8 in the human PTPRS gene are associated with UC. The presence of these SNPs is associated with novel splicing that removes the third immunoglobulin-like domain (exon 9) from the extracellular portion of PTPsigma, possibly altering dimerization or ligand recognition. We propose that polymorphisms in the human PTPRS gene lead to ulcerative colitis.

  6. Measuring residue associations in protein structures. Possible implications for protein folding.

    PubMed

    Karlin, S; Zuker, M; Brocchieri, L

    1994-06-03

    We propose a number of distance measures between residues in protein structures based on average, minimum and maximum distances of all atom (backbone and side-chain) coordinates or with respect to side-chain atom coordinates only. The d1-distance (D1-distance) refers to the average distance between side-chain (backbone and side-chain) atoms of a residue pair in a given structure. The dm-distance (Dm-distance) refers to the minimum distance between side-chain atoms (non-trivial minimum distance between all atoms of a residue pair). For each distance measure, averaging and normalizing over representative protein structures, association values and closeness orderings for all amino acid types are determined. The expected associations of side-chain interactions between oppositely charged residues, among hydrophobic residues and of cysteine with cysteine are confirmed. Several surprising associations are observed relative to (1) the aromatic residues tyrosine and tryptophan, but not phenylalanine; (2) multiple histidine residues; (3) asymmetries of arginine versus lysine, aspartate versus glutamate, alanine versus glycine, and asparagine versus glutamine; (4) absence of correlations of alpha-carbon distances with side-chain distances. The all atoms D1-distance attractions are dominated by steric relationships, with glycine and alanine significantly close to all amino acids, whereas large residues are under-associated with all residue types. In contrast, for the closeness ordering corresponding to the minimum side-chain dm-distance, glycine and alanine are among the least associated. However, in the d1-distance alanine is significantly close to all hydrophobic residues with the exception of tryptophan. The dm-distance preferences display a pervasive attraction for tyrosine by almost all residue types, the prominence of tyrosine and tryptophan in cation-aromatic interactions, and the versatility of histidine in functionality. The principal findings suggest a new

  7. Germ cell mitogenic activity is associated with nerve growth factor-like protein(s).

    PubMed

    Onoda, M; Pflug, B; Djakiew, D

    1991-12-01

    from pheochromocytoma cells (PC-12), a functional bioassay for NGF-like activity, was stimulated by addition of RSP and PSP to the culture media of the PC-12 cells. These results demonstrate mitogenic activity in germ cell proteins (RSP and PSP) and identify a NGF-like protein(s) which is associated with most of this activity.

  8. Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

    SciTech Connect

    Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A.

    2012-08-21

    DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

  9. Molecular Analysis of the Acinetobacter baumannii Biofilm-Associated Protein

    PubMed Central

    Goh, H. M. Sharon; Beatson, Scott A.; Totsika, Makrina; Moriel, Danilo G.; Phan, Minh-Duy; Szubert, Jan; Runnegar, Naomi; Sidjabat, Hanna E.; Paterson, David L.; Nimmo, Graeme R.; Lipman, Jeffrey

    2013-01-01

    Acinetobacter baumannii is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of A. baumannii to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the A. baumannii biofilm-associated protein (Bap) in 24 carbapenem-resistant A. baumannii ST92 strains isolated from a single institution over a 10-year period. The bap gene was highly prevalent, with 22/24 strains being positive for bap by PCR. Partial sequencing of bap was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that BapMS1968 clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the bapMS1968 gene was cloned, and its expression in a recombinant Escherichia coli strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of A. baumannii strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive A. baumannii strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by A. baumannii clinical isolates. PMID:23956398

  10. Association of C-reactive protein with mild cognitive impairment

    PubMed Central

    Roberts, Rosebud O.; Geda, Yonas E.; Knopman, David S.; Christianson, Teresa J.H.; Pankratz, V. Shane; Kullo, Iftikhar J.; Petersen, Ronald C.

    2010-01-01

    Background Inflammation is suggested to play a role in the development of Alzheimer’s disease, and may also be involved in the pathogenesis of mild cognitive impairment (MCI). This study examined the association of inflammatory markers in serum or plasma with prevalent MCI and MCI subtypes in a population-based sample. Methods Olmsted County, MN, residents aged 70–89 years on October 1, 2004, were evaluated using the Clinical Dementia Rating Scale, a neurological evaluation, and neuropsychological testing. Information ascertained for each participant was reviewed by an expert panel of neuropsychologists, physicians, and nurses, and a diagnosis of normal cognition, MCI, or dementia was made by consensus. C-reactive protein (CRP), interleukin 6 (IL-6), tumor necrosis alpha (TNFα), and adiponectin were measured at baseline. Results Among 313 subjects with MCI and 1,570 cognitively normal subjects, a CRP level in the upper quartile (> 3.3 mg/L) was significantly associated with MCI (odds ratio [OR], 1.42; 95% confidence interval [CI], 1.00–2.01) and with non-amnestic MCI (na-MCI; OR, 2.05; 95% CI, 1.12–3.78) after adjusting for age, sex, and years of education. However, there was no association with amnestic MCI (a-MCI; OR, 1.21; 95% CI, 0.81–1.82). No association was observed with the other inflammatory markers. Conclusions Plasma CRP is associated with prevalent MCI and with na-MCI in elderly, non-demented persons in the population-based setting. These findings suggest an involvement of inflammation in the pathogenesis of MCI. PMID:19751919

  11. Pregnancy-associated plasma protein A: spotlight on kidney diseases.

    PubMed

    Kalousová, Marta; Tesař, Vladimír; Muravská, Alexandra; Zima, Tomáš

    2012-03-24

    Pregnancy-associated plasma protein A (PAPP-A) is a biomarker routinely used in screening for Down syndrome in the first trimester of pregnancy. It is also present in very small amounts in men and non-pregnant women. PAPP-A is a key regulator of local insulin-like growth factor (IGF) bioavailability - IGFs are essential for normal body size during fetal development, but they are associated with aging and age-related diseases. Measurement of circulating PAPP-A can provide valuable information not only in pregnant women (chromosomal anomalies and adverse pregnancy outcomes) but also in patients with coronary artery disease (contribution to diagnosis, prognostic value) and in patients with kidney diseases. PAPP-A is associated with renal function and proteinuria, is increased mainly in dialysis patients and decreases after kidney transplantation. It is an independent mortality predictor of hemodialysis patients and indicator of adverse outcome of transplanted patients. PAPP-A levels can be influenced by various chemicals and drugs, among them mainly heparin. Various assays for PAPP-A exist and the type of assay used in a study should be considered. This article reviews the data summarizing basic information about PAPP-A with a particular focus on the significance of PAPP-A in renal diseases.

  12. Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomyces cerevisiae.

    PubMed

    Kennedy-Darling, Julia; Guillen-Ahlers, Hector; Shortreed, Michael R; Scalf, Mark; Frey, Brian L; Kendziorski, Christina; Olivier, Michael; Gasch, Audrey P; Smith, Lloyd M

    2014-08-01

    DNA-protein interactions play critical roles in the control of genome expression and other fundamental processes. An essential element in understanding how these systems function is to identify their molecular components. We present here a novel strategy, Hybridization Capture of Chromatin Associated Proteins for Proteomics (HyCCAPP), to identify proteins that are interacting with any given region of the genome. This technology identifies and quantifies the proteins that are specifically interacting with a genomic region of interest by sequence-specific hybridization capture of the target region from in vivo cross-linked chromatin, followed by mass spectrometric identification and quantification of associated proteins. We demonstrate the utility of HyCCAPP by identifying proteins associated with three multicopy and one single-copy loci in yeast. In each case, a locus-specific pattern of target-associated proteins was revealed. The binding of previously unknown proteins was confirmed by ChIP in 11 of 17 cases. The identification of many previously known proteins at each locus provides strong support for the ability of HyCCAPP to correctly identify DNA-associated proteins in a sequence-specific manner, while the discovery of previously unknown proteins provides new biological insights into transcriptional and regulatory processes at the target locus.

  13. Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomyces cerevisiae

    PubMed Central

    2015-01-01

    DNA–protein interactions play critical roles in the control of genome expression and other fundamental processes. An essential element in understanding how these systems function is to identify their molecular components. We present here a novel strategy, Hybridization Capture of Chromatin Associated Proteins for Proteomics (HyCCAPP), to identify proteins that are interacting with any given region of the genome. This technology identifies and quantifies the proteins that are specifically interacting with a genomic region of interest by sequence-specific hybridization capture of the target region from in vivo cross-linked chromatin, followed by mass spectrometric identification and quantification of associated proteins. We demonstrate the utility of HyCCAPP by identifying proteins associated with three multicopy and one single-copy loci in yeast. In each case, a locus-specific pattern of target-associated proteins was revealed. The binding of previously unknown proteins was confirmed by ChIP in 11 of 17 cases. The identification of many previously known proteins at each locus provides strong support for the ability of HyCCAPP to correctly identify DNA-associated proteins in a sequence-specific manner, while the discovery of previously unknown proteins provides new biological insights into transcriptional and regulatory processes at the target locus. PMID:24999558

  14. Tobacco mosaic virus movement protein functions as a structural microtubule-associated protein.

    PubMed

    Ashby, Jamie; Boutant, Emmanuel; Seemanpillai, Mark; Groner, Anna; Sambade, Adrian; Ritzenthaler, Christophe; Heinlein, Manfred

    2006-09-01

    The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

  15. Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

    PubMed Central

    Ashby, Jamie; Boutant, Emmanuel; Seemanpillai, Mark; Sambade, Adrian; Ritzenthaler, Christophe; Heinlein, Manfred

    2006-01-01

    The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection. PMID:16912284

  16. Genetic analysis of a Drosophila microtubule-associated protein

    PubMed Central

    1992-01-01

    The 205-kD microtubule-associated protein (205K MAP) is one of the principal MAPs in Drosophila. 205K MAP is similar to the HeLa 210K/MAP4 family of MAPs since it shares the following biochemical properties: it is present in several isoforms, has a molecular mass of approximately 200 kD, and is thermostable. Furthermore, immuno-crossreactivity has been observed between mouse MAP4, HeLa 210K, and Drosophila 205K MAP. Currently, there is little information concerning the biological function of this group of nonmotor MAPs. We have used a classical genetic approach to try to identify the role of the 205K MAP in Drosophila by isolating mutations in the 205K MAP gene. An F2 lethal screen was used to acquire deficiencies of 100EF, the chromosomal location of the 205K MAP gene. Drosophila bearing a homozygous deficiency for the 205K MAP region are fully viable and show no obvious phenotype. A recently developed polymerase chain reaction screen was also used to recover five P-element insertions upstream from the 205K MAP gene. Western blot analysis has shown that these insertions result in hypomorphic mutations of the 205K MAP gene. As was seen with animals that have no 205K MAP, these mutations appear to have no phenotype. These data unambiguously demonstrate that the 205K MAP gene is inessential for development. These results also suggest that there may exist protein(s) with redundant function that can substitute for 205K MAP. PMID:1309812

  17. Atomistic Simulation of Lignocellulosic Biomass and Associated Cellulosomal Protein Complexes

    SciTech Connect

    Petridis, Loukas; Crowley, Michael F; Smith, Jeremy C

    2010-01-01

    Computer simulations have been performed to obtain an atomic-level understanding of lignocellulose structure and the assembly of its associated cellulosomal protein complexes. First, a CHARMM molecular mechanics force field for lignin is derived and validated by performing a molecular dynamics simulation of a crystal of a lignin fragment molecule and comparing simulation-derived structural features with experimental results. Together with the existing force field for polysaccharides, this work provides the basis for full simulations of lignocellulose. Second, the underlying molecular mechanism governing the assembly of various cellulosomal modules is investigated by performing a novel free-energy calculation of the cohesin-dockerin dissociation. Our calculation indicates a free-energy barrier of ~17 kcal/mol and further reveals a stepwise dissociation pathway involving both the central -sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the -barrel structure.

  18. Altered cell-matrix associated ADAM proteins in Alzheimer disease.

    PubMed

    Gerst, J L; Raina, A K; Pirim, I; McShea, A; Harris, P L; Siedlak, S L; Takeda, A; Petersen, R B; Smith, M A

    2000-03-01

    Alterations in cell-matrix 'contact' are often related to a disruption of cell cycle regulation and, as such, occur variously in neoplasia. Given the recent findings showing cell cycle alterations in Alzheimer disease, we undertook a study of ADAM-1 and 2 (A Disintegrin And Metalloprotease), developmentally-regulated, integrin-binding, membrane-bound metalloproteases. Our results show that whereas ADAM-1 and 2 are found in susceptible hippocampal neurons in Alzheimer disease, these proteins were not generally increased in similar neuronal populations in younger or age-matched controls except in association with age-related neurofibrillary alterations. This increase in both ADAM-1 and 2 in cases of Alzheimer disease was verified by immunoblot analysis (P < 0.05). An ADAM-induced loss of matrix integration would effectively "reset" the mitotic clock and thereby stimulate re-entry into the cell cycle in neurons in Alzheimer disease. Furthermore, given the importance of integrins in maintaining short-term memory, alterations in ADAM proteins or their proteolytic activity could also play a proximal role in the clinico-pathological manifestations of Alzheimer disease. Copyright 2000 Wiley-Liss, Inc.

  19. Nanoscopic dynamics of bicontinous microemulsions: effect of membrane associated protein

    DOE PAGES

    Sharma, V. K.; Hayes, Douglas G.; Urban, Volker S.; ...

    2017-06-12

    Bicontinous microemulsions (BμE) generally consist of nanodomains formed by surfactant in a mixture of water and oil at nearly equal proportions and are potential candidates for the solubilization and purification of membrane proteins. In this paper, we present the first time report of nanoscopic dynamics of surfactant monolayers within BμEs formed by the anionic surfactant sodium dodecyl sulfate (SDS) measured on the nanosecond to picosecond time scale using quasielastic neutron scattering (QENS). BμEs investigated herein consisted of middle phases isolated from Winsor-III microemulsion systems that were formed by mixing aqueous and oil solutions under optimal conditions. QENS data indicates thatmore » surfactants undergo two distinct motions, namely (i) lateral motion along the surface of the oil nanodomains and (ii) localized internal motion. Lateral motion can be described using a continuous diffusion model, from which the lateral diffusion coefficient is obtained. Internal motion of surfactant is described using a model which assumes that a fraction of the surfactants’ hydrogens undergoes localized translational diffusion that could be considered confined within a spherical volume. The effect of cytochrome c, an archetypal membrane-associated protein known to strongly partition near the surfactant head groups in BμEs (a trend supported by small-angle X-ray scattering [SAXS] analysis), on the dynamics of BμE has also been investigated. QENS results demonstrated that cytochrome c significantly hindered both the lateral and the internal motions of surfactant. The lateral motion was more strongly affected: a reduction of the lateral diffusion coefficient by 33% was measured. This change is mainly attributable to the strong association of cytochrome c with oppositely charged SDS. In contrast, analysis of SAXS data suggested that thermal fluctuations (for a longer length and slower time scale compared to QENS) were increased upon incorporation of cytochrome

  20. Protein phosphorylation cascades associated with methamphetamine-induced glial activation.

    PubMed

    Hebert, M A; O'Callaghan, J P

    2000-09-01

    Reactive gliosis is the most prominent response to diverse forms of central nervous system (CNS) injury. The signaling events that mediate this characteristic response to neural injury are under intense investigation. Several studies have demonstrated the activation of phosphoproteins within the mitogen-activated protein kinase (MAPK) and Janus kinase (JAK) pathways following neural insult. These signaling pathways may be involved or responsible for the glial response following injury, by virtue of their ability to phosphorylate and dynamically regulate the activity of various transcription factors. This study sought to delineate, in vivo, the relative contribution of MAPK- and JAK-signaling components to reactive gliosis as measured by induction of glial-fibrillary acidic protein (GFAP), following chemical-induced neural damage. At time points (6, 24, and 48 h) following methamphetamine (METH, 10 mg/kg x 4, s.c.) administration, female C57BL/6J mice were sacrificed by focused microwave irradiation, a technique that preserves steady-state phosphorylation. Striatal (target) and nontarget (hippocampus) homogenates were assayed for METH-induced changes in markers of dopamine (DA) neuron integrity as well as differences in the levels of activated phosphoproteins. GFAP upregulation occurred as early as 6 h, reaching a threefold induction 48 h following METH exposure. Neurotoxicant-induced reductions in striatal levels of DA and tyrosine hydroxylase (TH) paralleled the temporal profile of GFAP induction. Blots of striatal homogenates, probed with phosphorylation-state specific antibodies, demonstrated significant changes in activated forms of extracellular-regulated kinase 1/2 (ERK 1/2), c-jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), MAPK/ERK kinase (MEK1/2), 70-kDa ribosomal S6 kinase (p70 S6), cAMP responsive element binding protein (CREB), and signal transducer and activator of transcription 3 (STAT3). MAPK-related phosphoproteins exhibited an

  1. Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis.

    PubMed

    Hong, Kyung Uk; Choi, Yong-Bock; Lee, Jung-Hwa; Kim, Hyun-Jun; Kwon, Hye-Rim; Seong, Yeon-Sun; Kim, Heung Tae; Park, Joobae; Bae, Chang-Dae; Hong, Kyeong-Man

    2008-08-31

    Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.

  2. Cdk1-cyclin B1-mediated phosphorylation of tumor-associated microtubule-associated protein/cytoskeleton-associated protein 2 in mitosis.

    PubMed

    Hong, Kyung Uk; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

    2009-06-12

    During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.

  3. Weighted-ensemble Brownian dynamics simulations for protein association reactions.

    PubMed Central

    Huber, G A; Kim, S

    1996-01-01

    A new method, weighted-ensemble Brownian dynamics, is proposed for the simulation of protein-association reactions and other events whose frequencies of outcomes are constricted by free energy barriers. The method features a weighted ensemble of trajectories in configuration space with energy levels dictating the proper correspondence between "particles" and probability. Instead of waiting a very long time for an unlikely event to occur, the probability packets are split, and small packets of probability are allowed to diffuse almost immediately into regions of configuration space that are less likely to be sampled. The method has been applied to the Northrup and Erickson (1992) model of docking-type diffusion-limited reactions and yields reaction rate constants in agreement with those obtained by direct Brownian simulation, but at a fraction of the CPU time (10(-4) to 10(-3), depending on the model). Because the method is essentially a variant of standard Brownian dynamics algorithms, it is anticipated that weighted-ensemble Brownian dynamics, in conjunction with biophysical force models, can be applied to a large class of association reactions of interest to the biophysics community. Images FIGURE 4 PMID:8770190

  4. Heavy path mining of protein-protein associations in the malaria parasite.

    PubMed

    Yu, Xinran; Korkmaz, Turgay; Lilburn, Timothy G; Cai, Hong; Gu, Jianying; Wang, Yufeng

    2015-07-15

    Annotating and understanding the function of proteins and other elements in a genome can be difficult in the absence of a well-studied and evolutionarily close relative. The causative agent of malaria, one of the oldest and most deadly global infectious diseases, is a good example of this problem. The burden of malaria is huge and there is a pressing need for new, more effective antimalarial strategies. However, techniques such as homology-dependent annotation transfer are severely impaired in this parasite because there are no well-understood close relatives. To circumvent this approach we developed a network-based method that uses a heavy path network-mining algorithm. We uncovered the protein-protein associations that are implicated in important cellular processes including genome integrity, DNA repair, transcriptional regulation, invasion, and pathogenesis, thus demonstrating the utility of this method. The URL of the source code for super-sequence mining method is http://www.cs.utsa.edu/~korkmaz/research/heavy-path-mining/.

  5. Huntingtin-Associated Protein 1 Interacts with Breakpoint Cluster Region Protein to Regulate Neuronal Differentiation

    PubMed Central

    Huang, Pai-Tsang; Chen, Chien-Ho; Hsu, I-Uen; Salim, Shaima’a Ahmad; Kao, Shu-Huei; Cheng, Chao-Wen; Lai, Chang-Hao; Lee, Cheng-Fan; Lin, Yung-Feng

    2015-01-01

    Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation. PMID:25671650

  6. Promyelocytic leukemia protein enhances apoptosis of gastric cancer cells through Yes-associated protein.

    PubMed

    Xu, Zhipeng; Chen, Jiamin; Shao, Liming; Ma, Wangqian; Xu, Dingting

    2015-09-01

    It has been shown that Yes-associated protein (YAP) acts as a transcriptional co-activator to regulate p73-dependent apoptosis in response to DNA damage in some cell types, and promyelocytic leukemia (PML) protein is involved in the regulation loop through stabilization of YAP through sumoylation. Although YAP has been shown to be significantly upregulated in gastric cancer, whether the YAP/PML/p73 regulation loop also functions in gastric cancer is unknown. Here, we show significantly higher levels of YAP and significantly lower levels of PML in the gastric cancer specimen. Overexpression of YAP in gastric cancer cells significantly increased cell growth, but did not affect apoptosis. However, overexpression of PML in gastric cancer cells significantly increased cell apoptosis, resulting in decreases in cell growth, which seemed to require the presence of YAP. The effect of PML on apoptosis appeared to be conducted through p73-mediated modulation of apoptosis-associated genes, Bcl-2, Bak, and caspase9. Thus, our study suggests the presence of a YAP/PML/p73 regulatory loop in gastric cancer, and highlights PML as a promising tumor suppressor in gastric cancer through YAP-coordinated cancer cell apoptosis.

  7. Revealing the potential pathogenesis of glioma by utilizing a glioma associated protein-protein interaction network.

    PubMed

    Pan, Weiran; Li, Gang; Yang, Xiaoxiao; Miao, Jinming

    2015-04-01

    This study aims to explore the potential mechanism of glioma through bioinformatic approaches. The gene expression profile (GSE4290) of glioma tumor and non-tumor samples was downloaded from Gene Expression Omnibus database. A total of 180 samples were available, including 23 non-tumor and 157 tumor samples. Then the raw data were preprocessed using robust multiarray analysis, and 8,890 differentially expressed genes (DEGs) were identified by using t-test (false discovery rate < 0.0005). Furthermore, 16 known glioma related genes were abstracted from Genetic Association Database. After mapping 8,890 DEGs and 16 known glioma related genes to Human Protein Reference Database, a glioma associated protein-protein interaction network (GAPN) was constructed. In addition, 51 sub-networks in GAPN were screened out through Molecular Complex Detection (score ≥ 1), and sub-network 1 was found to have the closest interaction (score = 3). What' more, for the top 10 sub-networks, Gene Ontology (GO) enrichment analysis (p value < 0.05) was performed, and DEGs involved in sub-network 1 and 2, such as BRMS1L and CCNA1, were predicted to regulate cell growth, cell cycle, and DNA replication via interacting with known glioma related genes. Finally, the overlaps of DEGs and human essential, housekeeping, tissue-specific genes were calculated (p value = 1.0, 1.0, and 0.00014, respectively) and visualized by Venn Diagram package in R. About 61% of human tissue-specific genes were DEGs as well. This research shed new light on the pathogenesis of glioma based on DEGs and GAPN, and our findings might provide potential targets for clinical glioma treatment.

  8. Establishing an osteosarcoma associated protein-protein interaction network to explore the pathogenesis of osteosarcoma

    PubMed Central

    2013-01-01

    Background The aim of this study was to establish an osteosarcoma (OS) associated protein-protein interaction network and explore the pathogenesis of osteosarcoma. Methods The gene expression profile GSE9508 was downloaded from the Gene Expression Omnibus database, including five samples of non-malignant bone (the control), seven samples for non-metastatic patients (six of which were analyzed in duplicate), and 11 samples for metastatic patients (10 of which were analyzed in duplicate). Differentially expressed genes (DEGs) between osteosarcoma and control samples were identified by packages in R with the threshold of |logFC (fold change)| > 1 and false discovery rate < 0.05. Osprey software was used to construct the interaction network of DEGs, and genes at protein-protein interaction (PPI) nodes with high degrees were identified. The Database for Annotation, Visualization and Integrated Discovery and WebGestalt software were then used to perform functional annotation and pathway enrichment analyses for PPI networks, in which P < 0.05 was considered statistically significant. Results Compared to the control samples, the expressions of 42 and 341 genes were altered in non-metastatic OS and metastatic OS samples, respectively. A total of 15 significantly enriched functions were obtained with Gene Ontology analysis (P < 0.05). The DEGs were classified and significantly enriched in three pathways, including the tricarboxylic acid cycle, lysosome and axon guidance. Genes such as HRAS, IDH3A, ATP6ap1, ATP6V0D2, SEMA3F and SEMA3A were involved in the enriched pathways. Conclusions The hub genes from metastatic OS samples are not only bio-markers of OS, but also help to improve therapies for OS. PMID:24330838

  9. Synaptosomal-associated protein 25 gene polymorphisms and antisocial personality disorder: association with temperament and psychopathy.

    PubMed

    Basoglu, Cengiz; Oner, Ozgur; Ates, Alpay; Algul, Ayhan; Bez, Yasin; Cetin, Mesut; Herken, Hasan; Erdal, Mehmet Emin; Munir, Kerim M

    2011-06-01

    The molecular genetic of personality disorders has been investigated in several studies; however, the association of antisocial behaviours with synaptosomal-associated protein 25 (SNAP25) gene polymorphisms has not. This association is of interest as SNAP25 gene polymorphism has been associated with attention-deficit hyperactivity disorder and personality. We compared the distribution of DdeI and MnII polymorphisms in 91 young male offenders and in 38 sex-matched healthy control subjects. We also investigated the association of SNAP25 gene polymorphisms with severity of psychopathy and with temperament traits: novelty seeking, harm avoidance, and reward dependence. The MnII T/T and DdeI T/T genotypes were more frequently present in male subjects with antisocial personality disorder (APD) than in sex-matched healthy control subjects. The association was stronger when the frequency of both DdeI and MnII T/T were taken into account. In the APD group, the genotype was not significantly associated with the Psychopathy Checklist-Revised scores, measuring the severity of psychopathy. However, the APD subjects with the MnII T/T genotype had higher novelty seeking scores; whereas, subjects with the DdeI T/T genotype had lower reward dependence scores. Again, the association between genotype and novelty seeking was stronger when both DdeI and MnII genotypes were taken into account. DdeI and MnII T/T genotypes may be a risk factor for antisocial behaviours. The association of the SNAP25 DdeI T/T and MnII T/T genotypes with lower reward dependence and higher novelty seeking suggested that SNAP25 genotype might influence other personality disorders, as well.

  10. Synaptosomal-Associated Protein 25 Gene Polymorphisms and Antisocial Personality Disorder: Association With Temperament and Psychopathy

    PubMed Central

    Basoglu, Cengiz; Oner, Ozgur; Ates, Alpay; Algul, Ayhan; Bez, Yasin; Cetin, Mesut; Herken, Hasan; Erdal, Mehmet Emin; Munir, Kerim M

    2011-01-01

    Objective The molecular genetic of personality disorders has been investigated in several studies; however, the association of antisocial behaviours with synaptosomal-associated protein 25 (SNAP25) gene polymorphisms has not. This association is of interest as SNAP25 gene polymorphism has been associated with attention-deficit hyperactivity disorder and personality. Methods We compared the distribution of DdeI and MnlI polymorphisms in 91 young male offenders and in 38 sex-matched healthy control subjects. We also investigated the association of SNAP25 gene polymorphisms with severity of psychopathy and with temperament traits: novelty seeking, harm avoidance, and reward dependence. Results The MnlI T/T and DdeI T/T genotypes were more frequently present in male subjects with antisocial personality disorder (APD) than in sex-matched healthy control subjects. The association was stronger when the frequency of both DdeI and MnlI T/T were taken into account. In the APD group, the genotype was not significantly associated with the Psychopathy Checklist–Revised scores, measuring the severity of psychopathy. However, the APD subjects with the MnlI T/T genotype had higher novelty seeking scores; whereas, subjects with the DdeI T/T genotype had lower reward dependence scores. Again, the association between genotype and novelty seeking was stronger when both DdeI and MnlI genotypes were taken into account. Conclusion DdeI and MnlI T/T genotypes may be a risk factor for antisocial behaviours. The association of the SNAP25 DdeI T/T and MnlI T/T genotypes with lower reward dependence and higher novelty seeking suggested that SNAP25 genotype might influence other personality disorders, as well. PMID:21756448

  11. Locating overlapping dense subgraphs in gene (protein) association networks and predicting novel protein functional groups among these subgraphs

    NASA Astrophysics Data System (ADS)

    Palla, Gergely; Derenyi, Imre; Farkas, Illes J.; Vicsek, Tamas

    2006-03-01

    Most tasks in a cell are performed not by individual proteins, but by functional groups of proteins (either physically interacting with each other or associated in other ways). In gene (protein) association networks these groups show up as sets of densely connected nodes. In the yeast, Saccharomyces cerevisiae, known physically interacting groups of proteins (called protein complexes) strongly overlap: the total number of proteins contained by these complexes by far underestimates the sum of their sizes (2750 vs. 8932). Thus, most functional groups of proteins, both physically interacting and other, are likely to share many of their members with other groups. However, current algorithms searching for dense groups of nodes in networks usually exclude overlaps. With the aim to discover both novel functions of individual proteins and novel protein functional groups we combine in protein association networks (i) a search for overlapping dense subgraphs based on the Clique Percolation Method (CPM) (Palla, G., et.al. Nature 435, 814-818 (2005), http://angel.elte.hu/clustering), which explicitly allows for overlaps among the groups, and (ii) a verification and characterization of the identified groups of nodes (proteins) with the help of standard annotation databases listing known functions.

  12. Staphylococcus saprophyticus surface-associated protein (Ssp) is associated with lifespan reduction in Caenorhabditis elegans.

    PubMed

    Szabados, Florian; Mohner, Amelie; Kleine, Britta; Gatermann, Sören G

    2013-10-01

    Staphylococcal lipases have been proposed as pathogenicity factors. In Staphylococcus saprophyticus the surface-associated protein (Ssp) has been previously characterized as a cell wall-associated true lipase. A S. saprophyticus Δssp::ermB mutant has been described as less virulent in an in vivo model of urinary tract infection compared with its wild-type. This is the first report showing that S. saprophyticus induced a lifespan reduction in Caenorhabditis elegans similar to that of S. aureus RN4220. In two S. saprophyticus Δssp::ermB mutants lifespan reduction in C. elegans was partly abolished. In order to attribute virulence to the lipase activity itself and distinguish this phenomenon from the presence of the Ssp-protein, the conserved active site of the lipase was modified by site-directed ligase-independent mutagenesis and lipase activity-deficient mutants were constructed. These results indicate that the Ssp is associated with pathogenicity in C. elegans and one could speculate that the lipase activity itself is responsible for this virulence.

  13. Therapeutic intervention based on protein prenylation and associated modifications

    PubMed Central

    Gelb, Michael H; Brunsveld, Lucas; Hrycyna, Christine A; Michaelis, Susan; Tamanoi, Fuyuhiko; Van Voorhis, Wesley C; Waldmann, Herbert

    2010-01-01

    In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome. PMID:16983387

  14. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases.

  15. Cellular proteins that associate with the middle and small T antigens of polyomavirus.

    PubMed

    Pallas, D C; Cherington, V; Morgan, W; DeAnda, J; Kaplan, D; Schaffhausen, B; Roberts, T M

    1988-11-01

    We have used two-dimensional gel electrophoresis to analyze in more detail the cellular proteins which associate with the middle and small tumor antigens (MT and ST, respectively) of polyomavirus. Proteins with molecular masses of 27, 29, 36, 51, 61, 63, and 85 kilodaltons (kDa) that specifically coimmunoprecipitated with MT were identified on these gels. The 36-, 51-, 61-, 63-, and 85-kDa proteins are probably the same as the proteins of similar sizes previously reported by a number of groups, whereas the 27- and 29-kDa proteins represent proteins that are heretofore undescribed. The 27- and 29-kDa proteins were abundant cellular proteins, whereas the others were minor cellular constituents. The association of each of these proteins with MT was sensitive to one or more mutations in MT that rendered it transformation defective. The association of the 85-kDa protein was the most sensitive indicator of the transformation competence of MT mutants. In addition, the 85-kDa protein was the only associated protein whose association with MT changed consistently in parallel with MT-associated phosphatidylinositol kinase activity. Furthermore, the fraction of the 85-kDa protein which was found associated with the MT complex contained 15 to 20% of its phosphate content on tyrosine. The 36- and 63-kDa proteins complexed with both polyomavirus MT and ST and comigrated on two-dimensional gels with two simian virus 40 ST-associated proteins originally described by Rundell and coworkers (K. Rundell, E. O. Major, and M. Lampert, J. Virol. 37:1090-1093, 1981). None of the other MT-associated proteins associated significantly with ST.

  16. Uncoupling protein 2 gene polymorphisms are associated with obesity

    PubMed Central

    2012-01-01

    Background Uncoupling protein 2 (UCP2) gene polymorphisms have been reported as genetic risk factors for obesity and type 2 diabetes mellitus (T2DM). We examined the association of commonly observed UCP2 G(−866)A (rs659366) and Ala55Val (C > T) (rs660339) single nucleotide polymorphisms (SNPs) with obesity, high fasting plasma glucose, and serum lipids in a Balinese population. Methods A total of 603 participants (278 urban and 325 rural subjects) were recruited from Bali Island, Indonesia. Fasting plasma glucose (FPG), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) were measured. Obesity was determined based on WHO classifications for adult Asians. Participants were genotyped for G(−866)A and Ala55Val polymorphisms of the UCP2 gene. Results Obesity prevalence was higher in urban subjects (51%) as compared to rural subjects (23%). The genotype, minor allele (MAF), and heterozygosity frequencies were similar between urban and rural subjects for both SNPs. All genotype frequencies were in Hardy-Weinberg equilibrium. A combined analysis of genotypes and environment revealed that the urban subjects carrying the A/A genotype of the G(−866)A SNP have higher BMI than the rural subjects with the same genotype. Since the two SNPs showed strong linkage disequilibrium (D’ = 0.946, r2 = 0.657), a haplotype analysis was performed. We found that the AT haplotype was associated with high BMI only when the urban environment was taken into account. Conclusions We have demonstrated the importance of environmental settings in studying the influence of the common UCP2 gene polymorphisms in the development of obesity in a Balinese population. PMID:22533685

  17. Fontan-associated protein-losing enteropathy and plastic bronchitis.

    PubMed

    Schumacher, Kurt R; Stringer, Kathleen A; Donohue, Janet E; Yu, Sunkyung; Shaver, Ashley; Caruthers, Regine L; Zikmund-Fisher, Brian J; Fifer, Carlen; Goldberg, Caren; Russell, Mark W

    2015-04-01

    To characterize the medical history, disease progression, and treatment of current-era patients with the rare diseases Fontan-associated protein-losing enteropathy (PLE) and plastic bronchitis. A novel survey that queried demographics, medical details, and treatment information was piloted and placed online via a Facebook portal, allowing social media to power the study. Participation regardless of PLE or plastic bronchitis diagnosis was allowed. Case control analyses compared patients with PLE and plastic bronchitis with uncomplicated control patients receiving the Fontan procedure. The survey was completed by 671 subjects, including 76 with PLE, 46 with plastic bronchitis, and 7 with both. Median PLE diagnosis was 2.5 years post-Fontan. Hospitalization for PLE occurred in 71% with 41% hospitalized ≥ 3 times. Therapy varied significantly. Patients with PLE more commonly had hypoplastic left ventricle (62% vs 44% control; OR 2.81, 95% CI 1.43-5.53), chylothorax (66% vs 41%; OR 2.96, CI 1.65-5.31), and cardiothoracic surgery in addition to staged palliation (17% vs 5%; OR 4.27, CI 1.63-11.20). Median plastic bronchitis diagnosis was 2 years post-Fontan. Hospitalization for plastic bronchitis occurred in 91% with 61% hospitalized ≥ 3 times. Therapy was very diverse. Patients with plastic bronchitis more commonly had chylothorax at any surgery (72% vs 51%; OR 2.47, CI 1.20-5.08) and seasonal allergies (52% vs 36%; OR 1.98, CI 1.01-3.89). Patient-specific factors are associated with diagnoses of PLE or plastic bronchitis. Treatment strategies are diverse without clear patterns. These results provide a foundation upon which to design future therapeutic studies and identify a clear need for forming consensus approaches to treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Mapping of domains on HIV envelope protein mediating association with calnexin and protein-disulfide isomerase.

    PubMed

    Papandréou, Marie-Jeanne; Barbouche, Rym; Guieu, Régis; Rivera, Santiago; Fantini, Jacques; Khrestchatisky, Michel; Jones, Ian M; Fenouillet, Emmanuel

    2010-04-30

    The cell catalysts calnexin (CNX) and protein-disulfide isomerase (PDI) cooperate in establishing the disulfide bonding of the HIV envelope (Env) glycoprotein. Following HIV binding to lymphocytes, cell-surface PDI also reduces Env to induce the fusogenic conformation. We sought to define the contact points between Env and these catalysts to illustrate their potential as therapeutic targets. In lysates of Env-expressing cells, 15% of the gp160 precursor, but not gp120, coprecipitated with CNX, whereas only 0.25% of gp160 and gp120 coprecipitated with PDI. Under in vitro conditions, which mimic the Env/PDI interaction during virus/cell contact, PDI readily associated with Env. The domains of Env interacting in cellulo with CNX or in vitro with PDI were then determined using anti-Env antibodies whose binding site was occluded by CNX or PDI. Antibodies against domains V1/V2, C2, and the C terminus of V3 did not bind CNX-associated Env, whereas those against C1, V1/V2, and the CD4-binding domain did not react with PDI-associated Env. In addition, a mixture of the latter antibodies interfered with PDI-mediated Env reduction. Thus, Env interacts with intracellular CNX and extracellular PDI via discrete, largely nonoverlapping, regions. The sites of interaction explain the mode of action of compounds that target these two catalysts and may enable the design of further new competitive agents.

  19. Complete protein-protein association kinetics in atomic detail revealed by molecular dynamics simulations and Markov modelling

    NASA Astrophysics Data System (ADS)

    Plattner, Nuria; Doerr, Stefan; de Fabritiis, Gianni; Noé, Frank

    2017-10-01

    Protein-protein association is fundamental to many life processes. However, a microscopic model describing the structures and kinetics during association and dissociation is lacking on account of the long lifetimes of associated states, which have prevented efficient sampling by direct molecular dynamics (MD) simulations. Here we demonstrate protein-protein association and dissociation in atomistic resolution for the ribonuclease barnase and its inhibitor barstar by combining adaptive high-throughput MD simulations and hidden Markov modelling. The model reveals experimentally consistent intermediate structures, energetics and kinetics on timescales from microseconds to hours. A variety of flexibly attached intermediates and misbound states funnel down to a transition state and a native basin consisting of the loosely bound near-native state and the tightly bound crystallographic state. These results offer a deeper level of insight into macromolecular recognition and our approach opens the door for understanding and manipulating a wide range of macromolecular association processes.

  20. Lipid droplet-associated proteins in atherosclerosis (Review)

    PubMed Central

    AYYAPPAN, JANEESH PLAKKAL; PAUL, ANTONI; GOO, YOUNG-HWA

    2016-01-01

    Accumulation of atherosclerotic plaques in arterial walls leads to major cardiovascular diseases and stroke. Macrophages/foam cells are central components of atherosclerotic plaques, which populate the arterial wall in order to remove harmful modified low-density lipoprotein (LDL) particles, resulting in the accumulation of lipids, mostly LDL-derived cholesterol ester, in cytosolic lipid droplets (LDs). At present, LDs are recognized as dynamic organelles that govern cellular metabolic processes. LDs consist of an inner core of neutral lipids surrounded by a monolayer of phospholipids and free cholesterol, and contain LD-associated proteins (LDAPs) that regulate LD functions. Foam cells are characterized by an aberrant accumulation of cytosolic LDs, and are considered a hallmark of atherosclerotic lesions through all stages of development. Previous studies have investigated the mechanisms underlying foam cell formation, aiming to discover therapeutic strategies that target foam cells and intervene against atherosclerosis. It is well established that LDAPs have a major role in the pathogenesis of metabolic diseases caused by dysfunction of lipid metabolism, and several studies have linked LDAPs to the development of atherosclerosis. In this review, several foam cell-targeting pathways have been described, with an emphasis on the role of LDAPs in cholesterol mobilization from macrophages. In addition, the potential of LDAPs as therapeutic targets to prevent the progression and/or facilitate the regression of the disease has been discussed. PMID:27082419

  1. Lipid droplet-associated proteins in atherosclerosis (Review).

    PubMed

    Plakkal Ayyappan, Janeesh; Paul, Antoni; Goo, Young-Hwa

    2016-06-01

    Accumulation of atherosclerotic plaques in arterial walls leads to major cardiovascular diseases and stroke. Macrophages/foam cells are central components of atherosclerotic plaques, which populate the arterial wall in order to remove harmful modified low‑density lipoprotein (LDL) particles, resulting in the accumulation of lipids, mostly LDL‑derived cholesterol ester, in cytosolic lipid droplets (LDs). At present, LDs are recognized as dynamic organelles that govern cellular metabolic processes. LDs consist of an inner core of neutral lipids surrounded by a monolayer of phospholipids and free cholesterol, and contain LD‑associated proteins (LDAPs) that regulate LD functions. Foam cells are characterized by an aberrant accumulation of cytosolic LDs, and are considered a hallmark of atherosclerotic lesions through all stages of development. Previous studies have investigated the mechanisms underlying foam cell formation, aiming to discover therapeutic strategies that target foam cells and intervene against atherosclerosis. It is well established that LDAPs have a major role in the pathogenesis of metabolic diseases caused by dysfunction of lipid metabolism, and several studies have linked LDAPs to the development of atherosclerosis. In this review, several foam cell‑targeting pathways have been described, with an emphasis on the role of LDAPs in cholesterol mobilization from macrophages. In addition, the potential of LDAPs as therapeutic targets to prevent the progression and/or facilitate the regression of the disease has been discussed.

  2. Death Associated Protein Kinases: Molecular Structure and Brain Injury

    PubMed Central

    Nair, Syam; Hagberg, Henrik; Krishnamurthy, Rajanikant; Thornton, Claire; Mallard, Carina

    2013-01-01

    Perinatal brain damage underlies an important share of motor and neurodevelopmental disabilities, such as cerebral palsy, cognitive impairment, visual dysfunction and epilepsy. Clinical, epidemiological, and experimental studies have revealed that factors such as inflammation, excitotoxicity and oxidative stress contribute considerably to both white and grey matter injury in the immature brain. A member of the death associated protein kinase (DAPk) family, DAPk1, has been implicated in cerebral ischemic damage, whereby DAPk1 potentiates NMDA receptor-mediated excitotoxicity through interaction with the NR2BR subunit. DAPk1 also mediate a range of activities from autophagy, membrane blebbing and DNA fragmentation ultimately leading to cell death. DAPk mRNA levels are particularly highly expressed in the developing brain and thus, we hypothesize that DAPk1 may play a role in perinatal brain injury. In addition to reviewing current knowledge, we present new aspects of the molecular structure of DAPk domains, and relate these findings to interacting partners of DAPk1, DAPk-regulation in NMDA-induced cerebral injury and novel approaches to blocking the injurious effects of DAPk1. PMID:23880846

  3. Death associated protein kinases: molecular structure and brain injury.

    PubMed

    Nair, Syam; Hagberg, Henrik; Krishnamurthy, Rajanikant; Thornton, Claire; Mallard, Carina

    2013-07-04

    Perinatal brain damage underlies an important share of motor and neurodevelopmental disabilities, such as cerebral palsy, cognitive impairment, visual dysfunction and epilepsy. Clinical, epidemiological, and experimental studies have revealed that factors such as inflammation, excitotoxicity and oxidative stress contribute considerably to both white and grey matter injury in the immature brain. A member of the death associated protein kinase (DAPk) family, DAPk1, has been implicated in cerebral ischemic damage, whereby DAPk1 potentiates NMDA receptor-mediated excitotoxicity through interaction with the NR2BR subunit. DAPk1 also mediate a range of activities from autophagy, membrane blebbing and DNA fragmentation ultimately leading to cell death. DAPk mRNA levels are particularly highly expressed in the developing brain and thus, we hypothesize that DAPk1 may play a role in perinatal brain injury. In addition to reviewing current knowledge, we present new aspects of the molecular structure of DAPk domains, and relate these findings to interacting partners of DAPk1, DAPk-regulation in NMDA-induced cerebral injury and novel approaches to blocking the injurious effects of DAPk1.

  4. JNK phosphorylates Yes-associated protein (YAP) to regulate apoptosis

    PubMed Central

    Tomlinson, V; Gudmundsdottir, K; Luong, P; Leung, K-Y; Knebel, A; Basu, S

    2010-01-01

    Yes-associated protein (YAP) regulates DNA damage and chemosensitivity, as well as functioning as a pro-growth, cell size regulator. For both of its roles, regulation by phosphorylation is crucial. We undertook an in vitro screen to identify novel YAP kinases to discover new signaling pathways to better understand YAP's function. We identified JNK1 and JNK2 as robust YAP kinases, as well as mapped multiple sites of phosphorylation. Using inhibitors and siRNA, we showed that JNK specifically phosphorylates endogenous YAP in a number of cell types. We show that YAP protects keratinocytes from UV irradiation but promotes UV-induced apoptosis in a squamous cell carcinoma. We defined the mechanism for this dual role to be YAP's ability to bind and stabilize the pro-proliferative ΔNp63α isoform in a JNK-dependent manner. Our report indicates that an evaluation of the expression of the different isoforms of p63 and p73 is crucial in determining YAP's function. PMID:21364637

  5. Protein Mediated Oxidative Stress in Patients with Diabetes and its Associated Neuropathy: Correlation with Protein Carbonylation and Disease Activity Markers

    PubMed Central

    Almogbel, Ebtehal

    2017-01-01

    Introduction Free radicals have been implicated as Diabetes Mellitus (DM) contributors in type 2 DM and its associated Diabetes Mellitus Neuropathy (DMN). However, the potential for protein mediated oxidative stress to contribute disease pathogenesis remains largely unexplored. Aim To investigate the status and contribution of protein mediated oxidative stress in patients with DM or DMN and to explore whether oxidative protein modification has a role in DM progression to DM associated neuropathy. Materials and Methods Sera from 42 DM and 37 DMN patients with varying levels of disease activities biomarkers (HbA1C, patients’ age or disease duration) and 21 age- and sex-matched healthy controls were evaluated for serum levels of protein mediated oxidative stress. Results Serum analysis showed significantly higher levels of protein carbonyl contents in both DM and DMN patients compared with healthy controls. Importantly, not only was there an increased number of subjects positive for protein carbonylation, but also the levels of protein carbonyl contents were significantly higher among DM and DMN patients, whose HbA1C were ≥8.8 as compared with patients with lower HbA1C (HbA1C<8.8). Similar pattern of protein carbonyls formation was also observed with patients’ ages or with patient’s disease durations, suggesting a possible relationship between protein oxidation and disease progression. Furthermore, sera from DMN patients had higher levels of protein carbonylation compared with non-neuropathic DM patients’ sera, suggesting an involvement of protein oxidation in the progression of diabetes to diabetes neuropathy. Conclusion These findings support an association between protein oxidation and DM or DMN progression. The stronger response observed in patients with higher HbA1C or patients’ ages or disease durations suggests, that protein mediated oxidative stress may be useful in evaluating the progression of DM and its associated DMN and in elucidating the

  6. Fusions involving protein kinase C and membrane-associated proteins in benign fibrous histiocytoma.

    PubMed

    Płaszczyca, Anna; Nilsson, Jenny; Magnusson, Linda; Brosjö, Otte; Larsson, Olle; Vult von Steyern, Fredrik; Domanski, Henryk A; Lilljebjörn, Henrik; Fioretos, Thoas; Tayebwa, Johnbosco; Mandahl, Nils; Nord, Karolin H; Mertens, Fredrik

    2014-08-01

    Benign fibrous histiocytoma (BFH) is a mesenchymal tumor that most often occurs in the skin (so-called dermatofibroma), but may also appear in soft tissues (so-called deep BFH) and in the skeleton (so-called non-ossifying fibroma). The origin of BFH is unknown, and it has been questioned whether it is a true neoplasm. Chromosome banding, fluorescence in situ hybridization, single nucleotide polymorphism arrays, RNA sequencing, RT-PCR and quantitative real-time PCR were used to search for recurrent somatic mutations in a series of BFH. BFHs were found to harbor recurrent fusions of genes encoding membrane-associated proteins (podoplanin, CD63 and LAMTOR1) with genes encoding protein kinase C (PKC) isoforms PRKCB and PRKCD. PKCs are serine-threonine kinases that through their many phosphorylation targets are implicated in a variety of cellular processes, as well as tumor development. When inactive, the amino-terminal, regulatory domain of PKCs suppresses the activity of their catalytic domain. Upon activation, which requires several steps, they typically translocate to cell membranes, where they interact with different signaling pathways. The detected PDPN-PRKCB, CD63-PRKCD and LAMTOR1-PRKCD gene fusions are all predicted to result in chimeric proteins consisting of the membrane-binding part of PDPN, CD63 or LAMTOR1 and the entire catalytic domain of the PKC. This novel pathogenetic mechanism should result in constitutive kinase activity at an ectopic location. The results show that BFH indeed is a true neoplasm, and that distorted PKC activity is essential for tumorigenesis. The findings also provide means to differentiate BFH from other skin and soft tissue tumors. This article is part of a Directed Issue entitled: Rare cancers.

  7. CAP2, cyclase-associated protein 2, is a dual compartment protein.

    PubMed

    Peche, V; Shekar, S; Leichter, M; Korte, H; Schröder, R; Schleicher, M; Holak, T A; Clemen, C S; Ramanath-Y, B; Pfitzer, G; Karakesisoglou, I; Noegel, A A

    2007-10-01

    Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins with roles in regulating the actin cytoskeleton and in signal transduction. Mammals have two CAP genes encoding the related CAP1 and CAP2. We studied the distribution and subcellular localization of CAP1 and CAP2 using specific antibodies. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. CAP2 is found in the nucleus in undifferentiated myoblasts and at the M-line of differentiated myotubes. In PAM212, a mouse keratinocyte cell line, CAP2 is enriched in the nucleus, and sparse in the cytosol. By contrast, CAP1 localizes to the cytoplasm in PAM212 cells. In human skin, CAP2 is present in all living layers of the epidermis localizing to the nuclei and the cell periphery. In in vitro studies, a C-terminal fragment of CAP2 interacts with actin, indicating that CAP2 has the capacity to bind to actin.

  8. Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions.

    PubMed

    Weiss, Jamie L; Evans, Nicholas A; Ahmed, Tanweer; Wrigley, Jonathan D J; Khan, Shukria; Wright, Charles; Keen, Jeffrey N; Holzenburg, Andreas; Findlay, John B C

    2005-03-01

    We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

  9. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  10. Association between serum pregnancy-associated plasma protein-A and bicarbonate in hemodialysis patients.

    PubMed

    Bicik, Zerrin; Coskun, Abdurrahman; Serteser, Mustafa; Bulur, Atilla; Mese, Meral; Unsal, Ibrahim

    2014-03-01

    Acidosis is associated with protein-energy malnutrition, inflammation, and bone disease, and low bicarbonate levels have been implicated in higher mortality rates in chronic kidney disease. Recently, the concentration of serum pregnancy-associated plasma protein-A (PAPP-A) has become accepted as a prognostic marker in hemodialysis patients. This study determined the relationship between PAPP-A and bicarbonate levels in these patients. The study enrolled 65 hemodialysis patients (41 males, 24 females) and 26 control subjects (11 males, 15 females). Serum PAPP-A, intact parathormone (iPTH), calcium, phosphorus (P), and bicarbonate levels were measured. Correlations between PAPP-A and bicarbonate, iPTH, calcium, and phosphorus were evaluated. Median PAPP-A levels were significantly higher in hemodialysis patients [15.1 (<0.03-158.8) ng/ml] than in control subjects [6.6 (<0.03-16.4) ng/ml] (P < 0.05). There were statistically significant correlations between serum PAPP-A and bicarbonate, iPTH, and P in hemodialysis patients but not in control subjects. Elevation of serum PAPP-A has been found in hemodialysis patients and its significant correlation with bicarbonate suggests that it may be a prognostic factor. © 2014 Wiley Periodicals, Inc.

  11. Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Tanase, Maya; Urbanska, Aleksandra M.; Zolla, Valerio; Clement, Cristina C.; Huang, Liling; Morozova, Kateryna; Follo, Carlo; Goldberg, Michael; Roda, Barbara; Reschiglian, Pierluigi; Santambrogio, Laura

    2016-01-01

    Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis.

  12. Novel intracellular proteins associated with cellular vitamin D action.

    PubMed

    Angelo, Giana; Wood, Richard J; Mayer, Jean

    2002-07-01

    Work with vitamin D-resistant New World primates has revealed novel cellular proteins involved in vitamin D action. An "intracellular vitamin D-binding protein" functions to bind vitamin D metabolites in the cell and enhances vitamin D action. By contrast, a "vitamin D response element-binding protein" inhibits vitamin D receptor binding to the DNA and is responsible for vitamin D resistance in New World primates.

  13. HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

    PubMed Central

    2016-01-01

    SUMMARY The HIV genome encodes a small number of viral proteins (i.e., 16), invariably establishing cooperative associations among HIV proteins and between HIV and host proteins, to invade host cells and hijack their internal machineries. As a known example, the HIV envelope glycoprotein GP120 is closely associated with GP41 for viral entry. From a genome-wide perspective, a hypothesis can be worked out to determine whether 16 HIV proteins could develop 120 possible pairwise associations either by physical interactions or by functional associations mediated via HIV or host molecules. Here, we present the first systematic review of experimental evidence on HIV genome-wide protein associations using a large body of publications accumulated over the past 3 decades. Of 120 possible pairwise associations between 16 HIV proteins, at least 34 physical interactions and 17 functional associations have been identified. To achieve efficient viral replication and infection, HIV protein associations play essential roles (e.g., cleavage, inhibition, and activation) during the HIV life cycle. In either a dispensable or an indispensable manner, each HIV protein collaborates with another viral protein to accomplish specific activities that precisely take place at the proper stages of the HIV life cycle. In addition, HIV genome-wide protein associations have an impact on anti-HIV inhibitors due to the extensive cross talk between drug-inhibited proteins and other HIV proteins. Overall, this study presents for the first time a comprehensive overview of HIV genome-wide protein associations, highlighting meticulous collaborations between all viral proteins during the HIV life cycle. PMID:27357278

  14. HIV Genome-Wide Protein Associations: a Review of 30 Years of Research.

    PubMed

    Li, Guangdi; De Clercq, Erik

    2016-09-01

    The HIV genome encodes a small number of viral proteins (i.e., 16), invariably establishing cooperative associations among HIV proteins and between HIV and host proteins, to invade host cells and hijack their internal machineries. As a known example, the HIV envelope glycoprotein GP120 is closely associated with GP41 for viral entry. From a genome-wide perspective, a hypothesis can be worked out to determine whether 16 HIV proteins could develop 120 possible pairwise associations either by physical interactions or by functional associations mediated via HIV or host molecules. Here, we present the first systematic review of experimental evidence on HIV genome-wide protein associations using a large body of publications accumulated over the past 3 decades. Of 120 possible pairwise associations between 16 HIV proteins, at least 34 physical interactions and 17 functional associations have been identified. To achieve efficient viral replication and infection, HIV protein associations play essential roles (e.g., cleavage, inhibition, and activation) during the HIV life cycle. In either a dispensable or an indispensable manner, each HIV protein collaborates with another viral protein to accomplish specific activities that precisely take place at the proper stages of the HIV life cycle. In addition, HIV genome-wide protein associations have an impact on anti-HIV inhibitors due to the extensive cross talk between drug-inhibited proteins and other HIV proteins. Overall, this study presents for the first time a comprehensive overview of HIV genome-wide protein associations, highlighting meticulous collaborations between all viral proteins during the HIV life cycle.

  15. Protein Z variants associated with protein Z plasma levels and with risk of idiopathic recurrent miscarriage.

    PubMed

    Al-Shaikh, Fatima S; Sater, Mai S; Finan, Ramzi R; Racoubian, Eddie; Abu-Hijleh, Tala M; Mustafa, Fekria E; Almawi, Wassim Y

    2013-09-01

    Protein Z (PZ) deficiency due to anti-PZ autoantibodies and/or mutations in PZgene was linked with adverse pregnancy outcomes, including idiopathic recurrent miscarriage (IRM). We investigated the association of rs3024718, rs3024719, rs3024731, rs3024778, rs3024772, and rs3024735 (G79A) PZ variants and changes in PZ levels in 287 women with IRM, and 308 control women. Of the 6 single nucleotide polymorphisms (SNPs) analyzed, higher minor allele frequency of rs3024735 (G79A) and rs3024731 were seen in IRM cases than in control women. Significantly higher frequencies of rs3024735/G79A G/A and A/A (P< .001), rs3024719 G/A (P= .009), and rs3024731 A/A (P = .012), but not rs3024718 (P= .12), rs3024778 (P = .76), or rs3024772 (P= .27) genotype carriers were seen between IRM cases versus control women, respectively, and was linked with reduced PZ levels. Six-locus (rs3024718/rs3024719/rs3024778/rs3024731/rs3024735/rs3024772) PZhaplotypes analysis demonstrated increased frequency of GAGAAG and AGGTAG and reduced frequency of AGGTGC haplotypes in IRM cases, thereby conferring disease susceptibility and protective nature to these haplotypes, respectively. These results demonstrate that specific PZSNPs and haplotypes are significantly associated with IRM.

  16. Proteomics analysis revealed changes in rat bronchoalveolar lavage fluid proteins associated with oil mist exposure.

    PubMed

    Lee, Yung-Shan; Chen, Pang-Wei; Tsai, Perng-Jy; Su, Shu-Hui; Liao, Pao-Chi

    2006-04-01

    Exposure to oil mist has been associated with a variety of acute and chronic respiratory effects. Using proteomics approaches to investigate exposure-associated proteins may provide useful information to understand the mechanisms of associated respiratory effects. The aim of this study was to investigate changes in rat bronchoalveolar lavage fluid proteins associated with oil mist exposure using nano-HPLC-ESI-MS/MS. The results revealed that 29 proteins exhibited significant changes after exposure. These proteins included surfactant-associated proteins (SP-A and SP-D), inflammatory proteins (complement component 3, immunoglobulins, lysozyme, etc.), growth factors (e.g., transforming growth factor alpha (TGF-alpha)), calcium-binding proteins (calcyclin, calgranulin A, calreticulin, and calvasculin), and other proteins (e.g., cathepsin D, saposin, and intestinal trefoil factor). To further evaluate changes in protein levels, a simple quantitative strategy was developed in this study. A large decrease in protein levels of SP-A and SP-D (0.24- and 0.38-fold, respectively) following exposure was observed. In contrast, protein levels of TGF-alpha and calcium-binding proteins were significantly increased (4.46- and 1.4-1.8-fold, respectively). Due to the diverse functions of these proteins, the results might contribute to understand the mechanisms involved in lung disorders induced by oil mist exposure.

  17. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein.

    PubMed

    Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

    2009-11-30

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.

  18. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

    PubMed Central

    Hong, Kyung Uk; Kim, Hyun-Jun

    2009-01-01

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions. PMID:19641375

  19. Cerebral infarction associated with protein C deficiency following allogeneic bone marrow transplantation.

    PubMed

    Gordon, B G; Saving, K L; McCallister, J A; Warkentin, P I; McConnell, J R; Roberts, W M; Coccia, P F; Haire, W D

    1991-10-01

    Hypercoagulable states associated with deficiencies in circulating anticoagulant protein C occur after chemotherapy for a variety of malignant diseases. Protein C deficiency also occurs following bone marrow transplantation (BMT) and may be responsible for a variety of transplantation-associated complications. We report the case of a child who suffered a stroke associated with low protein C antigen and activity occurring 11 months after allogeneic BMT. Protein C levels recovered spontaneously by 18 months after BMT. We speculate that the protein C deficiency and and resultant hypercoagulable state led to the stroke, and the deficiency of this anticoagulant was a sequela of the transplant.

  20. Third-Party Bioluminescence Resonance Energy Transfer Indicates Constitutive Association of Membrane Proteins: Application to Class A G-Protein-Coupled Receptors and G-Proteins

    PubMed Central

    Kuravi, Sudhakiranmayi; Lan, Tien-Hung; Barik, Arnab; Lambert, Nevin A.

    2010-01-01

    Abstract Many of the molecules that mediate G-protein signaling are thought to constitutively associate with each other in variably stable signaling complexes. Much of the evidence for signaling complexes has come from Förster resonance energy transfer and bioluminescence resonance energy transfer (BRET) studies. However, detection of constitutive protein association with these methods is hampered by nonspecific energy transfer that occurs when donor and acceptor molecules are in close proximity by chance. We show that chemically-induced recruitment of local third-party BRET donors or acceptors reliably separates nonspecific and specific BRET. We use this method to reexamine the constitutive association of class A G-protein-coupled receptors (GPCRs) with other GPCRs and with heterotrimeric G-proteins. We find that β2 adrenoreceptors constitutively associate with each other and with several other class A GPCRs. In contrast, GPCRs and G-proteins are unlikely to exist in stable constitutive preassembled complexes. PMID:20483349

  1. Evidence for direct association of Vpr and matrix protein p17 within the HIV-1 virion.

    PubMed

    Sato, A; Yoshimoto, J; Isaka, Y; Miki, S; Suyama, A; Adachi, A; Hayami, M; Fujiwara, T; Yoshie, O

    1996-06-01

    Vpr is one of the auxiliary proteins of HIV-1 and is selectively incorporated into the virion by a process involving the C-terminal p6 portion of the Gag precursor Pr55. Vpr and the matrix protein p17 are the components of the viral preintegration complex and appear to play important roles in the nuclear transport of proviral DNA in nondividing cells. In the present study, we have demonstrated by coimmunoprecipitation experiments that Vpr associates with matrix protein p17 but not with capsid protein p24 within the HIV-1 virion. Experiments employing the yeast two-hybrid GAL4 assay for protein-protein interactions also demonstrated a direct association between Vpr and the C-terminal region of matrix protein p17. Association of Vpr and the matrix protein p17 within the mature virion is consistent with their collaborative role in the nuclear transportation of the viral preintegration complex in nondividing cells such as macrophages.

  2. Proteomic analysis and identification of cell surface-associated proteins of Clostridium chauvoei.

    PubMed

    Jayaramaiah, Usharani; Singh, Neetu; Thankappan, Sabarinath; Mohanty, Ashok Kumar; Chaudhuri, Pallab; Singh, Vijendra Pal; Nagaleekar, Viswas Konasagara

    2016-06-01

    Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates.

  3. Roles of the Polymerase-Associated Protein Genes in Newcastle Disease Virus Virulence

    PubMed Central

    Yu, Xiao-hui; Cheng, Jin-long; Xue, Jia; Jin, Ji-hui; Song, Yang; Zhao, Jing; Zhang, Guo-zhong

    2017-01-01

    The virulence of Newcastle disease virus varies greatly and is determined by multiple genetic factors. In this study, we systematically evaluated the roles of the polymerase-associated (NP, P and L) protein genes in genotype VII NDV virulence after confirming the envelope-associated (F and HN) proteins contributed greatly to NDV virulence. The results revealed that the polymerase-associated protein genes individually had certain effect on virulence, while transfer of these three genes in combination significantly affected the chimeric virus virulence, especially when the L gene was involved. These results indicated that the L protein was a major contributor to NDV virulence when combined with the homologous NP and P proteins. We also investigated viral RNA synthesis using NDV minigenome systems to assess the interaction between the NP, P, and L proteins, which showed that the activity of the polymerase-associated proteins were directly related to viral RNA transcription and replication. PMID:28220114

  4. Overexpression of adenylate cyclase-associated protein 1 is associated with metastasis of lung cancer.

    PubMed

    Tan, Min; Song, Xiaolian; Zhang, Guoliang; Peng, Aimei; Li, Xuan; Li, Ming; Liu, Yang; Wang, Changhui

    2013-10-01

    Lung cancer ranks first in both prevalence and mortality rates among all types of cancer. Metastasis is the main cause of treatment failure. Biomarkers are critical to early diagnosis and prediction and monitoring of progressive lesions. Several biomarkers have been identified for lung cancer but none have been routinely used clinically. The present study assessed the diagnostic and prognostic value of cyclase-associated protein 1 (CAP1) for lung cancer. CAP1 mRNA abundance and protein content were determined by real-time PCR and western blot analysis and/or immunostaining in biopsy specimens (24 neoplastic and 6 non-neoplastic) freshly collected at surgical lung resection, in 82 pathologically banked lung cancer specimens and in cultured non-invasive (95-C) and invasive (95-D) lung cancer cells. Multivariate regression analysis was performed to correlate immunoreactive CAP1 signal with cancer type and stage. In vitro cell migration was performed to determine the effect of RNA interference-mediated CAP1 gene silencing on invasiveness of 95-D cells. These analyses collectively demonstrated that: i) both CAP1 mRNA abundance and protein content were significantly higher in neoplastic compared to non-neoplastic specimens and in metastatic compared to non-metastatic specimens but not different between adenocarcinoma and squamous cell carcinoma; ii) immunoreactive CAP1 signal was significantly stronger in metastatic specimens and 95-D cells compared to non-metastatic specimens and 95-C cells; and iii) RNA interference-mediated CAP1 gene silencing adequately attenuated the invasive capacity of 95-D cells in vitro. These findings suggest that overexpression of CAP1 in lung cancer cells, particularly at the metastatic stage, may have significant clinical implications as a diagnostic/prognostic factor for lung cancer.

  5. Proteome analysis of microtubule-associated proteins and their interacting partners from mammalian brain.

    PubMed

    Kozielski, Frank; Riaz, Tahira; DeBonis, Salvatore; Koehler, Christian J; Kroening, Mario; Panse, Isabel; Strozynski, Margarita; Donaldson, Ian M; Thiede, Bernd

    2011-07-01

    The microtubule (MT) cytoskeleton is essential for a variety of cellular processes. MTs are finely regulated by distinct classes of MT-associated proteins (MAPs), which themselves bind to and are regulated by a large number of additional proteins. We have carried out proteome analyses of tubulin-rich and tubulin-depleted MAPs and their interacting partners isolated from bovine brain. In total, 573 proteins were identified giving us unprecedented access to brain-specific MT-associated proteins from mammalian brain. Most of the standard MAPs were identified and at least 500 proteins have been reported as being associated with MTs. We identified protein complexes with a large number of subunits such as brain-specific motor/adaptor/cargo complexes for kinesins, dynein, and dynactin, and proteins of an RNA-transporting granule. About 25% of the identified proteins were also found in the synaptic vesicle proteome. Analysis of the MS/MS data revealed many posttranslational modifications, amino acid changes, and alternative splice variants, particularly in tau, a key protein implicated in Alzheimer's disease. Bioinformatic analysis of known protein-protein interactions of the identified proteins indicated that the number of MAPs and their associated proteins is larger than previously anticipated and that our database will be a useful resource to identify novel binding partners.

  6. Association of protein S p.Pro667Pro dimorphism with plasma protein S levels in normal individuals and patients with inherited protein S deficiency.

    PubMed

    Castaman, G; Biguzzi, E; Razzari, C; Tosetto, A; Fontana, G; Asti, D; Brancaccio, V; Castori, D; Lane, D A; Faioni, E M

    2007-01-01

    A dimorphism in PROS1 gene (c.A2,001G, p.Pro667Pro) has been associated with significantly reduced levels of both free and total protein S in carriers of the GG genotype. It is not known how the GG genotype could influence PS levels in normals, whether it could influence the levels of protein S in carriers of mutations in PROS1 gene and whether this genotype acts as an isolated or additive risk factor for venous thrombosis. With this as background, we evaluated the association of p.Pro667Pro dimorphism with free and total protein S centrally measured in a panel of 119 normal controls, 222 individuals with low protein S and 137 individuals with normal PS levels belonging to 76 families with protein S deficiency enrolled in the ProSIT study. Transient expression of recombinant wild type protein S and p.Pro667Pro protein S was performed to evaluate the role of the A to G transition at position 2001 in vitro. The p.Pro667Pro polymorphism was also expressed together with a p.Glu67Ala variant to assess a possible influence on protein S levels in protein S deficient subjects. Free and total protein S levels were significantly lower in normal women. In normal women only was the GG genotype associated with significantly lower free protein S levels in comparison to AA and AG genotypes (P=0.032). No significant influence of GG genotype was observed in patients, either with known mutations or with low protein S levels. These data were confirmed by in vitro transient expression, showing no difference in secretion levels of the p.Pro667Pro variant (even in association with the p.Glu67Ala mutation), compared to the wild type protein S. The genotype in itself was neither a significant risk factor for venous thrombosis nor a risk modifier in patients with known mutations.

  7. EP4 Receptor–Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated Tumorigenesis

    PubMed Central

    Nakatsuji, Masato; Yasui, Mika; Komekado, Hideyuki; Higuchi, Sei; Fujikawa, Risako; Nakanishi, Yuki; Fukuda, Akihisa; Kawada, Kenji; Sakai, Yoshiharu; Kita, Toru; Libby, Peter; Ikeuchi, Hiroki; Yokode, Masayuki; Chiba, Tsutomu

    2015-01-01

    Prostaglandin E2 plays important roles in the maintenance of colonic homeostasis. The recently identified prostaglandin E receptor (EP) 4–associated protein (EPRAP) is essential for an anti-inflammatory function of EP4 signaling in macrophages in vitro. To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis. In mice, EPRAP deficiency exacerbated colitis induced by dextran sodium sulfate (DSS) treatment. Wild-type (WT) or EPRAP-deficient recipients transplanted with EPRAP-deficient bone marrow developed more severe DSS-induced colitis than WT or EPRAP-deficient recipients of WT bone marrow. In the context of colitis-associated tumorigenesis, both systemic EPRAP null mutation and EPRAP-deficiency in the bone marrow enhanced intestinal polyp formation induced by azoxymethane (AOM)/DSS treatment. Administration of an EP4-selective agonist, ONO-AE1-329, ameliorated DSS-induced colitis in WT, but not in EPRAP-deficient mice. EPRAP deficiency increased the levels of the phosphorylated forms of p105, MEK, and ERK, resulting in activation of stromal macrophages in DSS-induced colitis. Macrophages of DSS-treated EPRAP-deficient mice exhibited a marked increase in the expression of pro-inflammatory genes, relative to WT mice. By contrast, forced expression of EPRAP in macrophages ameliorated DSS-induced colitis and AOM/DSS-induced intestinal polyp formation. These data suggest that EPRAP in macrophages functions crucially in suppressing colonic inflammation. Consistently, EPRAP-positive macrophages were also accumulated in the colonic stroma of ulcerative colitis patients. Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis. PMID:26439841

  8. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    PubMed

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. [Cytoskeletal actin and its associated proteins. Some examples in Protista].

    PubMed

    Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

    1998-06-01

    Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

  10. ASSOCIATIONS OF PROTEIN INTAKE AND PROTEIN SOURCE WITH BONE MINERAL DENSITY AND FRACTURE RISK: A POPULATION-BASED COHORT STUDY

    PubMed Central

    LANGSETMO, L.; BARR, S.I.; BERGER, C.; KREIGER, N.; RAHME, E.; ADACHI, J.D.; PAPAIOANNOU, A.; KAISER, S. M; PRIOR, J.C.; HANLEY, D.A.; KOVACS, C.S.; JOSSE, R.G.; GOLTZMAN, D.

    2016-01-01

    High dietary protein has been hypothesized to cause lower bone mineral density (BMD) and greater fracture risk. Previous results are conflicting and few studies have assessed potential differences related to differing protein sources. Objective To determine associations between total protein intake, and protein intake by source (dairy, non-dairy animal, plant) with BMD, BMD change, and incident osteoporotic fracture. Design/Setting Prospective cohort study (Canadian Multicentre Osteoporosis Study). Participants/Measures Protein intake was assessed as percent of total energy intake (TEI) at Year 2 (1997–99) using a food frequency questionnaire (N=6510). Participants were contacted annually to ascertain incident fracture. Total hip and lumbar spine BMD was measured at baseline and Year 5. Analyses were stratified by group (men 25–49 y, men 50+ y, premenopausal women 25–49 y, and postmenopausal women 50+ y) and adjusted for major confounders. Fracture analyses were limited to those 50+ y. Results Intakes of dairy protein (with adjustment for BMI) were positively associated with total hip BMD among men and women aged 50+ y, and in men aged 25–49. Among adults aged 50+ y, those with protein intakes of <12% TEI (women) and <11% TEI (men) had increased fracture risk compared to those with intakes of 15% TEI. Fracture risk did not significantly change as intake increased above 15% TEI, and was not significantly associated with protein source. Conclusions In contrast to hypothesized risk of high protein, we found that for adults 50+ y, low protein intake (below 15% TEI) may lead to increased fracture risk. Source of protein was a determinant of BMD, but not fracture risk. PMID:26412291

  11. Identification and Validation of PTEN Complex, Associated Proteins

    DTIC Science & Technology

    2005-11-01

    Rosalia construct was transcribed and translated using a wheat germ lysate transcription/translation system to generate an unphosphorylated protein...efficient using the wheat germ lysate transcription/translation, system the new antisera immunoprecipitated the protein as well as the C54 Ab, especially...pSGL-PTEN was in vitro translated in a Rabbit reticolocyte lysate system (A) or in a wheat germ system (B) in the presence of radioactively labeled

  12. Methyl-accepting protein associated with bacterial sensory phodopsin I

    SciTech Connect

    Spudich, E.N.; Hasselbacher, C.A. ); Spudich, J.L. )

    1988-09-01

    In vivo radiolabeling of Halaobacterium halobium phototaxis mutants and revertants with L-(methyl-{sup 3}H) methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in photoaxis. The lability of the radiolabeled bands to mild base treatment indicated the the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing photoaxis receptors in H. halobium. It was absent in a strain the contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based in the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By ({sup 3}H)retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced ({sup 3}H) retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the siginal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I.

  13. Methyl-accepting protein associated with bacterial sensory rhodopsin I.

    PubMed Central

    Spudich, E N; Hasselbacher, C A; Spudich, J L

    1988-01-01

    In vivo radiolabeling of Halobacterium halobium phototaxis mutants and revertants with L-[methyl-3H] methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in phototaxis. The lability of the radiolabeled bands to mild base treatment indicated that the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing phototaxis receptors in H. halobium. It was absent in a strain that contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based on the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By [3H]retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced [3H]retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the signal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I. Images PMID:3410829

  14. Matrix Gla Protein polymorphism, but not concentrations, is associated with radiographic hand osteoarthritis

    USDA-ARS?s Scientific Manuscript database

    Objective. Factors associated with mineralization and osteophyte formation in osteoarthritis (OA) are incompletely understood. Genetic polymorphisms of matrix Gla protein (MGP), a mineralization inhibitor, have been associated clinically with conditions of abnormal calcification. We therefore evalua...

  15. Inference of domain-disease associations from domain-protein, protein-disease and disease-disease relationships.

    PubMed

    Zhang, Wangshu; Coba, Marcelo P; Sun, Fengzhu

    2016-01-11

    Protein domains can be viewed as portable units of biological function that defines the functional properties of proteins. Therefore, if a protein is associated with a disease, protein domains might also be associated and define disease endophenotypes. However, knowledge about such domain-disease relationships is rarely available. Thus, identification of domains associated with human diseases would greatly improve our understanding of the mechanism of human complex diseases and further improve the prevention, diagnosis and treatment of these diseases. Based on phenotypic similarities among diseases, we first group diseases into overlapping modules. We then develop a framework to infer associations between domains and diseases through known relationships between diseases and modules, domains and proteins, as well as proteins and disease modules. Different methods including Association, Maximum likelihood estimation (MLE), Domain-disease pair exclusion analysis (DPEA), Bayesian, and Parsimonious explanation (PE) approaches are developed to predict domain-disease associations. We demonstrate the effectiveness of all the five approaches via a series of validation experiments, and show the robustness of the MLE, Bayesian and PE approaches to the involved parameters. We also study the effects of disease modularization in inferring novel domain-disease associations. Through validation, the AUC (Area Under the operating characteristic Curve) scores for Bayesian, MLE, DPEA, PE, and Association approaches are 0.86, 0.84, 0.83, 0.83 and 0.79, respectively, indicating the usefulness of these approaches for predicting domain-disease relationships. Finally, we choose the Bayesian approach to infer domains associated with two common diseases, Crohn's disease and type 2 diabetes. The Bayesian approach has the best performance for the inference of domain-disease relationships. The predicted landscape between domains and diseases provides a more detailed view about the disease

  16. Cancer-associated mutations are preferentially distributed in protein kinase functional sites.

    PubMed

    Izarzugaza, Jose M G; Redfern, Oliver C; Orengo, Christine A; Valencia, Alfonso

    2009-12-01

    Protein kinases are a superfamily involved in many crucial cellular processes, including signal transmission and regulation of cell cycle. As a consequence of this role, kinases have been reported to be associated with many types of cancer and are considered as potential therapeutic targets. We analyzed the distribution of pathogenic somatic point mutations (drivers) in the protein kinase superfamily with respect to their location in the protein, such as in structural, evolutionary, and functionally relevant regions. We find these driver mutations are more clearly associated with key protein features than other somatic mutations (passengers) that have not been directly linked to tumor progression. This observation fits well with the expected implication of the alterations in protein kinase function in cancer pathogenicity. To explain the relevance of the detected association of cancer driver mutations at the molecular level in the human kinome, we compare these with genetically inherited mutations (SNPs). We find that the subset of nonsynonymous SNPs that are associated to disease, but sufficiently mild to the point of being widespread in the population, tend to avoid those key protein regions, where they could be more detrimental for protein function. This tendency contrasts with the one detected for cancer associated-driver-mutations, which seems to be more directly implicated in the alteration of protein function. The detailed analysis of protein kinase groups and a number of relevant examples, confirm the relation between cancer associated-driver-mutations and key regions for protein kinase structure and function.

  17. The subunit interfaces of weakly associated homodimeric proteins.

    PubMed

    Dey, Sucharita; Pal, Arumay; Chakrabarti, Pinak; Janin, Joël

    2010-04-23

    We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the "weak" dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.

  18. Associations between milk-protein production and reproduction, health, and culling.

    PubMed

    Sargeant, J M; Martin, S W; Lissemore, K D; Leslie, K E; Gibson, J P; Scott, H M; Kelton, D F

    1998-04-16

    Associations between protein production and individual-cow reproductive performance, health, and culling were investigated in a 2-year observational study involving a convenience sample of 75 Ontario, 5 Alberta, and 3 Nova Scotia dairy farms. Protein production was defined by 305-day lactation protein yields and by estimated breeding values for protein yield. After controlling for the level of milk production, herd, parity, breed, and season of calving, there were no significant associations between either measure of protein production and days open or days to first breeding. The only associations between protein production and disease were small positive associations between the estimated breeding value for protein yield and cystic ovaries and mean lactation somatic cell count. The risk of culling, after controlling for the level of milk production, was negatively associated with previous-lactation 305-day protein yield for parity three animals only. The estimated breeding value for protein yield had a small negative association with the overall risk of culling, although the associations were not significant for individual lactations.

  19. Proteomic analysis of Col11a1-associated protein complexes

    PubMed Central

    Brown, Raquel J.; Mallory, Christopher; McDougal, Owen M.; Oxford, Julia Thom

    2012-01-01

    Cartilage plays an essential role during skeletal development within the growth plate and in articular joint function. Interactions between the collagen fibrils and other extracellular matrix molecules maintain structural integrity of cartilage, orchestrate complex dynamic events during embryonic development, and help to regulate fibrillogenesis. To increase our understanding of these events, affinity chromatography and liquid chromatography/tandem mass spectrometry were used to identify proteins that interact with the collagen fibril surface via the amino terminal domain of collagen alpha 1(XI) a protein domain that is displayed at the surface of heterotypic collagen fibrils of cartilage. Proteins extracted from fetal bovine cartilage using homogenization in high ionic strength buffer were selected based on affinity for the amino terminal noncollagenous domain of collagen alpha 1(XI). Mass spectrometry was used to determine the amino acid sequence of tryptic fragments for protein identification. Extracellular matrix molecules and cellular proteins that were identified as interacting with the amino terminal domain of collagen alpha 1(XI) directly or indirectly, included proteoglycans, collagens, and matricellular molecules, some of which also play a role in fibrillogenesis, while others are known to function in the maintenance of tissue integrity. Characterization of these molecular interactions will provide a more thorough understanding of how the extracellular matrix molecules of cartilage interact and what role collagen XI plays in the process of fibrillogenesis and maintenance of tissue integrity. Such information will aid tissue engineering and cartilage regeneration efforts to treat cartilage tissue damage and degeneration. PMID:22038862

  20. Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens*

    PubMed Central

    Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria O.; Eisenberg, Tobias; Harder, Lea M.; Schandorff, Søren; Farkas, Thomas; Kirkegaard, Thomas; Becker, Andrea C.; Schroeder, Sabrina; Vanselow, Katja; Lundberg, Emma; Nielsen, Mogens M.; Kristensen, Anders R.; Akimov, Vyacheslav; Bunkenborg, Jakob; Madeo, Frank; Jäättelä, Marja; Andersen, Jens S.

    2012-01-01

    Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection. PMID:22311637

  1. Detection of dysregulated protein-association networks by high-throughput proteomics predicts cancer vulnerabilities.

    PubMed

    Lapek, John D; Greninger, Patricia; Morris, Robert; Amzallag, Arnaud; Pruteanu-Malinici, Iulian; Benes, Cyril H; Haas, Wilhelm

    2017-09-11

    The formation of protein complexes and the co-regulation of the cellular concentrations of proteins are essential mechanisms for cellular signaling and for maintaining homeostasis. Here we use isobaric-labeling multiplexed proteomics to analyze protein co-regulation and show that this allows the identification of protein-protein associations with high accuracy. We apply this 'interactome mapping by high-throughput quantitative proteome analysis' (IMAHP) method to a panel of 41 breast cancer cell lines and show that deviations of the observed protein co-regulations in specific cell lines from the consensus network affects cellular fitness. Furthermore, these aberrant interactions serve as biomarkers that predict the drug sensitivity of cell lines in screens across 195 drugs. We expect that IMAHP can be broadly used to gain insight into how changing landscapes of protein-protein associations affect the phenotype of biological systems.

  2. Associations between heat shock protein 70 genetic polymorphisms and calving traits in crossbred Brahman cows

    USDA-ARS?s Scientific Manuscript database

    Stressors such as heat, cold, toxins, and oxygen deprivation are known to induce heat shock proteins. Genetic polymorphisms associated with heat shock protein genes have been associated with decreased male and female fertility. Our objectives were to 1) confirm single nucleotide polymorphisms (SNP) ...

  3. Rearrangement of microtubule associated protein parallels the morphological transformation of neurons from dorsal root ganglion.

    PubMed

    Hernández, M A; Avila, J; Moya, F; Alberto, C

    1989-01-01

    In primary cultures of dorsal root ganglion cells from rat embryos, neurons undergo a morphological transformation from a bipolar to a differentiated pseudo-unipolar shape, resembling their developmental stages in vivo. Cells present in these cultures are characterized here by immunological criteria using monoclonal and polyclonal antibodies against microtubule associated proteins MAP1 and MAP2 and against tubulin. After development for seven days in culture, antibodies against microtubule associated proteins MAP1 brightly labeled cells with neuronal morphology and lightly stained cells with the shape of Schwann cells. In addition, an extended network of neuronal processes was labeled with this antibody. Anti-microtubule associated protein MAP2 stained only neurons and a more restricted network of neuronal processes. The compartmentalization of microtubule associated protein MAP2 during the maturation process was followed by double-labeling with antibodies to microtubule associated proteins MAP1 and MAP2. Initially, microtubule associated protein MAP2 was present in the cell body and the two processes of bipolar neurons. Subsequently, the labeling of both processes changed, depending on neuronal morphology. In neurons in which both processes were approaching one another, one of these neurites was stained predominantly with anti-microtubule associated protein MAP2. Finally, in pseudo, unipolar neurons, anti-microtubule associated protein MAP2 labeling was found in the cell body and excluded from the more distal processes.

  4. Association of adaptor protein TRIP8b with clathrin.

    PubMed

    Popova, Nadezhda V; Deyev, Igor E; Petrenko, Alexander G

    2011-09-01

    TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  5. Heat shock proteins and their association with major pediatric malignancies.

    PubMed

    Skora, Dorota; Gorska, Magdalena

    2016-01-01

    Heat shock proteins belong to a group of molecular chaperones responsible for the regulation of many intracellular processes. HSPs play a pivotal role in the survival of cells under stressful conditions. Over-expression of these proteins have been found in both healthy and a great number of cancer cells. HSPs may be involved in numerous carcinogenic and chemoresistant processes. Due to that fact, they may be referred to as diagnostic biomarkers of oncogenesis and potential targets for anticancer drugs. Thus, we decided to review the involvement of major HSPs in the most malignant childhood cancers.

  6. Localized mRNA translation and protein association

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2014-08-01

    Recent direct observations of localization of mRNAs and proteins both in prokaryotic and eukaryotic cells can be related to slowdown of diffusion of these species due to macromolecular crowding and their ability to aggregate and form immobile or slowly mobile complexes. Here, a generic kinetic model describing both these factors is presented and comprehensively analyzed. Although the model is non-linear, an accurate self-consistent analytical solution of the corresponding reaction-diffusion equation has been constructed, the types of localized protein distributions have been explicitly shown, and the predicted kinetic regimes of gene expression have been classified.

  7. Herpesvirus saimiri protein StpB associates with cellular Src.

    PubMed

    Hör, S; Ensser, A; Reiss, C; Ballmer-Hofer, K; Biesinger, B

    2001-02-01

    Subgroup B isolates of Herpesvirus saimiri are less efficient in T lymphocyte transformation when compared with subgroups A or C. Here it is shown that subgroup B strain SMHI encodes a protein, StpB, at a position equivalent to those of the ORFs for the saimiri transforming proteins (Stp) of subgroups A and C. StpB shares little similarity with StpA or StpC, but interacts with the SH2 domain of cellular Src, as does StpA. Thus, factors other than c-Src binding determine the efficiency of primary T cell transformation by Herpesvirus saimiri.

  8. Is there a biological cost of protein disorder? Analysis of cancer-associated mutations.

    PubMed

    Pajkos, Mátyás; Mészáros, Bálint; Simon, István; Dosztányi, Zsuzsanna

    2012-01-01

    As many diseases can be traced back to altered protein function, studying the effect of genetic variations at the level of proteins can provide a clue to understand how changes at the DNA level lead to various diseases. Cellular processes rely not only on proteins with well-defined structure but can also involve intrinsically disordered proteins (IDPs) that exist as highly flexible ensembles of conformations. Disordered proteins are mostly involved in signaling and regulatory processes, and their functional repertoire largely complements that of globular proteins. However, it was also suggested that protein disorder entails an increased biological cost. This notion was supported by a set of individual IDPs involved in various diseases, especially in cancer, and the increased amount of disorder observed among disease-associated proteins. In this work, we tested if there is any biological risk associated with protein disorder at the level of single nucleotide mutations. Specifically, we analyzed the distribution of mutations within ordered and disordered segments. Our results demonstrated that while neutral polymorphisms were more likely to occur within disordered segments, cancer-associated mutations had a preference for ordered regions. Additionally, we proposed an alternative explanation for the association of protein disorder and the involvement in cancer with the consideration of functional annotations. Individual examples also suggested that although disordered segments are fundamental functional elements, their presence is not necessarily accompanied with an increased mutation rate in cancer. The presented study can help to understand how the different structural properties of proteins influence the consequences of genetic mutations.

  9. Discovery of circulating proteins associated to knee radiographic osteoarthritis.

    PubMed

    Lourido, Lucía; Ayoglu, Burcu; Fernández-Tajes, Juan; Oreiro, Natividad; Henjes, Frauke; Hellström, Cecilia; Schwenk, Jochen M; Ruiz-Romero, Cristina; Nilsson, Peter; Blanco, Francisco J

    2017-03-09

    Currently there are no sufficiently sensitive biomarkers able to reflect changes in joint remodelling during osteoarthritis (OA). In this work, we took an affinity proteomic approach to profile serum samples for proteins that could serve as indicators for the diagnosis of radiographic knee OA. Antibody suspension bead arrays were applied to analyze serum samples from patients with OA (n = 273), control subjects (n = 76) and patients with rheumatoid arthritis (RA, n = 244). For verification, a focused bead array was built and applied to an independent set of serum samples from patients with OA (n = 188), control individuals (n = 83) and RA (n = 168) patients. A linear regression analysis adjusting for sex, age and body mass index (BMI) revealed that three proteins were significantly elevated (P < 0.05) in serum from OA patients compared to controls: C3, ITIH1 and S100A6. A panel consisting of these three proteins had an area under the curve of 0.82 for the classification of OA and control samples. Moreover, C3 and ITIH1 levels were also found to be significantly elevated (P < 0.05) in OA patients compared to RA patients. Upon validation in additional study sets, the alterations of these three candidate serum biomarker proteins could support the diagnosis of radiographic knee OA.

  10. Plant Protein Intake and Dietary Diversity Are Independently Associated with Nutrient Adequacy in French Adults.

    PubMed

    Bianchi, Clélia M; Egnell, Manon; Huneau, Jean-François; Mariotti, François

    2016-11-01

    Plant protein intake, which is favorably associated with the intake of many nutrients, is a marker of a healthy diet. However, the higher nutrient adequacy of diets rich in plant protein may also originate from overarching factors associated with more healthful dietary behaviors, such as a greater dietary diversity. Our main objective was to determine whether the relation between plant protein intake and nutrient adequacy could be explained, at least in part, by an association with overall dietary diversity. We used data from 1330 adults participating in the French Nutrition and Health Survey [Etude Nationale Nutrition Santé (ENNS); 2006-2007]. With the use of global, integrative approaches, we assessed nutrient adequacy [by using the probabilistic PANDiet (Probability of Adequate Nutrient Intake) scoring system] and overall dietary diversity (by using a 100-point score that accounts for the relative number of subgroups consumed in 7 food groups). Linear multivariate modeling was used for the analysis. We found a positive association between plant protein (but not total or animal protein) intake and dietary diversity (β = 0.08) and a strong positive association between dietary diversity and nutrient adequacy (β = 0.33). However, the association between plant protein intake and nutrient adequacy was not explained by dietary diversity (r = 0.38 and partial r = 0.36, P < 0.0001). In multivariate analysis, nutrient adequacy was positively associated with dietary diversity (β = 0.44) and plant (β = 0.37) and animal (β = 0.15) protein intakes. Associations persisted after adjustment for potential confounders (total energy, energy density, sex, body mass index, income, occupational status, educational level, region, season, and smoking status). Overall dietary diversity is greater in French adults who consume more plant protein. Both plant protein intake and dietary diversity are associated with the nutrient adequacy of the diet. But the plant protein

  11. Association of adaptor protein TRIP8b with clathrin

    PubMed Central

    Popova, Nadezhda V.; Deyev, Igor E.; Petrenko, Alexander G.

    2011-01-01

    TRIP8b is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, HCN channels and G protein-coupled receptor CIRL. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat. PMID:21749376

  12. Computational Framework for Prediction of Peptide Sequences That May Mediate Multiple Protein Interactions in Cancer-Associated Hub Proteins

    PubMed Central

    Sarkar, Debasree; Patra, Piya; Ghosh, Abhirupa; Saha, Sudipto

    2016-01-01

    A considerable proportion of protein-protein interactions (PPIs) in the cell are estimated to be mediated by very short peptide segments that approximately conform to specific sequence patterns known as linear motifs (LMs), often present in the disordered regions in the eukaryotic proteins. These peptides have been found to interact with low affinity and are able bind to multiple interactors, thus playing an important role in the PPI networks involving date hubs. In this work, PPI data and de novo motif identification based method (MEME) were used to identify such peptides in three cancer-associated hub proteins—MYC, APC and MDM2. The peptides corresponding to the significant LMs identified for each hub protein were aligned, the overlapping regions across these peptides being termed as overlapping linear peptides (OLPs). These OLPs were thus predicted to be responsible for multiple PPIs of the corresponding hub proteins and a scoring system was developed to rank them. We predicted six OLPs in MYC and five OLPs in MDM2 that scored higher than OLP predictions from randomly generated protein sets. Two OLP sequences from the C-terminal of MYC were predicted to bind with FBXW7, component of an E3 ubiquitin-protein ligase complex involved in proteasomal degradation of MYC. Similarly, we identified peptides in the C-terminal of MDM2 interacting with FKBP3, which has a specific role in auto-ubiquitinylation of MDM2. The peptide sequences predicted in MYC and MDM2 look promising for designing orthosteric inhibitors against possible disease-associated PPIs. Since these OLPs can interact with other proteins as well, these inhibitors should be specific to the targeted interactor to prevent undesired side-effects. This computational framework has been designed to predict and rank the peptide regions that may mediate multiple PPIs and can be applied to other disease-associated date hub proteins for prediction of novel therapeutic targets of small molecule PPI modulators. PMID

  13. Associations between pre-eclampsia and protein C and protein S levels among pregnant Nigerian women.

    PubMed

    Okoye, Helen C; Eweputanna, Lisa I; Okpani, Anthony O U; Ejele, Oseikhuemen A

    2017-04-01

    To evaluate levels of protein C and free protein S among women with pre-eclampsia, and determine whether there is a relationship between deficiencies and pre-eclampsia. A cross-sectional study was conducted at a hospital in Nigeria from July 2013 to March 2014 among 90 pregnant women with pre-eclampsia (blood pressure ≥140/90 mm Hg, proteinuria ≥300 mg in 24 hours) and 90 normotensive pregnant women (control group). Plasma levels of protein C and free protein S were analyzed by enzyme-linked immunosorbent assay, and protein C activity by a chromogenic method. Mean protein C antigen and activity levels did not differ between groups (P=0.639 and P=0.444, respectively). The incidence of protein C antigen and activity deficiency also did not differ (P=0.288 and P>0.99, respectively). The mean free protein S antigen level was higher among women with pre-eclampsia (54.48%±19.58%) than in the control group (47.23%±10.27%; P=0.004). No woman in the control group had protein S deficiency, as compared with 2 (2%) of the women with pre-eclampsia (P=0.497). No association was found between deficiencies of these proteins and pre-eclampsia. Deficiencies of protein C and free protein S are unlikely to be etiopathogenetic for pre-eclampsia; therefore, therapeutic intervention should focus on other potential pathogenetic pathways. © 2016 International Federation of Gynecology and Obstetrics.

  14. Compartmentalization of Proteins in Epididymosomes Coordinates the Association of Epididymal Proteins with the Different Functional Structures of Bovine Spermatozoa1

    PubMed Central

    Girouard, Julie; Frenette, Gilles; Sullivan, Robert

    2009-01-01

    Epididymosomes are small membranous vesicles secreted by epithelial cells within the luminal compartment of the epididymis. In bovine, many proteins are associated with epididymosomes, and some of them, such as the glycosylphosphatidylinositol (GPI)-anchored protein P25b, macrophage migration inhibitory factor (MIF), and aldose reductase (AKR1B1), are transferred to spermatozoa during the epididymal maturation process. P25b is associated with detergent-resistant membrane (DRM) domains of epididymal spermatozoa, whereas MIF and AKR1B1 are cytosolic proteins associated with detergent-soluble fractions. In this study, we tested the hypothesis that DRM domains are also present in the epididymosomes and that P25b DRM-associated proteins in these vesicles are transferred to the DRMs of spermatozoa. The presence of DRMs in epididymosomes was confirmed by their insolubility in cold Triton X-100 and their low buoyant density in sucrose gradient. Furthermore, DRMs isolated from epididymosomes are characterized by the exclusive presence of ganglioside GM1 and by high levels of cholesterol and sphingomyelin. Biochemical analysis indicated that P25b is linked to DRM in epididymosomes, whereas MIF and AKR1B1 are completely excluded from these membrane domains. Proteolytic treatment of epididymosomes and immunoblotting studies showed that P25b is affected by trypsin or pronase proteolysis. In contrast, MIF and AKR1B1 are not degraded by proteases, suggesting that they are localized within epididymosomes. Interaction studies between epididymosomes and epididymal spermatozoa demonstrated that P25b is transferred from the DRM of epididymosomes to the DRM of the caput epididymal spermatozoa as a GPI-anchored protein. Together, these data suggest that specific localization and compartmentalization of proteins in the epididymosomes coordinate the association of epididymal proteins with the different functional structures of spermatozoa. PMID:19164173

  15. Effect of the microtubule-associated protein tau on dynamics of single-headed motor proteins KIF1A

    NASA Astrophysics Data System (ADS)

    Sparacino, J.; Farías, M. G.; Lamberti, P. W.

    2014-02-01

    Intracellular transport based on molecular motors and its regulation are crucial to the functioning of cells. Filamentary tracks of the cells are abundantly decorated with nonmotile microtubule-associated proteins, such as tau. Motivated by experiments on kinesin-tau interactions [Dixit et al., Science 319, 1086 (2008), 10.1126/science.1152993] we developed a stochastic model of interacting single-headed motor proteins KIF1A that also takes into account the interactions between motor proteins and tau molecules. Our model reproduces experimental observations and predicts significant effects of tau on bound time and run length which suggest an important role of tau in regulation of kinesin-based transport.

  16. Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling.

    PubMed

    Parag-Sharma, Kshitij; Leyme, Anthony; DiGiacomo, Vincent; Marivin, Arthur; Broselid, Stefan; Garcia-Marcos, Mikel

    2016-12-30

    GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gαi3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function.

  17. A maize protein associated with the G-box binding complex has homology to brain regulatory proteins.

    PubMed Central

    de Vetten, N C; Lu, G; Feri, R J

    1992-01-01

    The G-box element is a moderately conserved component of the promoter of many inducible genes, including the alcohol dehydrogenase genes of Arabidopsis and maize. We used monoclonal antibodies generated against partially purified G-box binding factor (GBF) activity to characterize maize proteins that are part of the DNA binding complex. Antibodies interacted with partially purified maize GBF complexes to produce a slower migrating complex in the gel retardation assay. Immunoprecipitation experiments suggested that the protein recognized by the antibody is not a DNA binding protein in and of itself, but rather is associated with the DNA binding complex. These monoclonal antibodies were used to isolate cDNA clones encoding a protein that we have designated GF14. Maize GF14 contains a region resembling a leucine zipper and acidic carboxy and amino termini, of which the latter can form an amphipathic alpha-helix similar to known transcriptional activators such as VP16 and GAL4. Protein gel blot analysis of cell culture extract showed that a single, major protein of approximately 30 kD is recognized by anti-GF14; the protein is also present predominantly in the kernel and root. The deduced amino acid sequence of maize GF14 is more than 80% identical to Arabidopsis GF14 and Oenothera PHP-O, and is more than 60% identical to a class of mammalian brain proteins described as both protein kinase C inhibitors and activators of tyrosine and tryptophan hydroxylases. GF14 is found in a variety of monocotyledons and dicotyledons, gymnosperms, and yeast. This suggests a deep evolutionary conservation of a potential regulatory protein associated with a core sequence found in the promoter region of many genes. PMID:1446170

  18. Retinal proteins associated with redox regulation and protein folding play central roles in response to high glucose conditions.

    PubMed

    Wang, Ssu-Han; Lee, Wen-Chi; Chou, Hsiu-Chuan

    2015-03-01

    Diabetic retinopathy typically causes poor vision and blindness. A previous study revealed that a high blood glucose concentration induces glycoxidation and weakens the retinal capillaries. Nevertheless, the molecular mechanisms underlying the effects of high blood glucose induced diabetic retinopathy remain to be elucidated. In the present study, we cultured the retinal pigmented epithelial cell line ARPE-19 in mannitol-balanced 5.5, 25, and 100 mM glucose media and investigated protein level alterations. Proteomic analysis revealed significant changes in 137 protein features, of which 124 demonstrated changes in a glucose concentration dependent manner. Several proteins functionally associated with redox regulation, protein folding, or the cytoskeleton are affected by increased glucose concentrations. Additional analyses also revealed that cellular oxidative stress, including endoplasmic reticulum stress, was significantly increased after treatment with high glucose concentrations. However, the mitochondrial membrane potential and cell survival remained unchanged during treatment with high glucose concentrations. To summarize, in this study, we used a comprehensive retinal pigmented epithelial cell based proteomic approach for identifying changes in protein expression associated retinal markers induced by high glucose concentrations. Our results revealed that a high glucose condition can induce cellular oxidative stress and modulate the levels of proteins with functions in redox regulation, protein folding, and cytoskeleton regulation; however, cell viability and mitochondrial integrity are not significantly disturbed under these high glucose conditions.

  19. RNA: protein interactions associated with satellites of panicum mosaic virus.

    PubMed

    Desvoyes, B; Scholthof, K B

    2000-11-17

    The interactions between satellite panicum mosaic virus (SPMV) capsid protein (CP) and its 824 nucleotide (nt) single stranded RNA were investigated by gel mobility shift assay and Northwestern blot assay. SPMV CP has specificity for its RNA at high affinity, but little affinity for non-viral RNA. The SPMV CP also bound a 350 nt satellite RNA (satRNA) that, like SPMV, is dependent on panicum mosaic virus for its replication. SPMV CP has the novel property of encapsidating SPMV RNA and satRNA. Competition gel mobility shift assays performed with a non-viral RNA and unlabeled SPMV RNA and satRNA revealed that these RNA:protein interactions were in part sequence specific.

  20. Characterization of Antibody Specific for Disease Associated Prion Protein

    DTIC Science & Technology

    2004-07-01

    Distribution Unlimited 13. ABSTRACT (Maximum 200 Words) Prion diseases are characterized by the presence of the abnormal scrapie isoform of prion protein...areas of Task 2. Our main research findings have been published recently (Zou W, Zheng J, Gray D, Gambetti P, Chen SG. Antibody to DNA detects scrapie ...chemiluminescence. (B) Immunocapture of PrP by OCD4 following incubation with nuclease and salmon DNA. 2 1 I Scrapie -infected hamster BH (2 pi each) was either

  1. Predicting Protein–protein Association Rates using Coarse-grained Simulation and Machine Learning

    PubMed Central

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

    2017-01-01

    Protein–protein interactions dominate all major biological processes in living cells. We have developed a new Monte Carlo-based simulation algorithm to study the kinetic process of protein association. We tested our method on a previously used large benchmark set of 49 protein complexes. The predicted rate was overestimated in the benchmark test compared to the experimental results for a group of protein complexes. We hypothesized that this resulted from molecular flexibility at the interface regions of the interacting proteins. After applying a machine learning algorithm with input variables that accounted for both the conformational flexibility and the energetic factor of binding, we successfully identified most of the protein complexes with overestimated association rates and improved our final prediction by using a cross-validation test. This method was then applied to a new independent test set and resulted in a similar prediction accuracy to that obtained using the training set. It has been thought that diffusion-limited protein association is dominated by long-range interactions. Our results provide strong evidence that the conformational flexibility also plays an important role in regulating protein association. Our studies provide new insights into the mechanism of protein association and offer a computationally efficient tool for predicting its rate. PMID:28418043

  2. Cytoskeletal scaffolding proteins interact with Lynch-Syndrome associated mismatch repair protein MLH1.

    PubMed

    Brieger, Angela; Adryan, Boris; Wolpert, Fabian; Passmann, Sandra; Zeuzem, Stefan; Trojan, Jörg

    2010-09-01

    The involvement of MLH1 in several mismatch repair-independent cellular processes has been reported. In an attempt to gain further insight into the protein's cellular functions, we screened for novel interacting partners of MLH1 utilizing a bacterial two-hybrid system. Numerous unknown interacting proteins were identified, suggesting novel biological roles of MLH1. The network of MLH1 and its partner proteins involves a multitude of cellular processes. Integration of our data with the "General Repository for Interaction Datasets" highlighted that MLH1 exhibits relationships to three interacting pairs of proteins involved in cytoskeletal and filament organization: Thymosin beta 4 and Actin gamma, Cathepsin B and Annexin A2 as well as Spectrin alpha and Desmin. Coimmunoprecipitation and colocalization experiments validated the interaction of MLH1 with these proteins. Differential mRNA levels of many of the identified proteins, detected by microarray analysis comparing MLH1-deficient and -proficient cell lines, support the assumed interplay of MLH1 and the identified candidate proteins. By siRNA knock down of MLH1, we demonstrated the functional impact of MLH1-Actin interaction on filament organization and propose that dysregulation of MLH1 plays an essential role in cytoskeleton dynamics. Our data suggest novel roles of MLH1 in cellular organization and colorectal cancerogenesis.

  3. Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

    PubMed

    Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

    2016-02-01

    Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation.

  4. Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics.

    PubMed

    Hüttenhain, Ruth; Soste, Martin; Selevsek, Nathalie; Röst, Hannes; Sethi, Atul; Carapito, Christine; Farrah, Terry; Deutsch, Eric W; Kusebauch, Ulrike; Moritz, Robert L; Niméus-Malmström, Emma; Rinner, Oliver; Aebersold, Ruedi

    2012-07-11

    The rigorous testing of hypotheses on suitable sample cohorts is a major limitation in translational research. This is particularly the case for the validation of protein biomarkers; the lack of accurate, reproducible, and sensitive assays for most proteins has precluded the systematic assessment of hundreds of potential marker proteins described in the literature. Here, we describe a high-throughput method for the development and refinement of selected reaction monitoring (SRM) assays for human proteins. The method was applied to generate such assays for more than 1000 cancer-associated proteins, which are functionally related to candidate cancer driver mutations. We used the assays to determine the detectability of the target proteins in two clinically relevant samples: plasma and urine. One hundred eighty-two proteins were detected in depleted plasma, spanning five orders of magnitude in abundance and reaching below a concentration of 10 ng/ml. The narrower concentration range of proteins in urine allowed the detection of 408 proteins. Moreover, we demonstrate that these SRM assays allow reproducible quantification by monitoring 34 biomarker candidates across 83 patient plasma samples. Through public access to the entire assay library, researchers will be able to target their cancer-associated proteins of interest in any sample type using the detectability information in plasma and urine as a guide. The generated expandable reference map of SRM assays for cancer-associated proteins will be a valuable resource for accelerating and planning biomarker verification studies.

  5. Association of Plasmodium berghei proteins with the host erythrocyte membrane: binding to inside-out vesicles.

    PubMed

    Wiser, M F; Sartorelli, A C; Patton, C L

    1990-01-01

    Two acidic phosphoproteins of Plasmodium berghei origin, of 65 and 46 kDa, are associated with the plasma membrane of the host mouse erythrocyte. The 65-kDa protein partitions between a soluble and particulate phase upon host cell lysis, whereas the 46-kDa protein is localized exclusively in the particulate fraction. Both proteins bind to inside-out vesicles derived from erythrocyte ghosts and the conditions of the reassociation reaction indicate that the binding is specific and that the proteins interact only with the cytoplasmic face of the erythrocyte membrane. The 65-kDa protein appears to exist in two membrane-associated states; one loosely bound, which readily dissociates from the membrane, and a more tightly associated state, which does not dissociate under non-denaturing conditions. The 46-kDa protein is tightly bound to the host erythrocyte membrane and does not dissociate. Cross-linking studies suggest that both of these parasite proteins interact with the submembrane cytoskeleton of the erythrocyte, and that the 65-kDa protein also appears to interact simultaneously with the lipid bilayer and erythrocyte membrane proteins. However, direct interaction between the malarial proteins and distinct erythrocyte membrane proteins could not be demonstrated. In summary, these findings indicate that the acidic phosphoproteins of the malarial parasite interact with the cytoplasmic face of the erythrocyte membrane both in vivo and in vitro.

  6. Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

    PubMed Central

    Lin, Qingqing; London, Erwin

    2013-01-01

    Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3α-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-Tm lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-Tm and low-Tm lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts. PMID:24359745

  7. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    PubMed

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

  8. Association equilibria for proteins interacted with crowders of short-range attraction in crowded environment

    NASA Astrophysics Data System (ADS)

    Wei, Jiachen; Song, Fan

    2017-01-01

    Based on a very simple coarse-grained colloidal model, here we implement an effective hard-sphere theory and numerical simulation to capture the general features of the association equilibria for globular proteins in crowded environment. We measure the activity coefficient, i.e., the deviation from ideal behavior of protein solution, and the crowding factor, i.e., the contribution of crowders to the association equilibria, for proteins in macromolecular crowding. The results show that the association balance in macromolecular crowding depends sensitively on the magnitude of protein-crowder attraction and the relative size of reactant to crowding agent. Since our coarse-grained model is irrelevant to the microscopic details of the molecules, it can be applied to the control of the association equilibria of many globular proteins such as bovine serum albumin, crystallin and lysozyme.

  9. M-Finder: Uncovering functionally associated proteins from interactome data integrated with GO annotations

    PubMed Central

    2013-01-01

    Background Protein-protein interactions (PPIs) play a key role in understanding the mechanisms of cellular processes. The availability of interactome data has catalyzed the development of computational approaches to elucidate functional behaviors of proteins on a system level. Gene Ontology (GO) and its annotations are a significant resource for functional characterization of proteins. Because of wide coverage, GO data have often been adopted as a benchmark for protein function prediction on the genomic scale. Results We propose a computational approach, called M-Finder, for functional association pattern mining. This method employs semantic analytics to integrate the genome-wide PPIs with GO data. We also introduce an interactive web application tool that visualizes a functional association network linked to a protein specified by a user. The proposed approach comprises two major components. First, the PPIs that have been generated by high-throughput methods are weighted in terms of their functional consistency using GO and its annotations. We assess two advanced semantic similarity metrics which quantify the functional association level of each interacting protein pair. We demonstrate that these measures outperform the other existing methods by evaluating their agreement to other biological features, such as sequence similarity, the presence of common Pfam domains, and core PPIs. Second, the information flow-based algorithm is employed to discover a set of proteins functionally associated with the protein in a query and their links efficiently. This algorithm reconstructs a functional association network of the query protein. The output network size can be flexibly determined by parameters. Conclusions M-Finder provides a useful framework to investigate functional association patterns with any protein. This software will also allow users to perform further systematic analysis of a set of proteins for any specific function. It is available online at http

  10. Co-Association of Cytochrome f Catabolites and Plastid-Lipid-Associated Protein with Chloroplast Lipid Particles1

    PubMed Central

    Smith, Matthew D.; Licatalosi, Donny D.; Thompson, John E.

    2000-01-01

    Distinguishable populations of lipid particles isolated from chloroplasts of yellow wax bean (Phaseolus vulgaris L. cv Kinghorn Wax) leaves have been found to contain plastid-lipid-associated protein (J. Pozueta-Romero, F. Rafia, G. Houlné, C. Cheniclet, J.P. Carde, M.-L. Schantz, R. Schantz [1997] Plant Physiol 115: 1185–1194). One population is comprised of plastoglobuli obtained from sonicated chloroplasts by flotation centrifugation. Higher density lipid-protein particles isolated from chloroplast stroma by ultrafiltration constitute a second population. Inasmuch as the stromal lipid-protein particles contain plastid-lipid-associated protein, but are distinguishable from plastoglobuli in terms of their lipid and protein composition, they appear to be plastoglobuli-like particles. Of particular interest is the finding that plastoglobuli and the higher density lipid-protein particles both contain catabolites of the thylakoid protein, cytochrome f. These observations support the view that there are distinguishable populations of plastoglobuli-like particles in chloroplasts. They further suggest that the formation of these particles may allow removal of protein catabolites from the thylakoid membrane that are destined for degradation as part of normal thylakoid turnover. PMID:10982436

  11. Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

    PubMed Central

    Bartee, Eric; Eyster, Craig A.; Viswanathan, Kasinath; Mansouri, Mandana; Donaldson, Julie G.; Früh, Klaus

    2010-01-01

    Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins. PMID:21151997

  12. Membrane-Associated RING-CH proteins associate with Bap31 and target CD81 and CD44 to lysosomes.

    PubMed

    Bartee, Eric; Eyster, Craig A; Viswanathan, Kasinath; Mansouri, Mandana; Donaldson, Julie G; Früh, Klaus

    2010-12-02

    Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

  13. Analysis of low-density lipoprotein-associated proteins using the method of digitized native protein mapping.

    PubMed

    Jin, Ya; Chen, Jin; Wang, Ahui; Zhang, Jun; Chen, Shumin; Manabe, Takashi; Tan, Wen

    2016-07-01

    The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS (in data-independent acquisition mode, or MS(E) ), was improved by using a new MS/MS mode, ion mobility separation enhanced-MS(E) (HDMS(E) ), and applied to analyze the area of human plasma low-density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid-cut into 72 square gel pieces and subjected to quantitative LC-MS/MS. Compared with MS(E) , HDMS(E) showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC-HDMS(E) and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B-100 was the most abundant protein in the grid-cut area, concentrated at pI ca. 5.4-6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39-42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B-100. Twenty-two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL.

  14. Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

    PubMed Central

    Piñol-Roma, Serafín

    1999-01-01

    rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits. PMID:9880328

  15. The ankyrin repeat containing SOCS box protein 5: a novel protein associated with arteriogenesis.

    PubMed

    Boengler, Kerstin; Pipp, Frederic; Fernandez, Borja; Richter, Alexandra; Schaper, Wolfgang; Deindl, Elisabeth

    2003-02-28

    Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbit by occlusion of the femoral artery. In order to identify and characterize genes differentially expressed during the early phase of arteriogenesis, cDNA of collateral arteries 24h after femoral ligation or sham operation was subjected to suppression subtractive hybridization. We identified the ankyrin repeat containing SOCS box protein 5 (asb5) and cloned the rabbit full-length cDNA. Asb5 was demonstrated to be a single-copy gene. We localized the asb5 protein in vivo in endothelial and smooth muscle cells of collateral arteries as well as in satellite cells. Asb5 was significantly upregulated in growing collateral arteries on mRNA and protein level. The infusion of doxorubicin in rabbit led to a significant decrease of the asb5 mRNA. In summary, our data show that asb5 is a novel protein implicated in the initiation of arteriogenesis.

  16. Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit.

    PubMed

    Bañó-Polo, Manuel; Martínez-Garay, Carlos A; Grau, Brayan; Martínez-Gil, Luis; Mingarro, Ismael

    2017-05-01

    Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.

  17. Associations between milk protein concentration at various stages of lactation and reproductive performance in dairy cows.

    PubMed

    Morton, J M; Auldist, M J; Douglas, M L; Macmillan, K L

    2016-12-01

    Milk protein concentration has been positively associated with a range of measures of reproductive performance in dairy cows. These beneficial associations are most likely due to factors affecting both milk protein concentration and reproductive performance possibly being mediated, in part, by energy balance during early lactation. However, it is likely that factors other than energy balance are also involved in these relationships. A retrospective single cohort study was conducted using subsets of data collected from 74 dairy herds with seasonal or split calving patterns. Associations between milk protein concentration at various stages of lactation and reproductive performance in Holstein dairy cows were assessed using random effects logistic regression and survival analysis with milk protein concentration during the cow's breeding period fitted as a time-varying covariate. The beneficial associations between milk protein concentration and each of the 4 selected indices for measuring reproductive performance were evident when milk protein concentration was derived for each 30-d period from calving up to 300d in milk. For the first 150d of lactation the adjusted odds ratios were highest from 31 to 60d and only slightly lower for all periods up to 150d of lactation. Estimated associations for 31 to 60d were stronger than for 0 to 30d. In addition, milk protein concentration during a cow's breeding period was positively associated with the subsequent daily hazard of conception, even after adjusting for milk protein concentration in the cow's first or second month of lactation. Milk protein concentrations from 0 to 30d of lactation were less closely correlated with concentrations measured at subsequent 30-d intervals; correlations were closer between other periods in lactation. These results indicate that the association between milk protein concentration and reproductive performance is partly due to factors other than the extent of negative energy balance in early

  18. Axoplasmic transport of microtubule-associated proteins in the rat sciatic nerve

    SciTech Connect

    Takenaka, T.; Inomata, K.

    1981-09-01

    /sup 32/P-ATP was injected into the L5 dorsal root ganglion and axoplasmic transport of the phosphorylate MA proteins 2, microtubule-associated proteins 2, was observed. After the injection of /sup 32/P-ATP, the nerve was dissected out at prescribed time intervals and sliced into 5-mm pieces. Each segment was electrophoresed on an SDS-polyacrylamide slab gel and subjected to autoradiography. A protein of 310,000 dalton was transported at a velocity of 6.6-10.6 mm/day in the axon with the electrophoretic mobility identical to that of MA proteins 2, one of the key components associated with the microtubules.

  19. Surfactant protein A2 mutations associated with pulmonary fibrosis lead to protein instability and endoplasmic reticulum stress.

    PubMed

    Maitra, Meenakshi; Wang, Yongyu; Gerard, Robert D; Mendelson, Carole R; Garcia, Christine Kim

    2010-07-16

    Rare heterozygous mutations in the gene encoding surfactant protein A2 (SP-A2, SFTPA2) are associated with adult-onset pulmonary fibrosis and adenocarcinoma of the lung. We have previously shown that two recombinant SP-A2 mutant proteins (G231V and F198S) remain within the endoplasmic reticulum (ER) of A549 cells and are not secreted into the culture medium. The pathogenic mechanism of the mutant proteins is unknown. Here we analyze all common and rare variants of the surfactant protein A2, SP-A2, in both A549 cells and in primary type II alveolar epithelial cells. We show that, in contrast with all other SP-A2 variants, the mutant proteins are not secreted into the medium with wild-type SP-A isoforms, form fewer intracellular dimer and trimer oligomers, are partially insoluble in 0.5% Nonidet P-40 lysates of transfected A549 cells, and demonstrate greater protein instability in chymotrypsin proteolytic digestions. Both the G231V and F198S mutant SP-A2 proteins are destroyed via the ER-association degradation pathway. Expression of the mutant proteins increases the transcription of a BiP-reporter construct, expression of BiP protein, and production of an ER stress-induced XBP-1 spliced product. Human bronchoalveolar wash samples from individuals who are heterozygous for the G231V mutation have similar levels of total SP-A as normal family members, which suggests that the mechanism of disease does not involve an overt lack of secreted SP-A but instead involves an increase in ER stress of resident type II alveolar epithelial cells.

  20. Ligand induced galectin-3 protein self-association.

    PubMed

    Lepur, Adriana; Salomonsson, Emma; Nilsson, Ulf J; Leffler, Hakon

    2012-06-22

    Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in cross-linking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here we studied interaction of galectin-3 with the model glycoprotein asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concentration of free galectin carbohydrate recognition sites in solution. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concentrations, showing that many more galectin-3 molecules were engaged than expected due to the about nine known glycan-based binding sites per ASF molecule. This suggests that after ASF-induced nucleation, galectin-3 associates with itself by the carbohydrate recognition site binding to another galectin-3 molecule, possibly forming oligomers. We named this type-C self-association to distinguish it from the previously proposed models (type-N) where galectin-3 molecules bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-association can result in precipitates, as measured here by turbidimetry and dynamic light scattering. Type-C self-association and precipitation occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required much higher concentration (∼50 μM) as compared with wild type (∼1 μM). ASF also induced weaker type-C self-association of galectin-3 lacking its N-terminal domains, but as expected, no precipitation. Neither a monovalent nor a divalent N-acetyl-D-lactosamine-containing glycan induced type-C self-association, even if the latter gave precipitates with high concentrations of galectin-3 (>∼50 μM) in agreement with published results and perhaps due to type-N self-association.

  1. Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin

    SciTech Connect

    Leiman, Petr G.; Basler, Marek; Ramagopal, Udupi A.; Bonanno, Jeffrey B.; Sauder, J. Michael; Pukatzki, Stefan; Burley, Stephen K.; Almo, Steven C.; Mekalanos, John J.

    2009-04-22

    Protein secretion is a common property of pathogenic microbes. Gram-negative bacterial pathogens use at least 6 distinct extracellular protein secretion systems to export proteins through their multilayered cell envelope and in some cases into host cells. Among the most widespread is the newly recognized Type VI secretion system (T6SS) which is composed of 15--20 proteins whose biochemical functions are not well understood. Using crystallographic, biochemical, and bioinformatic analyses, we identified 3 T6SS components, which are homologous to bacteriophage tail proteins. These include the tail tube protein; the membrane-penetrating needle, situated at the distal end of the tube; and another protein associated with the needle and tube. We propose that T6SS is a multicomponent structure whose extracellular part resembles both structurally and functionally a bacteriophage tail, an efficient machine that translocates proteins and DNA across lipid membranes into cells.

  2. Proteomic Study of Retinal Proteins Associated with Transcorneal Electric Stimulation in Rats

    PubMed Central

    Kanamoto, Takashi; Souchelnytskyi, Nazariy; Kurimoto, Takuji; Ikeda, Yasuhiro; Sakaue, Hiroaki; Munemasa, Yasunari; Kiuchi, Yoshiaki

    2015-01-01

    Background. To investigate how transcorneal electric stimulation (TES) affects the retina, by identifying those proteins up- and downregulated by transcorneal electric stimulation (TES) in the retina of rats. Methods. Adult Wistar rats received TES on the left eyes at different electrical currents while the right eyes received no treatment and served as controls. After TES, the eye was enucleated and the retina was isolated. The retinas were analyzed by proteomics. Results. Proteomics showed that twenty-five proteins were upregulated by TES. The identified proteins included cellular signaling proteins, proteins associated with neuronal transmission, metabolic proteins, immunological factors, and structural proteins. Conclusions. TES induced changes in expression of various functional proteins in the retina. PMID:25821588

  3. Proteomic study of retinal proteins associated with transcorneal electric stimulation in rats.

    PubMed

    Kanamoto, Takashi; Souchelnytskyi, Nazariy; Kurimoto, Takuji; Ikeda, Yasuhiro; Sakaue, Hiroaki; Munemasa, Yasunari; Kiuchi, Yoshiaki

    2015-01-01

    Background. To investigate how transcorneal electric stimulation (TES) affects the retina, by identifying those proteins up- and downregulated by transcorneal electric stimulation (TES) in the retina of rats. Methods. Adult Wistar rats received TES on the left eyes at different electrical currents while the right eyes received no treatment and served as controls. After TES, the eye was enucleated and the retina was isolated. The retinas were analyzed by proteomics. Results. Proteomics showed that twenty-five proteins were upregulated by TES. The identified proteins included cellular signaling proteins, proteins associated with neuronal transmission, metabolic proteins, immunological factors, and structural proteins. Conclusions. TES induced changes in expression of various functional proteins in the retina.

  4. Homologs of the yeast Tvp38 vesicle-associated protein are conserved in chloroplasts and cyanobacteria

    PubMed Central

    Keller, Rebecca; Schneider, Dirk

    2013-01-01

    Vesicle transfer processes in eukaryotes depend on specific proteins, which mediate the selective packing of cargo molecules for subsequent release out of the cells after vesicle fusion to the plasma membrane. The protein Tvp38 is conserved in yeasts and higher eukaryotes and potentially involved in vesicle transfer processes at the Golgi membrane. Members of the so-called “SNARE-associated proteins of the Tvp38-family” have also been identified in prokaryotes and those belong to the DedA protein family. Tvp38/DedA proteins are also conserved in cyanobacteria and chloroplasts. While only a single member of this family appears to be present in chloroplasts, cyanobacterial genomes typically encode multiple homologous proteins. Mainly based on our understanding of the DedA-homologous proteins of Escherichia coli, it appears likely that the function of these proteins in chloroplast and cyanobacteria involves stabilizing and organizing the structure of internal membrane systems. PMID:24312110

  5. Effects of a high protein diet on body weight and comorbidities associated with obesity.

    PubMed

    Clifton, Peter

    2012-08-01

    Red meat intake has been frequently associated with the development of coronary artery disease and type 2 diabetes but vegetable protein has been associated with protection from these diseases. Whether this is related to the protein per se or to the increased polyunsaturated fat or higher fibre levels associated with more vegetarian diets is not clear. High protein diets are associated with greater satiety and in some studies are associated with greater weight loss compared with high carbohydrate diets especially in an ad libitum design. These diets also lower plasma triglyceride and blood pressure and sometimes spare lean mass. There appear to be no harmful effects of high protein diets on bone density or renal function in weight loss studies.

  6. A Golgi-associated protein 4.1B variant is required for assimilation of proteins in the membrane.

    PubMed

    Kang, Qiaozhen; Wang, Ting; Zhang, Huizheng; Mohandas, Narla; An, Xiuli

    2009-04-15

    The archetypal membrane skeleton is that of the erythrocyte, consisting predominantly of spectrin, actin, ankyrin R and protein 4.1R. The presence in the Golgi of a membrane skeleton with a similar structure has been inferred, based on the identification of Golgi-associated spectrin and ankyrin. It has long been assumed that a Golgi-specific protein 4.1 must also exist, but it has not previously been found. We demonstrate here that a hitherto unknown form of protein 4.1, a 200 kDa 4.1B, is associated with the Golgi of Madin-Darby canine kidney (MDCK) and human bronchial epithelial (HBE) cells. This 4.1B variant behaves like a Golgi marker after treatment with Brefeldin A and during mitosis. Depletion of the protein in HBE cells by siRNA resulted in disruption of the Golgi structure and failure of Na(+)/K(+)-ATPase, ZO-1 and ZO-2 to migrate to the membrane. Thus, this newly identified Golgi-specific protein 4.1 appears to have an essential role in maintaining the structure of the Golgi and in assembly of a subset of membrane proteins.

  7. Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

    PubMed Central

    Huang, Da Wei; Fanti, Laura; Pak, Daniel T.S.; Botchan, Michael R.; Pimpinelli, Sergio; Kellum, Rebecca

    1998-01-01

    The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae. PMID:9679132

  8. Identification of genes and proteins associated with anagen wool growth.

    PubMed

    Zhao, J; Liu, N; Liu, K; He, J; Yu, J; Bu, R; Cheng, M; De, W; Liu, J; Li, H

    2017-02-01

    Identifying genes of major effect for wool growth would offer strategies for improving the quality and increasing the yield of fine wool. In this study, we employed the Agilent Sheep Gene Expression Microarray and proteomic technology to investigate the gene expression patterns of body side skin (more wool growing) in Aohan fine wool sheep (a Chinese indigenous breed) in comparison with groin skin (no wool growing) at the anagen stage of the wool follicle. A microarray study revealed that 4772 probes were differentially expressed, including 2071 upregulated and 2701 downregulated probes, in the comparisons of body side skin vs. groin skin (S/G). The microarray results were verified by means of quantitative PCR. A total of 1099 probes were assigned to unique genes/transcripts. The number of distinct genes/transcripts (annotated) was 926, of which 352 were upregulated and 574 were downregulated. In S/G, 13 genes were upregulated by more than 10 fold, whereas 60 genes were downregulated by more than 10 fold. Further analysis revealed that the majority of the genes possibly related to the wool growth could be assigned to categories including regulation of cell division, intermediate filament, cytoskeletal part and growth factor activity. Several potential gene families may participate in hair growth regulation, including fibroblast growth factors, transforming growth factor-β, WNTs, insulin-like growth factor, vascular endothelial growth factors and so on. Proteomic analysis also revealed 196 differentially expressed protein points, of which 121 were identified as single protein points.

  9. Proteins Associated with SF3a60 in T. brucei

    PubMed Central

    Nyambega, Benson; Helbig, Claudia; Masiga, Daniel K.; Clayton, Christine; Levin, Mariano J.

    2014-01-01

    Trypanosoma brucei relies on Spliced leader trans splicing to generate functional messenger RNAs. Trans splicing joins the specialized SL exon from the SL RNA to pre-mRNAs and is mediated by the trans-spliceosome, which is made up of small nuclear ribonucleoprotein particles and non-snRNP factors. Although the trans spliceosome is essential for trypanosomatid gene expression, not all spliceosomal protein factors are known and of these, only a few are completely characterized. In this study, we have characterized the trypanosome Splicing Factor, SF3a60, the only currently annotated SF3a component. As expected, epitope-tagged SF3a60 localizes in the trypanosome nucleus. SF3a60 is essential for cell viability but its depletion seem to have no detectable effect on trans-splicing. In addition, we used SF3a60 as bait in a Yeast-2-hybrid system screen and identified its interacting protein factors. The interactions with SF3a120, SF3a66 and SAP130 were confirmed by tandem affinity purification and mass spectrometry. PMID:24651488

  10. Finding undetected protein associations in cell signaling by belief propagation

    PubMed Central

    Bailly-Bechet, M.; Borgs, C.; Braunstein, A.; Chayes, J.; Dagkessamanskaia, A.; François, J.-M.; Zecchina, R.

    2011-01-01

    External information propagates in the cell mainly through signaling cascades and transcriptional activation, allowing it to react to a wide spectrum of environmental changes. High-throughput experiments identify numerous molecular components of such cascades that may, however, interact through unknown partners. Some of them may be detected using data coming from the integration of a protein–protein interaction network and mRNA expression profiles. This inference problem can be mapped onto the problem of finding appropriate optimal connected subgraphs of a network defined by these datasets. The optimization procedure turns out to be computationally intractable in general. Here we present a new distributed algorithm for this task, inspired from statistical physics, and apply this scheme to alpha factor and drug perturbations data in yeast. We identify the role of the COS8 protein, a member of a gene family of previously unknown function, and validate the results by genetic experiments. The algorithm we present is specially suited for very large datasets, can run in parallel, and can be adapted to other problems in systems biology. On renowned benchmarks it outperforms other algorithms in the field. PMID:21187432

  11. A large solvent isotope effect on protein association thermodynamics.

    PubMed

    Eginton, Christopher; Beckett, Dorothy

    2013-09-24

    Solvent reorganization can contribute significantly to the energetics of protein-protein interactions. However, our knowledge of the magnitude of the energetic contribution is limited, in part, by a dearth of quantitative experimental measurements. The biotin repressor forms a homodimer as a prerequisite to DNA binding to repress transcription initiation. At 20 °C, the dimerization reaction, which is thermodynamically coupled to binding of a small ligand, bio-5'-AMP, is characterized by a Gibbs free energy of -7 kcal/mol. This modest net dimerization free energy reflects underlying, very large opposing enthalpic and entropic driving forces of 41 ± 3 and -48 ± 3 kcal/mol, respectively. The thermodynamics have been interpreted as indicating coupling of solvent release to dimerization. In this work, this interpretation has been investigated by measuring the effect of replacing H2O with D2O on the dimerization thermodynamics. Sedimentation equilibrium measurements performed at 20 °C reveal a solvent isotope effect of -1.5 kcal/mol on the Gibbs free energy of dimerization. Analysis of the temperature dependence of the reaction in D2O indicates enthalpic and entropic contributions of 28 and -37 kcal/mol, respectively, considerably smaller than the values measured in H2O. These large solvent isotope perturbations to the thermodynamics are consistent with a significant contribution of solvent release to the dimerization reaction.

  12. An inventory of yeast proteins associated with nucleolar and ribosomal components

    PubMed Central

    Staub, Eike; Mackowiak, Sebastian; Vingron, Martin

    2006-01-01

    Background Although baker's yeast is a primary model organism for research on eukaryotic ribosome assembly and nucleoli, the list of its proteins that are functionally associated with nucleoli or ribosomes is still incomplete. We trained a naïve Bayesian classifier to predict novel proteins that are associated with yeast nucleoli or ribosomes based on parts lists of nucleoli in model organisms and large-scale protein interaction data sets. Phylogenetic profiling and gene expression analysis were carried out to shed light on evolutionary and regulatory aspects of nucleoli and ribosome assembly. Results We predict that, in addition to 439 known proteins, a further 62 yeast proteins are associated with components of the nucleolus or the ribosome. The complete set comprises a large core of archaeal-type proteins, several bacterial-type proteins, but mostly eukaryote-specific inventions. Expression of nucleolar and ribosomal genes tends to be strongly co-regulated compared to other yeast genes. Conclusion The number of proteins associated with nucleolar or ribosomal components in yeast is at least 14% higher than known before. The nucleolus probably evolved from an archaeal-type ribosome maturation machinery by recruitment of several bacterial-type and mostly eukaryote-specific factors. Not only expression of ribosomal protein genes, but also expression of genes encoding the 90S processosome, are strongly co-regulated and both regulatory programs are distinct from each other. PMID:17067374

  13. Detergent-associated Solution Conformations of Helical and Beta-barrel Membrane Proteins

    SciTech Connect

    Mo, Yiming; Lee, Byung-Kwon; Ankner, John Francis; Becker, Jeffrey Marvin; Heller, William T

    2008-01-01

    Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the ?-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

  14. Expression of synaptosomal-associated protein-25 in the rat brain after subarachnoid hemorrhage

    PubMed Central

    Chen, Gang; Hu, Tong; Li, Qi; Li, Jianke; Jia, Yang; Wang, Zhong

    2013-01-01

    Synaptosomal-associated protein-25 is an important factor for synaptic functions and cognition. In this study, subarachnoid hemorrhage models with spatial learning disorder were established through a blood injection into the chiasmatic cistern. Immunohistochemical staining and western blot analysis results showed that synaptosomal-associated protein-25 expression in the temporal lobe, hippocampus, and cerebellum significantly lower at days 1 and 3 following subarachnoid morrhage. Our findings indicate that synaptosomal-associated protein-25 expression was down-regulated in the rat brain after subarachnoid hemorrhage. PMID:25206580

  15. The Associations of Plant Protein Intake With All-Cause Mortality in CKD.

    PubMed

    Chen, Xiaorui; Wei, Guo; Jalili, Thunder; Metos, Julie; Giri, Ajay; Cho, Monique E; Boucher, Robert; Greene, Tom; Beddhu, Srinivasan

    2016-03-01

    Plant protein intake is associated with lower production of uremic toxins and lower serum phosphorus levels. Therefore, at a given total protein intake, a higher proportion of dietary protein from plant sources might be associated with lower mortality in chronic kidney disease. Observational study. 14,866 NHANES III participants 20 years or older without missing data for plant and animal protein intake and mortality. Plant protein to total protein ratio and total plant protein intake. Patients were stratified by estimated glomerular filtration rate (eGFR)<60 or ≥60mL/min/1.73m(2). All-cause mortality. Plant and total protein intakes were estimated from 24-hour dietary recalls. Mortality was ascertained by probabilistic linkage with National Death Index records through December 31, 2000. Mean values for plant protein intake and plant protein to total protein ratio were 24.6±13.2 (SD) g/d and 33.0% ± 14.0%, respectively. The prevalence of eGFRs<60mL/min/1.73m(2) was 4.9%. There were 2,163 deaths over an average follow-up of 8.4 years. Adjusted for demographics, smoking, alcohol use, comorbid conditions, body mass index, calorie and total protein intake, and physical inactivity, each 33% increase in plant protein to total protein ratio was not associated with mortality (HR, 0.88; 95% CI, 0.74-1.04) in the eGFR≥60mL/min/1.73m(2) subpopulation, but was associated with lower mortality risk (HR, 0.77; 95% CI, 0.61-0.96) in the eGFR<60mL/min/1.73m(2) subpopulation. In sensitivity analyses, results were similar in those with eGFR<60mL/min/1.73m(2) defined by serum cystatin C level. Whether results are related to plant protein itself or to other factors associated with more plant-based diets is difficult to establish. A diet with a higher proportion of protein from plant sources is associated with lower mortality in those with eGFR<60mL/min/1.73m(2). Future studies are warranted to determine the causal role of plant protein intake in reducing mortality in those with e

  16. Deficiency of cyclase-associated protein 2 promotes arrhythmias associated with connexin43 maldistribution and fibrosis

    PubMed Central

    Peche, Vivek Shahaji; Linhart, Markus; Nickenig, Georg; Noegel, Angelika Anna; Schrickel, Jan Wilko

    2016-01-01

    Introduction Cyclase-associated protein 2 (CAP2) plays a major role in regulating the actin cytoskeleton. Since inactivation of CAP2 in a mouse model by a gene trap approach (Cap2gt/gt) results in cardiomyopathy and increased mortality, we hypothesized that CAP2 has a major impact on arrhythmias and electrophysiological parameters. Material and methods We performed long-term-ECG recordings in transgenic CAP2 deficient mice (C57BL/6) to detect spontaneous arrhythmias. In vivo electrophysiological studies by right heart catheterization and ex vivo epicardial mapping were used to analyze electrophysiological parameters, the inducibility of arrhythmias, and conduction velocities. Expression and distribution of cardiac connexins and the amount of cardiac fibrosis were evaluated. Results Spontaneous ventricular arrhythmias could be detected in Cap2gt/gt during the long-term-ECG recording. Cap2gt/gt showed marked conduction delays at atrial and ventricular levels, including a reduced heart rate (421.0 ±40.6 bpm vs. 450.8 ±27.9 bpm; p < 0.01), and prolongations of PQ (46.3 ±4.1 ms vs. 38.6 ±6.5 ms; p < 0.01), QRS (16.2 ±2.6 ms vs. 12.6 ±1.4 ms; p < 0.01), and QTc interval (55.8 ±6.0 ms vs. 45.2 ±3.3 ms; p = 0.02) in comparison to wild type mice. The PQ prolongation was due to an infra-Hisian conduction delay (HV: 9.7 ±2.1 ms vs. 6.5 ±3.1 ms; p = 0.02). The inducibility of ventricular tachycardias during the electrophysiological studies was significantly elevated in the mutant mice (inducible animals: 88% vs. 33%; p = 0.04). Cap2gt/gt showed more abnormal distribution of connexin43 compared to WT (23.0 ±4.7% vs. 2.9 ±0.8%; p < 0.01). Myocardial fibrosis was elevated in Cap2gt/gt hearts (9.1 ±6.7% vs. 5.5 ±3.3%; p < 0.01). Conclusions Loss of CAP2 results in marked electrophysiological disturbances including impaired sinus node function, conduction delays, and susceptibility to malignant arrhythmias. Structural changes in Cap2gt/gt are associated with

  17. Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics.

    PubMed

    Xiao, Lin; Xian, Hongxu; Lee, Kit Yee; Xiao, Bin; Wang, Hongyan; Yu, Fengwei; Shen, Han-Ming; Liou, Yih-Cherng

    2015-10-09

    Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

    PubMed

    Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E

    1996-07-19

    We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

  19. The myeloid leukemia-associated protein SET is a potent inhibitor of protein phosphatase 2A.

    PubMed

    Li, M; Makkinje, A; Damuni, Z

    1996-05-10

    Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor proteins designated I1PP2A and I2PP2A have been purified to apparent homogeneity from extracts of bovine kidney (Li, M., Guo, H., and Damuni, Z. (1995) Biochemistry 34, 1988-1996). N-terminal and internal amino acid sequencing indicated that I2PP2A was a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia. Experiments using purified preparations of recombinant human SET confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 2 nM SET. By contrast, SET (up to 20 nM) did not affect the activities of purified preparations of protein phosphatases 1, 2B, and 2C. The results indicate that SET is a potent and specific inhibitor of PP2A and suggest that impaired regulation of PP2A may contribute to acute myeloid leukemogenesis.

  20. Identification and partial characterization of mitotic centromere- associated kinesin, a kinesin-related protein that associates with centromeres during mitosis

    PubMed Central

    1995-01-01

    Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message. PMID:7822426

  1. Vaccinia virus p37 interacts with host proteins associated with LE-derived transport vesicle biogenesis.

    PubMed

    Chen, Yali; Honeychurch, Kady M; Yang, Guang; Byrd, Chelsea M; Harver, Chris; Hruby, Dennis E; Jordan, Robert

    2009-04-28

    Proteins associated with the late endosome (LE) appear to play a central role in the envelopment of a number of taxonomically diverse viruses. How viral proteins interact with LE-associated proteins to facilitate envelopment is not well understood. LE-derived transport vesicles form through the interaction of Rab9 GTPase with cargo proteins, and TIP47, a Rab9-specific effector protein. Vaccinia virus (VV) induces a wrapping complex derived from intracellular host membranes to envelope intracellular mature virus particles producing egress-competent forms of virus. We show that VV p37 protein associates with TIP47-, Rab9-, and CI-MPR-containing membranes. Mutation of a di-aromatic motif in p37 blocks association with TIP47 and inhibits plaque formation. ST-246, a specific inhibitor of p37 function, inhibits these interactions and also blocks wrapped virus particle formation. Vaccinia virus expressing p37 variants with reduced ST-246 susceptibility associates with Rab9 and co-localizes with CI-MPR in the presence and absence of compound. These results suggest that p37 localizes to the LE and interacts with proteins associated with LE-derived transport vesicle biogenesis to facilitate assembly of extracellular forms of virus.

  2. Vaccinia virus p37 interacts with host proteins associated with LE-derived transport vesicle biogenesis

    PubMed Central

    Chen, Yali; Honeychurch, Kady M; Yang, Guang; Byrd, Chelsea M; Harver, Chris; Hruby, Dennis E; Jordan, Robert

    2009-01-01

    Background Proteins associated with the late endosome (LE) appear to play a central role in the envelopment of a number of taxonomically diverse viruses. How viral proteins interact with LE-associated proteins to facilitate envelopment is not well understood. LE-derived transport vesicles form through the interaction of Rab9 GTPase with cargo proteins, and TIP47, a Rab9-specific effector protein. Vaccinia virus (VV) induces a wrapping complex derived from intracellular host membranes to envelope intracellular mature virus particles producing egress-competent forms of virus. Results We show that VV p37 protein associates with TIP47-, Rab9-, and CI-MPR-containing membranes. Mutation of a di-aromatic motif in p37 blocks association with TIP47 and inhibits plaque formation. ST-246, a specific inhibitor of p37 function, inhibits these interactions and also blocks wrapped virus particle formation. Vaccinia virus expressing p37 variants with reduced ST-246 susceptibility associates with Rab9 and co-localizes with CI-MPR in the presence and absence of compound. Conclusion These results suggest that p37 localizes to the LE and interacts with proteins associated with LE-derived transport vesicle biogenesis to facilitate assembly of extracellular forms of virus. PMID:19400954

  3. RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction.

    PubMed

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-07-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. © 2014 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  4. Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation

    PubMed Central

    Kauwe, John S. K.; Bailey, Matthew H.; Ridge, Perry G.; Perry, Rachel; Wadsworth, Mark E.; Hoyt, Kaitlyn L.; Staley, Lyndsay A.; Karch, Celeste M.; Harari, Oscar; Cruchaga, Carlos; Ainscough, Benjamin J.; Bales, Kelly; Pickering, Eve H.; Bertelsen, Sarah; Fagan, Anne M.; Holtzman, David M.; Morris, John C.; Goate, Alison M.

    2014-01-01

    Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10−10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal

  5. Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus ▿

    PubMed Central

    Chen, Wuguo; Dittmer, Dirk P.

    2011-01-01

    The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA-interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA. PMID:21734034

  6. Specific Association of Growth-associated Protein 43 with Calcium Release Units in Skeletal Muscles of Lower Vertebrates

    PubMed Central

    Caprara, G.A.; Perni, S.; Morabito, C.; Mariggiò, M.A.; Guarnieri, S.

    2014-01-01

    Growth-associated protein 43 (GAP43), is a strictly conserved protein among vertebrates implicated in neuronal development and neurite branching. Since GAP43 structure contains a calmodulin-binding domain, this protein is able to bind calmodulin and gather it nearby membrane network, thus regulating cytosolic calcium and consequently calcium-dependent intracellular events. Even if for many years GAP43 has been considered a neuronal-specific protein, evidence from different laboratories described its presence in myoblasts, myotubes and adult skeletal muscle fibers. Data from our laboratory showed that GAP43 is localized between calcium release units (CRUs) and mitochondria in mammalian skeletal muscle suggesting that, also in skeletal muscle, this protein can be a key player in calcium/calmodulin homeostasis. However, the previous studies could not clearly distinguish between a mitochondrion- or a triad-related positioning of GAP43. To solve this question, the expression and localization of GAP43 was studied in skeletal muscle of Xenopus and Zebrafish known to have triads located at the level of the Z-lines and mitochondria not closely associated with them. Western blotting and immunostaining experiments revealed the expression of GAP43 also in skeletal muscle of lower vertebrates (like amphibians and fishes), and that the protein is localized closely to the triad junction. Once more, these results and GAP43 structural features, support an involvement of the protein in the dynamic intracellular Ca2+ homeostasis, a common conserved role among the different species. PMID:25578978

  7. Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins.

    PubMed

    Mackenzie, Kimberly D; Lim, Yoon; Duffield, Michael D; Chataway, Timothy; Zhou, Xin-Fu; Keating, Damien J

    2017-07-01

    Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1(-/-) cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. ERD2 proteins mediate ER retention of the HNEL signal of LRP's receptor-associated protein (RAP).

    PubMed

    Bu, G; Rennke, S; Geuze, H J

    1997-01-01

    The 39 kDa receptor-associated protein (RAP) is a receptor antagonist that interacts with several members of the low density lipoprotein (LDL) receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. Our recent studies have demonstrated that RAP is an endoplasmic reticulum (ER) resident protein and an intracellular chaperone for the LDL receptor-related protein (LRP). The HNEL sequence at the carboxyl terminus of RAP represents a novel ER retention signal that shares homology with the well-characterized KDEL signal. In the present study, using immunoelectron microscopy we demonstrate that cells stably transfected with human growth hormone (GH) tagged with either KDEL (GH + KDEL) or HNEL (GH + HNEL) signals exhibit ER and cis-Golgi localization typical of ER-retained proteins. Overexpression of not only GH + HNEL but also GH + KDEL cDNA in transfected cells results in saturation of ER retention receptors and secretion of endogenous RAP indicating that the two signals interact with the same ER retention receptor(s). The role of RAP in the maturation of LRP is further supported by the observation that functional LRP is reduced about 60% as a result of decreased intracellular RAP. Pulse-chase labeling and immunolocalization studies of ERD2.1 and ERD2.2 proteins in transfected cells demonstrate a long half-life and Golgi localization for both receptors. Finally, overexpression of either ERD2.1 or ERD2.2 proteins significantly increases the capacity of cells to retain both KDEL and HNEL-containing proteins. Taken together, our results thus demonstrate that ERD2 proteins are capable of retaining the novel ER retention signal associated with RAP.

  9. Integrins and Integrin-Associated Proteins in the Cardiac Myocyte

    PubMed Central

    Ross, Robert S.

    2014-01-01

    Integrins are heterodimeric, transmembrane receptors that are expressed in all cells, including those in the heart. They participate in multiple critical cellular processes including adhesion, extracellular matrix organization, signaling, survival, and proliferation. Particularly relevant for a contracting muscle cell, integrins are mechanotransducers, translating mechanical to biochemical information. While it is likely that cardiovascular clinicians and scientists have highest recognition of integrins in the cardiovascular system from drugs used to inhibit platelet aggregation, the focus of this article will be on the role of integrins specifically in the cardiac myocyte. Following a general introduction to integrin biology, the manuscript will discuss important work on integrin signaling, mechanotransduction, and lessons learned about integrin function from a range of model organisms. Then we will detail work on integrin-related proteins in the myocyte, how integrins may interact with ion channels and mediate viral uptake into cells, and also play a role in stem cell biology. Finally, we will discuss directions for future study. PMID:24481847

  10. [Relationship between tight junction proteins and Helicobacter pylori-associated gastric diseases].

    PubMed

    Li, Wei; Jiang, Mi-Zu

    2014-03-01

    Helicobacter pylori (Hp) infection is an important cause of chronic gastritis and peptic ulcer, but their pathogenesis is unclear. The role of gastric mucosal barrier dysfunction induced by impaired structure and function of tight junction in the pathogenesis of Hp-associated gastric diseases has received considerable attention in recent years. Tight junction is composed of a variety of proteins and molecules, including 3 integral membrane proteins (occludin, claudins, and junctional adhesion molecules) and a cytoplasmic protein (zonula occludens). This paper mainly describes the composition and function of various tight junction proteins, changes in tight junction protein function induced by Hp infection and their relationship with the incidence of gastric diseases, and the significance of enhancing the tight junction protein function in the prevention and treatment of Hp-associated gastric diseases.

  11. Protein homeostasis of a metastable subproteome associated with Alzheimer’s disease

    PubMed Central

    Kundra, Rishika; Ciryam, Prajwal; Morimoto, Richard I.; Dobson, Christopher M.; Vendruscolo, Michele

    2017-01-01

    Alzheimer’s disease is the most common cause of dementia. A hallmark of this disease is the presence of aberrant deposits containing by the Aβ peptide (amyloid plaques) and the tau protein (neurofibrillary tangles) in the brains of affected individuals. Increasing evidence suggests that the formation of these deposits is closely associated with the age-related dysregulation of a large set of highly expressed and aggregation-prone proteins, which make up a metastable subproteome. To understand in more detail the origins of such dysregulation, we identify specific components of the protein homeostasis system associated with these metastable proteins by using a gene coexpression analysis. Our results reveal the particular importance of the protein trafficking and clearance mechanisms, including specific branches of the endosomal–lysosomal and ubiquitin–proteasome systems, in maintaining the homeostasis of the metastable subproteome associated with Alzheimer’s disease. PMID:28652376

  12. Quantification of protein group coherence and pathway assignment using functional association

    PubMed Central

    2011-01-01

    Background Genomics and proteomics experiments produce a large amount of data that are awaiting functional elucidation. An important step in analyzing such data is to identify functional units, which consist of proteins that play coherent roles to carry out the function. Importantly, functional coherence is not identical with functional similarity. For example, proteins in the same pathway may not share the same Gene Ontology (GO) terms, but they work in a coordinated fashion so that the aimed function can be performed. Thus, simply applying existing functional similarity measures might not be the best solution to identify functional units in omics data. Results We have designed two scores for quantifying the functional coherence by considering association of GO terms observed in two biological contexts, co-occurrences in protein annotations and co-mentions in literature in the PubMed database. The counted co-occurrences of GO terms were normalized in a similar fashion as the statistical amino acid contact potential is computed in the protein structure prediction field. We demonstrate that the developed scores can identify functionally coherent protein sets, i.e. proteins in the same pathways, co-localized proteins, and protein complexes, with statistically significant score values showing a better accuracy than existing functional similarity scores. The scores are also capable of detecting protein pairs that interact with each other. It is further shown that the functional coherence scores can accurately assign proteins to their respective pathways. Conclusion We have developed two scores which quantify the functional coherence of sets of proteins. The scores reflect the actual associations of GO terms observed either in protein annotations or in literature. It has been shown that they have the ability to accurately distinguish biologically relevant groups of proteins from random ones as well as a good discriminative power for detecting interacting pairs of

  13. Microtubule-Associated Protein Expression and Predicting Taxane Response

    DTIC Science & Technology

    2009-10-01

    taxanes. Our results indicate that MAP- tau functions as a prognostic factor in both the Yale cohort and the TAX 307 cohort with high MAP- tau ...expression associated with longer overall survival and TTP. Tau does NOT behave as a predictor of response to taxane-based chemotherapy since differences...between low and high MAP- tau groups by treatment arm and response rate were not observed in the TAX 307 clinical trial cohort. Our data supports the

  14. Enhanced vulnerability of human proteins towards disease-associated inactivation through divergent evolution.

    PubMed

    Medina-Carmona, Encarnación; Fuchs, Julian E; Gavira, Jose A; Mesa-Torres, Noel; Neira, Jose L; Salido, Eduardo; Palomino-Morales, Rogelio; Burgos, Miguel; Timson, David J; Pey, Angel L

    2017-09-15

    Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine:glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please

  15. High dietary protein intake is associated with an increased body weight and total death risk.

    PubMed

    Hernández-Alonso, Pablo; Salas-Salvadó, Jordi; Ruiz-Canela, Miguel; Corella, Dolores; Estruch, Ramón; Fitó, Montserrat; Arós, Fernando; Gómez-Gracia, Enrique; Fiol, Miquel; Lapetra, José; Basora, Josep; Serra-Majem, Lluis; Muñoz, Miguel Ángel; Buil-Cosiales, Pilar; Saiz, Carmen; Bulló, Mònica

    2016-04-01

    High dietary protein diets are widely used to manage overweight and obesity. However, there is a lack of consensus about their long-term efficacy and safety. Therefore, the aim of this study was to assess the effect of long-term high-protein consumption on body weight changes and death outcomes in subjects at high cardiovascular risk. A secondary analysis of the PREDIMED trial was conducted. Dietary protein was assessed using a food-frequency questionnaire during the follow-up. Cox proportional hazard models were used to estimate the multivariate-adjusted hazard ratio (HR) and 95% confidence intervals (95%CI) for protein intake in relation to the risk of body weight and waist circumference changes, cardiovascular disease, cardiovascular death, cancer death and total death. Higher total protein intake, expressed as percentage of energy, was significantly associated with a greater risk of weight gain when protein replaced carbohydrates (HR: 1.90; 95%CI: 1.05, 3.46) but not when replaced fat (HR: 1.69; 95%CI: 0.94, 3.03). However, no association was found between protein intake and waist circumference. Contrary, higher total protein intake was associated with a greater risk of all-cause death in both carbohydrate and fat substitution models (HR: 1.59; 95%CI: 1.08, 2.35; and HR: 1.66; 95%CI: 1.13, 2.43, respectively). A higher consumption of animal protein was associated with an increased risk of fatal and non-fatal outcomes when protein substituted carbohydrates or fat. Higher dietary protein intake is associated with long-term increased risk of body weight gain and overall death in a Mediterranean population at high cardiovascular risk. Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  16. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    SciTech Connect

    Bricker, T.M.; Boyer, M.J.; Keith, J.; Watson-McKown, R.; Wise, K.S.

    1988-02-01

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 (/sup 35/S)methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of (/sup 3/H)palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from /sup 125/I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of (/sup 3/H)palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins.

  17. Presence of proteolipid protein in coelacanth brain myelin demonstrates tetrapod affinities and questions a chondrichthyan association.

    PubMed

    Waehneldt, T V; Malotka, J

    1989-06-01

    The protein and glycoprotein compositions of CNS myelin from the living coelacanth (Latimeria chalumnae) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An unglycosylated component of 25 kilodaltons showed substantially stronger immunoblot reactivity with antibodies against mammalian proteolipid protein (PLP) than lungfish glycosylated PLP. DM-20 (intermediate protein) was not detectable in either fish. The presence of unglycosylated PLP in CNS myelin of the actinistian coelacanth contradicts an association with cartilaginous fishes but supports tetrapod affinities closer than those of lungfish.

  18. COP9-Associated CSN5 Regulates Exosomal Protein Deubiquitination and Sorting

    PubMed Central

    Liu, Yuelong; Shah, Spandan V.; Xiang, Xiaoyu; Wang, Jianhua; Deng, Zhong-bin; Liu, Cunren; Zhang, Liming; Wu, Jianming; Edmonds, Tara; Jambor, Christina; Kappes, John C.; Zhang, Huang-Ge

    2009-01-01

    Ubiquitinated endosomal proteins that are deposited into the lumens of multivesicular bodies are either sorted for lysosomal-mediated degradation or secreted as exosomes into the extracellular milieu. The mechanisms that underlie the sorting of cellular cargo proteins are currently unknown. In this study, we show that the COP9 signalosome (CSN)-associated protein CSN5 quantitatively regulated proteins that were sorted into exosomes. Western blot analysis of exosomal proteins indicated that small interfering (si)RNA knockdown of CSN5 results in increased levels of both ubiquitinated and non-ubiquitinated exosomal proteins, including heat shock protein 70, in comparison with exosomes isolated from the supernatants of 293 cells transfected with scrambled siRNA. Furthermore, 293 cells transfected with JAB1/MPN/Mov34 metalloenzyme domain-deleted CSN5 produced exosomes with higher levels of ubiquitinated heat shock protein 70, which did not affect non-ubiquitinated heat shock protein 70 levels. The loss of COP9-associated deubiquitin activity of CSN5 also led to the enhancement of HIV Gag that was sorted into exosomes as well as the promotion of HIV-1 release, suggesting that COP9-associated CSN5 regulates the sorting of a number of exosomal proteins in both a CSN5 JAB1/MPN/Mov34 metalloenzyme domain-dependent and -independent manner. We propose that COP9-associated CSN5 regulates exosomal protein sorting in both a deubiquitinating activity-dependent and -independent manner, which is contrary to the current idea of ubiquitin-dependent sorting of proteins to exosomes. PMID:19246649

  19. Decoding the disease-associated proteins encoded in the human chromosome 4.

    PubMed

    Chen, Lien-Chin; Liu, Mei-Ying; Hsiao, Yung-Chin; Choong, Wai-Kok; Wu, Hsin-Yi; Hsu, Wen-Lian; Liao, Pao-Chi; Sung, Ting-Yi; Tsai, Shih-Feng; Yu, Jau-Song; Chen, Yu-Ju

    2013-01-04

    Chromosome 4 is the fourth largest chromosome, containing approximately 191 megabases (~6.4% of the human genome) with 757 protein-coding genes. A number of marker genes for many diseases have been found in this chromosome, including genetic diseases (e.g., hepatocellular carcinoma) and biomedical research (cardiac system, aging, metabolic disorders, immune system, cancer and stem cell) related genes (e.g., oncogenes, growth factors). As a pilot study for the chromosome 4-centric human proteome project (Chr 4-HPP), we present here a systematic analysis of the disease association, protein isoforms, coding single nucleotide polymorphisms of these 757 protein-coding genes and their experimental evidence at the protein level. We also describe how the findings from the chromosome 4 project might be used to drive the biomarker discovery and validation study in disease-oriented projects, using the examples of secretomic and membrane proteomic approaches in cancer research. By integrating with cancer cell secretomes and several other existing databases in the public domain, we identified 141 chromosome 4-encoded proteins as cancer cell-secretable/shedable proteins. Additionally, we also identified 54 chromosome 4-encoded proteins that have been classified as cancer-associated proteins with successful selected or multiple reaction monitoring (SRM/MRM) assays developed. From literature annotation and topology analysis, 271 proteins were recognized as membrane proteins while 27.9% of the 757 proteins do not have any experimental evidence at the protein-level. In summary, the analysis revealed that the chromosome 4 is a rich resource for cancer-associated proteins for biomarker verification projects and for drug target discovery projects.

  20. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

    PubMed

    Kammula, Ellen C; Mötter, Jessica; Gorgels, Alexandra; Jonas, Esther; Hoffmann, Silke; Willbold, Dieter

    2012-01-01

    HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  1. Cardiac arrhythmias associated with a liquid protein diet for the treatment of obesity

    SciTech Connect

    Lantigua, R.A.; Amatruda, J.M.; Biddle, T.L.; Forbes, G.B.; Lockwood, D.H.

    1980-09-25

    Our data demonstrate that a liquid protein diet is frequently associated with potentially life-threatening arrhythmias that are not detected on routine electrocardiography. Several studies of metabolic balance failed to reveal a cause for these arrhythmias. We recommended that the use of liquid protein diets should be terminated pending further investigation of the causes and prevention of the cardiac toxicity.

  2. Separation of tubulin and microtubule-associated proteins by ion exchange chromatography.

    PubMed

    Sloboda, Roger D

    2015-01-05

    Conventional liquid chromatography on phosphocellulose (PC) can be used to separate tubulin and microtubule-associated proteins (MAPs). Tubulin is a highly acidic protein and thus does not bind to PC. MAPs, however, do bind to PC and can be eluted with a subsequent salt wash of the column.

  3. The surface-associated proteins of wheat starch granules: suitability of wheat starch for celiac patients

    USDA-ARS?s Scientific Manuscript database

    Wheat starch is used to make baked products for celiac patients in several European countries, but is avoided in the US because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye and barley proteins and products that...

  4. Identification of proteins associated with the Pseudomonas aeruginosa biofilm extracellular matrix.

    PubMed

    Toyofuku, Masanori; Roschitzki, Bernd; Riedel, Katharina; Eberl, Leo

    2012-10-05

    Biofilms are surface-associated bacteria that are embedded in a matrix of self-produced polymeric substances (EPSs). The EPS is composed of nucleic acids, polysaccharides, lipids, and proteins. While polysaccharide components have been well studied, the protein content of the matrix is largely unknown. Here we conducted a comprehensive proteomic study to identify proteins associated with the biofilm matrix of Pseudomonas aeruginosa PAO1 (the matrix proteome). This analysis revealed that approximately 30% of the identified matrix proteins were outer membrane proteins, which are also typically found in outer membrane vesicles (OMVs). Electron microscopic inspection confirmed the presence of large amounts of OMVs within the biofilm matrix, supporting previous notions that OMVs are abundant constituents of P. aeruginosa biofilms. Our results demonstrate that while some proteins associated with the P. aeruginosa matrix are derived from secreted proteins and lysed cells, the large majority of the matrix proteins originate from OMVs. Furthermore, we demonstrate that the protein content of planktonic and biofilm OMVs is surprisingly different and may reflect the different physiological states of planktonic and sessile cells.

  5. Flagellar mitochondrial association of the male-specific Don Juan protein in Drosophila spermatozoa.

    PubMed

    Santel, A; Blümer, N; Kämpfer, M; Renkawitz-Pohl, R

    1998-11-01

    The Drosophila don juan gene encodes a basic protein (Don Juan protein), which is solely expressed postmeiotically during spermiogenesis in elongated spermatids and in mature sperm. Transgenic expression of a GFP-tagged Don Juan protein (DJ-GFP) in the male germ line showed an association of the fusion protein with the sperm tail. Detailed examination of DJ-GFP localization revealed novel insights into its distinct temporal and spatial distribution along the sperm tail during the last phase of spermatid maturation. Co-localization of DJ-GFP with actin-labeled cysts demonstrated its emergence in elongated spermatids during individualization. Additionally, the endogenous Don Juan protein was detected with epitope-specific antibodies in finally elongated nuclei of spermatids. After completion of nuclear shaping Don Juan is no longer detectable in the sperm heads with the onset of individualization. Mislocalization of the DJ-GFP protein in flagella of a mutant with defective mitochondrial differentiation provides evidence of mitochondrial association of the fusion protein with flagellar mitochondrial arrays. Ectopically expressed DJ-GFP in premeiotic germ cells as well as salivary gland cells confirmed the capability of the fusion protein to associate with mitochondria. Therefore we suppose that Don Juan is a nuclear-encoded, germ-cell specifically expressed mitochondrial protein, which might be involved in the final steps of mitochondrial differentiation within the flagellum.

  6. Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage.

    PubMed

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-15

    The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1×, DE3×, ME3×, NEL3×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P=0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P=0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P=0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P=0.09), RD of PB2 (29 vs. 62 g/kg CP, P=0.02), RD of CB2 (251 vs. 198 g/kg DM, P=0.06), RD of CB3 (236 vs. 261 g/kg CHO, P=0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P=0.06), and metabolic bioenergy (P=0.095). As to protein molecular structure, there were differences in protein structure 1st and 2nd amide groups (P

  7. Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage

    NASA Astrophysics Data System (ADS)

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-01

    The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1 ×, DE3 ×, ME3 ×, NEL3 ×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P = 0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P = 0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P = 0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P = 0.09), RD of PB2 (29 vs. 62 g/kg CP, P = 0.02), RD of CB2 (251 vs. 198 g/kg DM, P = 0.06), RD of CB3 (236 vs. 261 g/kg CHO, P = 0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P = 0.06), and metabolic bioenergy (P = 0.095). As to protein molecular structure, there were differences in protein structure 1st

  8. Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization.

    PubMed

    Lemos, C L; Sampaio, P; Maiato, H; Costa, M; Omel'yanchuk, L V; Liberal, V; Sunkel, C E

    2000-07-17

    Through mutational analysis in Drosopjila we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein-Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle.

  9. Biased random walk model for the prioritization of drug resistance associated proteins

    PubMed Central

    Guo, Hao; Dong, Jiaqiang; Hu, Sijun; Cai, Xiqiang; Tang, Guangbo; Dou, Jianhua; Tian, Miaomiao; He, Fuchu; Nie, Yongzhan; Fan, Daiming

    2015-01-01

    Multi-drug resistance is the main cause of treatment failure in cancer patients. How to identify molecules underlying drug resistance from multi-omics data remains a great challenge. Here, we introduce a data biased strategy, ProteinRank, to prioritize drug-resistance associated proteins in cancer cells. First, we identified differentially expressed proteins in Adriamycin and Vincristine resistant gastric cancer cells compared to their parental cells using iTRAQ combined with LC-MS/MS experiments, and then mapped them to human protein-protein interaction network; second, we applied ProteinRank to analyze the whole network and rank proteins similar to known drug resistance related proteins. Cross validations demonstrated a better performance of ProteinRank compared to the method without usage of MS data. Further validations confirmed the altered expressions or activities of several top ranked proteins. Functional study showed PIM3 or CAV1 silencing was sufficient to reverse the drug resistance phenotype. These results indicated ProteinRank could prioritize key proteins related to drug resistance in gastric cancer and provided important clues for cancer research. PMID:26039373

  10. A PII-Like Protein Regulated by Bicarbonate: Structural and Biochemical Studies of the Carboxysome-Associated CPII Protein.

    PubMed

    Wheatley, Nicole M; Eden, Kevin D; Ngo, Joanna; Rosinski, Justin S; Sawaya, Michael R; Cascio, Duilio; Collazo, Michael; Hoveida, Hamidreza; Hubbell, Wayne L; Yeates, Todd O

    2016-10-09

    Autotrophic bacteria rely on various mechanisms to increase intracellular concentrations of inorganic forms of carbon (i.e., bicarbonate and CO2) in order to improve the efficiency with which they can be converted to organic forms. Transmembrane bicarbonate transporters and carboxysomes play key roles in accumulating bicarbonate and CO2, but other regulatory elements of carbon concentration mechanisms in bacteria are less understood. In this study, after analyzing the genomic regions around α-type carboxysome operons, we characterize a protein that is conserved across these operons but has not been previously studied. On the basis of a series of apo- and ligand-bound crystal structures and supporting biochemical data, we show that this protein, which we refer to as the carboxysome-associated PII protein (CPII), represents a new and distinct subfamily within the broad superfamily of previously studied PII regulatory proteins, which are generally involved in regulating nitrogen metabolism in bacteria. CPII undergoes dramatic conformational changes in response to ADP binding, and the affinity for nucleotide binding is strongly enhanced by the presence of bicarbonate. CPII therefore appears to be a unique type of PII protein that senses bicarbonate availability, consistent with its apparent genomic association with the carboxysome and its constituents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Characterization of Mediator Complex and its Associated Proteins from Rice.

    PubMed

    Samanta, Subhasis; Thakur, Jitendra Kumar

    2017-01-01

    The Mediator complex is a multi-protein complex that acts as a molecular bridge conveying transcriptional messages from the cis element-bound transcription factor to the RNA Polymerase II machinery. It is found in all eukaryotes including members of the plant kingdom. Increasing number of reports from plants regarding different Mediator subunits involved in a multitude of processes spanning from plant development to environmental interactions have firmly established it as a central hub of plant regulatory networks. Routine isolation of Mediator complex in a particular species is a necessity because of many reasons. First, composition of the Mediator complex varies from species to species. Second, the composition of the Mediator complex in a particular species is not static under all developmental and environmental conditions. Besides this, at times, Mediator complex is used in in vitro transcription systems. Rice, a staple food crop of the world, is used as a model monocot crop. Realizing the need of a reliable protocol for the isolation of Mediator complex from plants, we describe here the isolation of Mediator complex from rice.

  12. G-protein from Medicago sativa: functional association to photoreceptors.

    PubMed Central

    Muschietti, J P; Martinetto, H E; Coso, O A; Farber, M D; Torres, H N; Flawia, M M

    1993-01-01

    G-protein subunits were characterized from Medicago sativa (alfalfa) seedlings. Crude membranes and GTP-Sepharose-purified fractions were electrophoresed on SDS/polyacrylamide gels and analysed by Western blotting with 9193 (anti-alpha common) and AS/7 (anti-alpha t, anti-alpha i1 and anti-alpha i2) polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 (anti-beta) antibody, of about 37 kDa, was also detected. The 43 kDa polypeptide bound specifically [alpha-32P]GTP by a photoaffinity reaction and was ADP-ribosylated by activated cholera toxin, but not by pertussis toxin. Irradiation of etiolated Medicago sativa protoplast preparations at 660 nm for 1 min produced a maximal increase in the guanosine 5'-[gamma-thio]triphosphate (GTP[35S])-binding rate. After this period of irradiation, the binding rate tended to decrease. The effect of a red-light (660 nm) pulse on the binding rate was reversed when it was immediately followed by a period of far-red (> 730 nm) illumination. These results may suggest that activation of GTP[S]-binding rate was a consequence of conversion of phytochrome Pr into the Ptr form. Images Figure 1 Figure 2 Figure 3 PMID:8484719

  13. A model of protein association based on their hydrophobic and electric interactions.

    PubMed

    Mozo-Villarías, Angel; Cedano, Juan; Querol, Enrique

    2014-01-01

    The propensity of many proteins to oligomerize and associate to form complex structures from their constituent monomers, is analyzed in terms of their hydrophobic (H), and electric pseudo-dipole (D) moment vectors. In both cases these vectors are defined as the product of the distance between their positive and negative centroids, times the total hydrophobicity or total positive charge of the protein. Changes in the magnitudes and directions of H and D are studied as monomers associate to form larger complexes. We use these descriptors to study similarities and differences in two groups of associations: a) open associations such as polymers with an undefined number of monomers (i.e. actin polymerization, amyloid and HIV capsid assemblies); b) closed symmetrical associations of finite size, like spherical virus capsids and protein cages. The tendency of the hydrophobic moments of the monomers in an association is to align in parallel arrangements following a pattern similar to those of phospholipids in a membrane. Conversely, electric dipole moments of monomers tend to align in antiparallel associations. The final conformation of a given assembly is a fine-tuned combination of these forces, limited by steric constraints. This determines whether the association will be open (indetermined number of monomers) or closed (fixed number of monomers). Any kinetic, binding or molecular peculiarities that characterize a protein assembly, comply with the vector rules laid down in this paper. These findings are also independent of protein size and shape.

  14. A Model of Protein Association Based on Their Hydrophobic and Electric Interactions

    PubMed Central

    Mozo-Villarías, Angel; Cedano, Juan; Querol, Enrique

    2014-01-01

    The propensity of many proteins to oligomerize and associate to form complex structures from their constituent monomers, is analyzed in terms of their hydrophobic (H), and electric pseudo-dipole (D) moment vectors. In both cases these vectors are defined as the product of the distance between their positive and negative centroids, times the total hydrophobicity or total positive charge of the protein. Changes in the magnitudes and directions of H and D are studied as monomers associate to form larger complexes. We use these descriptors to study similarities and differences in two groups of associations: a) open associations such as polymers with an undefined number of monomers (i.e. actin polymerization, amyloid and HIV capsid assemblies); b) closed symmetrical associations of finite size, like spherical virus capsids and protein cages. The tendency of the hydrophobic moments of the monomers in an association is to align in parallel arrangements following a pattern similar to those of phospholipids in a membrane. Conversely, electric dipole moments of monomers tend to align in antiparallel associations. The final conformation of a given assembly is a fine-tuned combination of these forces, limited by steric constraints. This determines whether the association will be open (indetermined number of monomers) or closed (fixed number of monomers). Any kinetic, binding or molecular peculiarities that characterize a protein assembly, comply with the vector rules laid down in this paper. These findings are also independent of protein size and shape. PMID:25329830

  15. GMAP-210, A Cis-Golgi Network-associated Protein, Is a Minus End Microtubule-binding Protein

    PubMed Central

    Infante, Carlos; Ramos-Morales, Francisco; Fedriani, Concepción; Bornens, Michel; Rios, Rosa M.

    1999-01-01

    We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997–1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1,979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210–encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus. PMID:10189370

  16. Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated, Actin-Binding Protein1[W][OPEN

    PubMed Central

    Jimenez-Lopez, Jose C.; Wang, Xia; Kotchoni, Simeon O.; Huang, Shanjin; Szymanski, Daniel B.; Staiger, Christopher J.

    2014-01-01

    The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction. PMID:25201878

  17. Protein kinase Cβ activates fat mass and obesity-associated protein by influencing its ubiquitin/proteasome degradation.

    PubMed

    Tai, Haoran; Wang, Xiaobo; Zhou, Jiao; Han, Xiaojuan; Fang, Tingting; Gong, Hui; Huang, Ning; Chen, Honghan; Qin, Jianqiong; Yang, Ming; Wei, Xiawei; Yang, Li; Xiao, Hengyi

    2017-10-01

    Protein kinase Cβ (PKCβ) is a serine-threonine kinase associated with obesity and diabetic complications; its activation contributes to weight gain, and deletion of its gene results in resistance to genetic- and diet-induced obesity. Fat mass and obesity-associated (FTO) protein is a recently identified RNA demethylase, and its overexpression in mice leads to increased body weight as well as fat mass. Although sharing some features in anabolism regulation, PKCβ and FTO have not been investigated together; therefore, their relationship has not been established. We report that PKCβ positively regulates FTO on the posttranslation level, evidenced by the facts that PKCβ activation contributes to high-glucose-induced FTO up-regulation, and overexpression of PKCβ suppresses ubiquitin-proteasome degradation of FTO, whereas PKCβ inactivation acts in the opposite manner. It was also found that PKCβ can phosphorylate FTO on threonine, and this phosphorylation requires both catalytic and regulatory domains of PKCβ. Moreover, PKCβ inhibition can suppress 3T3-L1 cell differentiation in normal and FTO-overexpressing cells but not in FTO-silenced or -inhibited cells. We propose that PKCβ acts to suppress the degradation of FTO protein and reveals the associated role of PKCβ and FTO in adipogenesis, suggesting a new pathway that affects the development of obesity and metabolic diseases.-Tai, H., Wang, X., Zhou, J., Han, X., Fang, T., Gong, H., Huang, N., Chen, H., Qin, J., Yang, M., Wei, X., Yang, L., Xiao, H. Protein kinase Cβ activates fat mass and obesity-associated protein by influencing its ubiquitin/proteasome degradation. © FASEB.

  18. Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

    SciTech Connect

    Klinefelter, G.R.; Hamilton, D.W.

    1985-11-01

    We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-(35S)methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-(35S)methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB(3H)4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis.

  19. Automatic extraction of protein point mutations using a graph bigram association.

    PubMed

    Lee, Lawrence C; Horn, Florence; Cohen, Fred E

    2007-02-02

    Protein point mutations are an essential component of the evolutionary and experimental analysis of protein structure and function. While many manually curated databases attempt to index point mutations, most experimentally generated point mutations and the biological impacts of the changes are described in the peer-reviewed published literature. We describe an application, Mutation GraB (Graph Bigram), that identifies, extracts, and verifies point mutations from biomedical literature. The principal problem of point mutation extraction is to link the point mutation with its associated protein and organism of origin. Our algorithm uses a graph-based bigram traversal to identify these relevant associations and exploits the Swiss-Prot protein database to verify this information. The graph bigram method is different from other models for point mutation extraction in that it incorporates frequency and positional data of all terms in an article to drive the point mutation-protein association. Our method was tested on 589 articles describing point mutations from the G protein-coupled receptor (GPCR), tyrosine kinase, and ion channel protein families. We evaluated our graph bigram metric against a word-proximity metric for term association on datasets of full-text literature in these three different protein families. Our testing shows that the graph bigram metric achieves a higher F-measure for the GPCRs (0.79 versus 0.76), protein tyrosine kinases (0.72 versus 0.69), and ion channel transporters (0.76 versus 0.74). Importantly, in situations where more than one protein can be assigned to a point mutation and disambiguation is required, the graph bigram metric achieves a precision of 0.84 compared with the word distance metric precision of 0.73. We believe the graph bigram search metric to be a significant improvement over previous search metrics for point mutation extraction and to be applicable to text-mining application requiring the association of words.

  20. Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas.

    PubMed

    Kingtong, Sutin; Kellner, Kristell; Bernay, Benoît; Goux, Didier; Sourdaine, Pascal; Berthelin, Clothilde Heude

    2013-04-26

    Knowledge of sperm maturation process is limited in the Pacific oyster Crassostrea gigas and major factors of fertilization success of this free spawning animal are unknown. We investigated proteins associated to spermatozoa by analyzing two cellular fractions obtained from a 40-80% Percoll gradient fractioning of germ cell of mature male gonads. Mature spermatozoa were enriched in the lower Percoll fraction while the upper fraction contained less mature or earlier germ cells. A 2-DE proteomic approach was used to identify differentially expressed proteins in both fractions. We screened out 31 differential proteins (P<0.05) which included 14 up-regulated and 17 down-regulated proteins. Using MALDI-TOF/TOF MS and bioinformatics search against a C. gigas database, 13 and 8 proteins were identified for the up-regulated and down-regulated groups, respectively. In the spermatozoa enriched fraction, proteins regarding flagellum formation and control, energy production and Proteosome subunit beta were increased. In less mature germ cell fraction, proteins regarding developmental processes and chaperon molecules were mainly increased. Our results improve current knowledge of proteins associated with spermatozoa maturation related to zootechnical practices used in mollusk hatcheries. This is the revised version of the manuscript "Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas" by Kingtong et al. to the Journal of Proteomics. The corrections have been done by the team carefully. This work highlight the enrichment method of spermatozoa of Pacific oyster from stripped complex sample using Percoll gradient. The results reflexed developmental stages of germ cells in gonadal tubules of this species. We have used proteomic approach to identify differentially expressed proteins in mature spermatozoa fraction compared to less mature spermatozoa fraction which provided candidates of protein associated to mature spermatozoa

  1. Association between protein C levels and mortality in patients with advanced prostate, lung and pancreatic cancer.

    PubMed

    Wilts, I T; Hutten, B A; Meijers, J C M; Spek, C A; Büller, H R; Kamphuisen, P W

    2017-06-01

    Procoagulant factors promote cancer progression and metastasis. Protein C is involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. In mice, administration of activated protein C reduced experimental metastasis. We assessed the association between protein C and mortality in patients with three types of cancer. The study population consisted of patients with advanced prostate, non-small cell lung or pancreatic cancer, who participated in the INPACT trial (NCT00312013). The trial evaluated the addition of nadroparin to chemotherapy in patients with advanced malignancy. Patients were divided into tertiles based on protein C at baseline. The association between protein C levels and mortality was evaluated with Cox proportional hazard models. We analysed 477 patients (protein C tertiles: <97, 97-121 and ≥121%). Mean age was 65±9years; 390 (82%) were male; 191 patients (40%) had prostate cancer, 161 (34%) had lung cancer, and 125 (26%) pancreatic cancer. During a median follow-up of 10.4months, 291 patients (61%) died. Median protein C level was 107% (IQR 92-129). In the lowest tertile, 75 patients per 100 patient-years died, as compared to 60 and 54 in the middle and high tertile, respectively. Lower levels of protein C were associated with increased mortality (in tertiles: HR for trend 1.18, 95%CI 1.02-1.36, adjusted for age, sex and nadroparin use; as a continuous variable: HR 1.004, 95%CI 1.00-1.008, p=0.07). Protein C seems inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. Further research should validate protein C as a biomarker for mortality, and explore the effects of protein C on progression of cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies.

    PubMed

    Cordwell, Stuart J; Len, Alice C L; Touma, Rachel G; Scott, Nichollas E; Falconer, Linda; Jones, David; Connolly, Angela; Crossett, Ben; Djordjevic, Steven P

    2008-01-01

    Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.

  3. Association of elongation factor 1 alpha and ribosomal protein L3 with the proline-rich region of yeast adenylyl cyclase-associated protein CAP.

    PubMed

    Yanagihara, C; Shinkai, M; Kariya, K; Yamawaki-Kataoka, Y; Hu, C D; Masuda, T; Kataoka, T

    1997-03-17

    CAP is a multifunctional protein; the N-terminal region binds adenylyl cyclase and controls its response to Ras while the C-terminal region is involved in cytoskeletal regulation. In between the two regions, CAP possesses two proline-rich segments, P1 and P2, resembling a consensus sequence for binding SH3 domains. We have identified two yeast proteins with molecular sizes of 48 and 46 kDa associated specifically with P2. Determination of partial protein sequences demonstrated that the 48-kDa and 46-kDa proteins correspond to EF1 alpha and rL3, respectively, neither of which contains any SH3-domain-like sequence. Deletion of P2 from CAP resulted in loss of the activity to bind the two proteins either in vivo or in vitro. Yeast cells whose chromosomal CAP was replaced by the P2-deletion mutant displayed an abnormal phenotype represented by dissociated localizations of CAP and F-actin, which were colocalized in wild-type cells. These results suggest that these associations may have functional significance.

  4. Phylogenetic and functional analyses of a plant protein related to human B-cell receptor-associated proteins.

    PubMed

    Atabekova, Anastasia K; Pankratenko, Anna V; Makarova, Svetlana S; Lazareva, Ekaterina A; Owens, Robert A; Solovyev, Andrey G; Morozov, Sergey Y

    2017-01-01

    Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  5. Proteomic analysis of membrane-associated proteins from rat liver autophagosomes.

    PubMed

    Øverbye, Anders; Fengsrud, Monica; Seglen, Per O

    2007-01-01

    Proteins associated with membranes from purified rat liver autophagosomes were separated by two-dimensional (2D) gel electrophoresis (zoom gels, pl 4-7 and 6-9), silver-stained and identified by MALDI-TOF mass spectrometry. Among >1,500 detectable protein spots, 58 (derived from 39 different known proteins) were at least twofold (and significantly) enriched in autophagosomal membranes relative to cytoplasmic membranes. All of these membrane-associated proteins were also present in the cytosol, many of them being truncated enzyme variants that would be expected to serve a binding rather than an enzymatic function. Eleven proteins were highly enriched (consistent with the theoretical maximum of 25x), corresponding to an exclusive membrane localization in the delimiting membrane of the autophagosome. Three of these were methyltransferases: betaine:homocysteine methyltransferase (five variants); catechol O-methyltransferase (one phosphorylated and one unphosphorylated variant) and methionine adenosyltransferase, perhaps indicating that methylation/demethylation of membrane components could play a role in autophagy. A fourth highly enriched autophagosomal protein, phosphatidylethanolamine binding protein, is particularly interesting considering that the autophagic marker protein, LC3/ Atg8, is linked to autophagosomal membranes through its covalent conjugation with phosphatidylethanolamine (as the form LC3-II). LC3-II was not detectable on silver-stained 2D-gels, but could be shown by immunoblotting to be highly enriched in autophagosomal membranes. Other highly enriched proteins were heat shock cognate protein Hsc70 (one short and one long variant), peroxiredoxin 2, peroxiredoxin 6 (two variants), fructose 1,6-bisphosphatase (one phosphorylated and one unphosphorylated variant), adenosine kinase, inorganic pyrophosphatase and selenium-binding protein 2. Hsc70, a chaperonin that plays an important role in the recognition and proteasomal degradation of aggregated

  6. Huntington's Disease Protein Huntingtin Associates with its own mRNA.

    PubMed

    Culver, Brady P; DeClercq, Josh; Dolgalev, Igor; Yu, Man Shan; Ma, Bin; Heguy, Adriana; Tanese, Naoko

    2016-01-01

    The Huntington's disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-spliced Htt mRNA encoding a truncated protein comprised of exon 1 and a portion of the downstream intron in the immunoprecipitates containing mutant Htt protein. We show that Htt protein co-localizes with Htt mRNA and that wild-type Htt reduces expression of a reporter construct harboring the Htt 3' UTR. HD protein is found in a complex with its own mRNA and RNA binding proteins and translation factors. Htt may be involved in modulating its expression through post-transcriptional pathways. It is possible that Htt shares mechanistic properties similar to RNA binding proteins such as TDP-43 and FUS implicated in other neurodegenerative diseases.

  7. Effect of Acidic pH on Expression of Surface-Associated Proteins of Streptococcus oralis

    PubMed Central

    Wilkins, Joanna C.; Beighton, David; Homer, Karen A.

    2003-01-01

    Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions. PMID:12957916

  8. Genetic dissection of peroxisome-associated matrix protein degradation in Arabidopsis thaliana.

    PubMed

    Burkhart, Sarah E; Lingard, Matthew J; Bartel, Bonnie

    2013-01-01

    Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H(2)O(2), which can damage proteins. However, little is known about how damaged or obsolete peroxisomal proteins are degraded. We exploit developmentally timed peroxisomal content remodeling in Arabidopsis thaliana to elucidate peroxisome-associated protein degradation. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. We previously found that ICL is stabilized when a peroxisome-associated ubiquitin-conjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, we mutagenized a line expressing GFP-ICL, which is degraded similarly to endogenous ICL, and identified persistent GFP-ICL fluorescence (pfl) mutants. We found three pfl mutants that were defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix, indicating that proteins must enter the peroxisome for efficient degradation. One pfl mutant was missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, suggesting that peroxisomal metabolism influences the rate of matrix protein degradation. Finally, one pfl mutant that displayed normal matrix protein import carried a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. The isolation of pex6-2 as a pfl mutant supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation.

  9. Huntington’s Disease Protein Huntingtin Associates with its own mRNA

    PubMed Central

    Culver, Brady P.; DeClercq, Josh; Dolgalev, Igor; Yu, Man Shan; Ma, Bin; Heguy, Adriana; Tanese, Naoko

    2016-01-01

    Background: The Huntington’s disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. Objective: To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. Methods: RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. Results: Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-spliced Htt mRNA encoding a truncated protein comprised of exon 1 and a portion of the downstream intron in the immunoprecipitates containing mutant Htt protein. We show that Htt protein co-localizes with Htt mRNA and that wild-type Htt reduces expression of a reporter construct harboring the Htt 3’ UTR. Conclusions: HD protein is found in a complex with its own mRNA and RNA binding proteins and translation factors. Htt may be involved in modulating its expression through post-transcriptional pathways. It is possible that Htt shares mechanistic properties similar to RNA binding proteins such as TDP-43 and FUS implicated in other neurodegenerative diseases. PMID:26891106

  10. Axonal transport of calmodulin: a physiologic approach to identification of long-term associations between proteins

    PubMed Central

    1981-01-01

    Calmodulin is a soluble, heat-stable protein which has been shown to modulate both membrane-bound and soluble enzymes, but relatively little has been known about the in vivo associations of calmodulin. A 17,000- dalton heat-stable protein was found to move in axonal transport in the guinea pig visual system with the proteins of slow component b (SCb; 2 mm/d) along with actin and the bulk of the soluble proteins of the axon. Co-electrophoresis of purified calmodulin and radioactively labeled SCb proteins in two dimensional polyacrylamide gel electrophoresis (PAGE) demonstrated the identity of the heat-stable SCb protein and calmodulin on the basis of pI, molecular weight, and anomalous migration in the presence of Ca2+-chelating agents. No proteins co-migrating with calmodulin in two-dimensional PAGE could be detected among the proteins of slow component a (SCa; 0.3 mm/d, microtubules and neurofilaments) or fast component (FC; 250 mm/d, membrane-associated proteins). We conclude that calmodulin is transported solely as part of the SCb complex of proteins, the axoplasmic matrix. Calmodulin moves in axonal transport independent of the movements of microtubules (SCa) and membranes (FC), which suggests that the interactions of calmodulin with these structures may represent a transient interaction between groups of proteins moving in axonal transport at different rates. Axonal transport has been shown to be an effective tool for the demonstration of long-term in vivo protein associations. PMID:6166619

  11. Analysis of disease-associated protein expression using quantitative proteomics—fibulin-5 is expressed in association with hepatic fibrosis.

    PubMed

    Bracht, Thilo; Schweinsberg, Vincent; Trippler, Martin; Kohl, Michael; Ahrens, Maike; Padden, Juliet; Naboulsi, Wael; Barkovits, Katalin; Megger, Dominik A; Eisenacher, Martin; Borchers, Christoph H; Schlaak, Jörg F; Hoffmann, Andreas-Claudius; Weber, Frank; Baba, Hideo A; Meyer, Helmut E; Sitek, Barbara

    2015-05-01

    Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n = 77). Finally, we performed a targeted proteomics approach, multiple reaction monitoring (MRM), to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM), and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.

  12. Selective visualization of GLUT4 storage vesicles and associated Rab proteins using IRAP-pHluorin.

    PubMed

    Chen, Yu; Lippincott-Schwartz, Jennifer

    2015-01-01

    Fluorescence microscopy and fluorescent protein (FP)-tagged GLUT4 molecule have been great tools to characterize GLUT4 localization and dynamics inside the cell. However, it was difficult to distinguish GLUT4 storage vesicles (GSVs) from other intracellular compartments containing GLUT4 in live cells. Here, we describe the use of IRAP-pHluorin and total internal reflection fluorescence (TIRF) microscopy to selectively visualize GSVs and Rab proteins that associate with GSVs. This assay is also valuable to further defining GSV identity by unraveling other GSV-associated proteins.

  13. Genetic variation in heat shock protein 70 is associated with septic shock: narrowing the association to a specific haplotype.

    PubMed

    Kee, C; Cheong, K Y; Pham, K; Waterer, G W; Temple, S E L

    2008-12-01

    Heat shock protein 70 (HSP70) plays a major role in immune responses. Polymorphisms within the gene have been associated with development of septic shock. This study refines the region of the HSP70 gene associated with development of septic shock and confirms its functionality. Subjects (n = 31) were grouped into one of three haplotypes based on their HSPA1B-179C>T and HSPA1B1267A>G genotypes. Mononuclear cells from these subjects were stimulated with heat-killed bacteria (10(7 )colony-forming units/mL Escherichia coli or Streptococcus pneumoniae) for 8 and 21 h. HSP70 and tumour necrosis factor (TNF) mRNA and protein levels were measured by reverse transcriptase-polymerase chain reaction and ELISA, respectively. The HSPA1B-179*C:1267*A haplotype was associated with significantly lower levels of HSPA1B mRNA and protein and higher production of TNF mRNA and protein compared to the other haplotypes. Induction of HSP70 was TNF independent. These results suggest that the HSPA1B-179C>T:1267A>G haplotype is functional and may explain the association of the HSP70 gene with development of septic shock.

  14. The Role of Lectin-Carbohydrate Interactions in the Regulation of ER-Associated Protein Degradation.

    PubMed

    Słomińska-Wojewódzka, Monika; Sandvig, Kirsten

    2015-05-27

    Proteins entering the secretory pathway are translocated across the endoplasmic reticulum (ER) membrane in an unfolded form. In the ER they are restricted to a quality control system that ensures correct folding or eventual degradation of improperly folded polypeptides. Mannose trimming of N-glycans on newly synthesized proteins plays an important role in the recognition and sorting of terminally misfolded glycoproteins for ER-associated protein degradation (ERAD). In this process misfolded proteins are retrotranslocated into the cytosol, polyubiquitinated, and eventually degraded by the proteasome. The mechanism by which misfolded glycoproteins are recognized and recruited to the degradation machinery has been extensively studied during last decade. In this review, we focus on ER degradation-enhancing α-mannosidase-like protein (EDEM) family proteins that seem to play a key role in the discrimination between proteins undergoing a folding process and terminally misfolded proteins directed for degradation. We describe interactions of EDEM proteins with other components of the ERAD machinery, as well as with various protein substrates. Carbohydrate-dependent interactions together with N-glycan-independent interactions seem to regulate the complex process of protein recognition and direction for proteosomal degradation.

  15. Identification of a new class of lipid droplet-associated proteins in plants.

    PubMed

    Horn, Patrick J; James, Christopher N; Gidda, Satinder K; Kilaru, Aruna; Dyer, John M; Mullen, Robert T; Ohlrogge, John B; Chapman, Kent D

    2013-08-01

    Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.

  16. Function of plasma membrane microdomain-associated proteins during legume nodulation.

    PubMed

    Qiao, Zhenzhen; Libault, Marc

    2017-08-17

    Plasma membrane microdomains are plasma membrane sub-compartments enriched in sphingolipids and sterols, and composed by a specific set of proteins. They are involved in recognizing signal molecules, transducing these signals, and controlling endocytosis and exocytosis processes. In a recent study, applying biochemical and microscopic methods, we characterized the soybean GmFWL1 protein, a major regulator of soybean nodulation, as a new membrane microdomain-associated protein. Interestingly, upon rhizobia inoculation of the soybean root system, GmFWL1 and one of its interacting partners, GmFLOT2/4, both translocate to the root hair cell tip, the primary site of interaction and infection between soybean and Rhizobium. The role of GmFWL1 as a plasma membrane microdomain-associated protein is also supported by immunoprecipitation assays performed on soybean nodules, which revealed 178 GmFWL1 protein partners including a large number of microdomain-associated proteins such as GmFLOT2/4. In this addendum, we provide additional information about the identity of the soybean proteins repetitively identified as GmFWL1 protein partners. Their function is discussed especially in regard to plant-microbe interactions and microbial symbiosis. This addendum will provide new insights in the role of plasma membrane microdomains in regulating legume nodulation.

  17. [A case of protein-losing enteropathy associated with small bowel villous atrophy].

    PubMed

    Han, Sung Hee; Lee, Oh Young; Eun, Chang Su; Roh, Byoung Joo; Sohn, Won; Baeg, Seung Sam; Yoon, Byung Chul; Choi, Ho Soon

    2007-01-01

    Protein losing enteropathy is described as a diverse group of disorders associated with excessive loss of serum proteins into the gastrointestinal (GI) tract. The etiology of protein losing enteropathy is various. Increased mucosal permeability to protein as a result of cell damage, mucosal erosion, or lymphatic obstruction may develop protein losing enteropathy. Celiac disease is a common cause of protein losing enteropathy associated with small bowel villous atrophy in Europe. We experienced a case of protein losing enteropathy associated with small bowel villous atrophy of unknown origin. A 36-year-old woman was admitted due to chronic watery diarrhea and weight loss. Laboratory findings showed total protein 4.7 g/dL, albumin 2.7 g/dL, cholesterol 100 mg/dL, WBC 6,000/mm3 (lymphocyte 13.6%) with the absence of proteinuria. On esophagogastroduodenoscopic examination, duodenal ulcer scar was noted on the bulb and colonoscopic finding was nonspecific. On small bowel enteroscopy, jejunal and ileal villi was scantly noticed. Small bowel biopsy showed marked villous atrophy. Her symptoms did not improve after supportive care. Gluten free diet was tried because celiac disease could not be ruled out completely. Diarrhea ceased and body weight regained after gluten free diet.

  18. Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes.

    PubMed

    Cubeñas-Potts, Caelin; Srikumar, Tharan; Lee, Christine; Osula, Omoruyi; Subramonian, Divya; Zhang, Xiang-Dong; Cotter, Robert J; Raught, Brian; Matunis, Michael J

    2015-02-01

    Sumoylation is essential for progression through mitosis, but the specific protein targets and functions remain poorly understood. In this study, we used chromosome spreads to more precisely define the localization of SUMO-2/3 (small ubiquitin-related modifier) to the inner centromere and protein scaffold of mitotic chromosomes. We also developed methods to immunopurify proteins modified by endogenous, untagged SUMO-2/3 from mitotic chromosomes. Using these methods, we identified 149 chromosome-associated SUMO-2/3 substrates by nLC-ESI-MS/MS. Approximately one-third of the identified proteins have reported functions in mitosis. Consistent with SUMO-2/3 immunolocalization, we identified known centromere- and kinetochore-associated proteins, as well as chromosome scaffold associated proteins. Notably, >30 proteins involved in chromatin modification or remodeling were identified. Our results provide insights into the roles of sumoylation as a regulator of chromatin structure and other diverse processes in mitosis. Furthermore, our purification and fractionation methodologies represent an important compliment to existing approaches to identify sumoylated proteins using exogenously expressed and tagged SUMOs.

  19. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

    PubMed

    Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

    2005-07-01

    Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

  20. Prediction of nanoparticles-cell association based on corona proteins and physicochemical properties.

    PubMed

    Liu, Rong; Jiang, Wen; Walkey, Carl D; Chan, Warren C W; Cohen, Yoram

    2015-06-07

    Cellular association of nanoparticles (NPs) in biological fluids is affected by proteins adsorbed onto the NP surface, forming a "protein corona", thereby impacting cellular bioactivity. Here we investigate, based on an extensive gold NPs protein corona dataset, the relationships between NP-cell association and protein corona fingerprints (PCFs) as well as NP physicochemical properties. Accordingly, quantitative structure-activity relationships (QSARs) were developed based on both linear and non-linear support vector regression (SVR) models making use of a sequential forward floating selection of descriptors. The SVR model with only 6 serum proteins and zeta potential had higher accuracy (R(2) = 0.895) relative to the linear model (R(2) = 0.850) with 11 PCFs. Considering the initial pool of 148 descriptors, the APOB, A1AT, ANT3, and PLMN serum proteins along with NP zeta potential were identified as most significant to correlating NP-cell association. The present study suggests that QSARs exploration of NP-cell association data, considering the role of both NP protein corona and physicochemical properties, can support the planning and interpretation of toxicity studies and guide the design of NPs for biomedical applications.

  1. EFHD2 IS A NOVEL AMYLOID PROTEIN ASSOCIATED TO PATHOLOGICAL TAU IN ALZHEIMER’S DISEASE

    PubMed Central

    Ferrer-Acosta, Yancy; Rodríguez-Cruz, Eva N.; Orange, François; De Jesús-Cortés, Hector; Madera, Bismark; Vaquer-Alicea, Jaime; Ballester, Juan; Guinel, Maxime J-F.; Bloom, George S.; Vega, Irving E.

    2013-01-01

    EFhd2 is a conserved calcium binding protein, abundant within the central nervous system. Previous studies identified EFhd2 associated with pathological forms of tau proteins in the tauopathy mouse model JNPL3, which expresses the human tauP301L mutant. This association was validated in human tauopathies, such as Alzheimer’s disease (AD). However, the role that EFhd2 may play in tauopathies is still unknown. Here, we show that EFhd2 formed amyloid structures in vitro, a capability that is reduced by calcium ions. Electron microscopy (EM) analyses demonstrated that recombinant EFhd2 formed filamentous structures. EM analyses of sarkosyl insoluble fractions derived from human AD brains also indicated that EFhd2 co-localizes with aggregated tau proteins and formed granular structures. Immunohistological analyses of brain slices demonstrated that EFhd2 co-localizes with pathological tau proteins in AD brains, confirming the co-aggregation of EFhd2 and pathological tau. Furthermore, EFhd2’s coiled-coil domain mediated its self-oligomerization in vitro and its association with tau proteins in JNPL3 mouse brain extracts. The results demonstrate that EFhd2 is a novel amyloid protein associated with pathological tau proteins in AD brain and that calcium binding may regulate the formation of EFhd2’s amyloid structures. Hence, EFhd2 may play an important role in the pathobiology of tau-mediated neurodegeneration. PMID:23331044

  2. EFhd2 is a novel amyloid protein associated with pathological tau in Alzheimer's disease.

    PubMed

    Ferrer-Acosta, Yancy; Rodríguez-Cruz, Eva N; Orange, François; De Jesús-Cortés, Hector; Madera, Bismark; Vaquer-Alicea, Jaime; Ballester, Juan; Guinel, Maxime J-F; Bloom, George S; Vega, Irving E

    2013-06-01

    EFhd2 is a conserved calcium-binding protein, abundant within the central nervous system. Previous studies identified EFhd2 associated with pathological forms of tau proteins in the tauopathy mouse model JNPL3, which expresses the human tau(P301L) mutant. This association was validated in human tauopathies, such as Alzheimer's disease (AD). However, the role that EFhd2 may play in tauopathies is still unknown. Here, we show that EFhd2 formed amyloid structures in vitro, a capability that is reduced by calcium ions. Electron microscopy (EM) analyses demonstrated that recombinant EFhd2 formed filamentous structures. EM analyses of sarkosyl-insoluble fractions derived from human AD brains also indicated that EFhd2 co-localizes with aggregated tau proteins and formed granular structures. Immunohistological analyses of brain slices demonstrated that EFhd2 co-localizes with pathological tau proteins in AD brains, confirming the co-aggregation of EFhd2 and pathological tau. Furthermore, EFhd2's coiled-coil domain mediated its self-oligomerization in vitro and its association with tau proteins in JNPL3 mouse brain extracts. The results demonstrate that EFhd2 is a novel amyloid protein associated with pathological tau proteins in AD brain and that calcium binding may regulate the formation of EFhd2's amyloid structures. Hence, EFhd2 may play an important role in the pathobiology of tau-mediated neurodegeneration. © 2013 International Society for Neurochemistry.

  3. Alpha-Dystrobrevin and its associated proteins in human promyelocytic leukemia cells induced to apoptosis.

    PubMed

    Navakauskienė, Rūta; Treigytė, Gražina; Borutinskaitė, Veronika-Viktorija; Matuzevičius, Dalius; Navakauskas, Dalius; Magnusson, Karl-Eric

    2012-06-18

    Dystrobrevin is a dystrophin-related component of the dystrophin-associated protein complex (DAPC). Using alpha-dystrobrevin as indicator, we aimed to elucidate the interaction network of the DAPC with other proteins during apoptosis of promyelocytic HL-60 cells. The precise role(s) of DBs are not known, but we and others have shown that they play a role in intracellular signal transduction and cellular organization. Apoptosis was induced with etoposide in the absence or presence of Z-VAD to block caspase activity, and we then followed the cellular distribution of α-DB and its association with other proteins, using confocal imaging and cell fractions analyses after immune-precipitation with anti-α-DB and mass spectrometry. Confocal imaging revealed distinct spatial relocalizations of α-DB between the cell membrane, cytosol and nucleus after induction of apoptosis. The expression levels of the identified proteins were evaluated with computer-assisted image analysis of the gels. We thus identified associations with structural and transport proteins (tropomyosin, myosin), membrane (ADAM21, syntrophin), ER-Golgi (TGN51, eIF38) and nuclear (Lamins, ribonucleoprotein C1/C2) proteins. These results suggest that apoptosis-induction in HL-60 cells involves not only classical markers of apoptosis but also a network α-DB-associated proteins at the cell membrane, the cytoplasm and nucleus, affecting key cellular transport processes and cellular structure.

  4. Dietary protein intake is associated with lean body mass in community-dwelling older adults.

    PubMed

    Geirsdottir, Olof G; Arnarson, Atli; Ramel, Alfons; Jonsson, Palmi V; Thorsdottir, Inga

    2013-08-01

    Lean body mass (LBM) is important to maintain physical function during aging. We hypothesized that dietary protein intake and leisure-time physical activity are associated with LBM in community-dwelling older adults. To test the hypothesis, participants (n = 237; age, 65-92 years) did 3-day weighed food records and reported physical activity. Body composition was assessed using dual-energy x-ray absorptiometry. Protein intake was 0.98 ± 0.28 and 0.95 ± 0.29 g/kg body weight in male and female participants, respectively. Protein intake (in grams per kilogram of body weight) was associated with LBM (in kilograms); that is, the differences in LBM were 2.3 kg (P < .05) and 2.0 kg (P = .054) between the fourth vs the first and the fourth vs the second quartiles of protein intake, respectively. Only a minor part of this association was explained by increased energy intake, which follows an increased protein intake. Our study shows that dietary protein intake was positively associated with LBM in older adults with a mean protein intake higher than the current recommended daily allowance of 0.8 g/kg per day. Leisure-time physical activity, predominantly consisting of endurance type exercises, was not related to LBM in this group.

  5. Protein Profiles Associated With Context Fear Conditioning and Their Modulation by Memantine*

    PubMed Central

    Ahmed, Md. Mahiuddin; Dhanasekaran, A. Ranjitha; Block, Aaron; Tong, Suhong; Costa, Alberto C. S.; Gardiner, Katheleen J.

    2014-01-01

    Analysis of the molecular basis of learning and memory has revealed details of the roles played by many genes and the proteins they encode. Because most individual studies focus on a small number of proteins, many complexities of the relationships among proteins and their dynamic responses to stimulation are not known. We have used the technique of reverse phase protein arrays (RPPA) to assess the levels of more than 80 proteins/protein modifications in subcellular fractions from hippocampus and cortex of mice trained in Context Fear Conditioning (CFC). Proteins include components of signaling pathways, several encoded by immediate early genes or involved in apoptosis and inflammation, and subunits of glutamate receptors. At one hour after training, levels of more than half the proteins had changed in one or more fractions, among them multiple components of the Mitogen-activated protein kinase, MAPK, and Mechanistic Target of Rapamycin, MTOR, pathways, subunits of glutamate receptors, and the NOTCH pathway modulator, NUMB homolog (Drosophila). Levels of 37 proteins changed in the nuclear fraction of hippocampus alone. Abnormalities in levels of thirteen proteins analyzed have been reported in brains of patients with Alzheimer's Disease. We therefore further investigated the protein profiles of mice treated with memantine, a drug approved for treatment of AD. In hippocampus, memantine alone induced many changes similar to those seen after CFC and altered the levels of seven proteins associated with Alzheimer's Disease abnormalities. Lastly, to further explore the relevance of these datasets, we superimposed responses to CFC and memantine onto components of the long term potentiation pathway, a process subserving learning and memory formation. Fourteen components of the long term potentiation pathway and 26 proteins interacting with components responded to CFC and/or memantine. Together, these datasets provide a novel view of the diversity and complexity in protein

  6. [Hepatocellular carcinoma associated antigen HCA520, a novel Ca2+-binding protein].

    PubMed

    Yang, Mei-xiang; Han, Ke-jun; Qu, Xun; Ma, Dao-xin; Chen, Wei-feng

    2004-03-01

    To analyze the binding activity of the hepatocellular carcinoma associated antigen HCA520 to Ca2+. The HCA520 gene was gained by PCR. Then, it was cloned into the prokaryotic expression vector pGEX-4T-3. The GST-HCA520 fusion gene was induced to express in E. coli and the expressed product was purified via GST-agarose affinity resin. The fusion protein was confirmed by Western blot analysis. The Ca2+ binding capability of the fusion protein was analyzed by dot blot. The GST-HCA520 fusion gene was constructed and the protein was successfully expressed and purified, which was identified by DNA sequencing and Western blot respectively. The fusion protein could bind to Ca2+ depend on dosages. HCA520, a novel hepatocellular carcinoma associated antigen, is a novel Ca2+ binding protein.

  7. The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery

    PubMed Central

    Toriyama, Michinori; Lee, Chanjae; Taylor, S. Paige; Duran, Ivan; Cohn, Daniel H.; Bruel, Ange-Line; Tabler, Jacqueline M.; Drew, Kevin; Kelley, Marcus R.; Kim, Sukyoung; Park, Tae Joo; Braun, Daniella; Pierquin, Ghislaine; Biver, Armand; Wagner, Kerstin; Malfroot, Anne; Panigrahi, Inusha; Franco, Brunella; Al-lami, Hadeel Adel; Yeung, Yvonne; Choi, Yeon Ja; Duffourd, Yannis; Faivre, Laurence; Rivière, Jean-Baptiste; Chen, Jiang; Liu, Karen J.; Marcotte, Edward M.; Hildebrandt, Friedhelm; Thauvin-Robinet, Christel; Krakow, Deborah; Jackson, Peter K.; Wallingford, John B.

    2016-01-01

    Summary Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. The ciliopathies are a spectrum of human disease resulting from defects in cilia structure or function. Mechanisms regulating assembly of ciliary multiprotein complexes and their transport to the base of cilia remain largely unknown. Combine proteomics, in vivo imaging, and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy, WDPCP), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector) and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme, and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with novel ciliopathies in human patients. PMID:27158779

  8. Human fallopian tube proteome shows high coverage of mesenchymal stem cells associated proteins

    PubMed Central

    Wang, Chenyuan; Liu, Yang; Chang, Cheng; Wu, Songfeng; Gao, Jie; Zhang, Yang; Chen, Yingjie; Zhong, Fan; Deng, Gaopi

    2016-01-01

    The object of this research was to report a draft proteome of human fallopian tube (hFT) comprises 5416 identified proteins, which could be considered as a physiological reference to complement Human Proteome Draft. The proteomic raw data and metadata were stored in an integrated proteome resources centre iProX (IPX00034300). This hFT proteome contains many hFT markers newly identified by mass spectrum. This hFT proteome comprises 660 high-, 3605 medium- and 1181 low-abundant proteins. Ribosome, cytoskeleton, vesicle and protein folding associated proteins showed obvious tendency to be higher abundance in hFT. The extraordinary high coverage of mesenchymal stem cells (MSCs)-associated proteins were identified in this hFT proteome, which highly supported that hFT should contain a plenty of MSCs. PMID:26759384

  9. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease

    PubMed Central

    Filimonenko, Maria; Stuffers, Susanne; Raiborg, Camilla; Yamamoto, Ai; Malerød, Lene; Fisher, Elizabeth M.C.; Isaacs, Adrian; Brech, Andreas; Stenmark, Harald; Simonsen, Anne

    2007-01-01

    The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations. PMID:17984323

  10. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease.

    PubMed

    Filimonenko, Maria; Stuffers, Susanne; Raiborg, Camilla; Yamamoto, Ai; Malerød, Lene; Fisher, Elizabeth M C; Isaacs, Adrian; Brech, Andreas; Stenmark, Harald; Simonsen, Anne

    2007-11-05

    The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations.

  11. A Bayesian network model of proteins' association with promyelocytic leukemia (PML) nuclear bodies.

    PubMed

    Bodén, Mikael; Dellaire, Graham; Burrage, Kevin; Bailey, Timothy L

    2010-04-01

    The modularity that nuclear organization brings has the potential to explain the function of aggregates of proteins and RNA. Promyelocytic leukemia nuclear bodies are implicated in important regulatory processes. To understand the complement of proteins associated with these intra-nuclear bodies, we construct a Bayesian network model that integrates sequence and protein-protein interaction data. The model predicts association with promyelocytic leukemia nuclear bodies accurately when interaction data is available. At a false positive rate of 10%, the true positive rate is almost 50%, indicated by an independent nuclear proteome reference set. The model provides strong support for further expanding the protein complement with several important regulators and a richer functional repertoire. Using special support vector machine (SVM)-nodes (equipped with string kernels), the Bayesian network is also able to produce predictions on the basis of sequence only, with an accuracy superior to that of baseline models. Supplementary Material is available online at www.liebertonline.com.

  12. Evidence for protein self-association induced by excluded volume. Myoglobin in the presence of globular proteins.

    PubMed

    Wilf, J; Minton, A P

    1981-10-28

    The fluorescence polarization of fluorescent derivatives of hemoglobin and myoglobin was measured as a function of the concentration of added polymers (PEG-6 000, PEG-20 000) and globular proteins (lysozyme, ribonuclease A, beta-lactoglobulin). The results indicated that the effective size and shape of 1-anilino-9-naphthalene sulfonate myoglobin are unaltered in the presence of up to 25 g/dl poly(ethylene glycol), whereas they are significantly altered in the presence of comparable concentrations of other proteins. The results are consistent with the hypothesis that in the presence of high concentrations of added protein, 1-anilino-9-naphthalene sulfonate myoglobin self-associates to form a dimer similar in size and shape to 1-anilino-9-naphthalene sulfonate hemoglobin.

  13. The Entropic Cost of Protein-Protein Association: A Case Study on Acetylcholinesterase Binding to Fasciculin-2

    PubMed Central

    Minh, David D. L.; Bui, Jennifer M.; Chang, Chia-en; Jain, Tushar; Swanson, Jessica M. J.; McCammon, J. Andrew

    2005-01-01

    Protein-protein association is accompanied by a large reduction in translational and rotational (external) entropy. Based on a 15 ns molecular dynamics simulation of acetylcholinesterase (AChE) in complex with fasciculin 2 (Fas2), we estimate the loss in external entropy using quasiharmonic analysis and histogram-based approximations of the probability distribution function. The external entropy loss of AChE-Fas2 binding, ∼30 cal/mol K, is found to be significantly larger than most previously characterized protein-ligand systems. However, it is less than the entropy loss estimated in an earlier study by A. V. Finkelstein and J. Janin, which was based on atomic motions in crystals. PMID:16100267

  14. RNA-binding protein RBM14 regulates dissociation and association of non-homologous end joining proteins.

    PubMed

    Simon, Nicholas E; Yuan, Ming; Kai, Mihoko

    2017-06-18

    Defects in the DNA damage response (DDR) are associated with multiple diseases, including cancers and neurodegenerative disorders. Emerging evidence indicates involvement of RNA-binding proteins (RBPs) in DDR. However, functions of RBPs in the DDR pathway remain elusive. We have shown previously that the RNA-binding protein RBM14 is required for non-homologous end joining (NHEJ). Here we show that RBM14 is required for efficient recruitment of XRCC4 and XLF to chromatin and the release of KU proteins from chromatin upon DNA damage. Failure of this process leads to accumulation of double-strand breaks (DSBs) in cells. Thus RBM14 plays crucial role in regulation of NHEJ upon DNA damage.

  15. Heavy metal-associated isoprenylated plant protein (HIPP): characterization of a family of proteins exclusive to plants.

    PubMed

    de Abreu-Neto, João Braga; Turchetto-Zolet, Andreia C; de Oliveira, Luiz Felipe Valter; Zanettini, Maria Helena Bodanese; Margis-Pinheiro, Marcia

    2013-04-01

    Metallochaperones are key proteins for the safe transport of metallic ions inside the cell. HIPPs (heavy metal-associated isoprenylated plant proteins) are metallochaperones that contain a metal binding domain (HMA) and a C-terminal isoprenylation motif. In this study, we provide evidence that proteins of this family are found only in vascular plants and may be separated into five distinct clusters. HIPPs may be involved in (a) heavy metal homeostasis and detoxification mechanisms, especially those involved in cadmium tolerance, (b) transcriptional responses to cold and drought, and (c) plant-pathogen interactions. In particular, our results show that the rice (Oryza sativa) HIPP OsHIPP41 gene is highly expressed in response to cold and drought stresses, and its product is localized in the cytosol and the nucleus. The results suggest that HIPPs play an important role in the development of vascular plants and in plant responses to environmental changes.

  16. Proteomic analysis of cytoskeleton-associated RNA binding proteins in developing rice seed.

    PubMed

    Doroshenk, Kelly A; Crofts, Andrew J; Morris, Robert T; Wyrick, John J; Okita, Thomas W

    2009-10-01

    In eukaryotes, RNA binding proteins (RBPs) play an integral role not only in RNA processing within the nucleus, but also in the cytoplasmic events of RNA transport, localization, translation, storage and degradation. While many studies have been done, relatively little is known about RBPs in plants. As part of our continuing efforts to understand cytoplasmic gene expression events in developing rice seed (Oryza sativa L.), a proteomics approach was used to identify cytoplasmic-localized, cytoskeletal-associated RBPs. The nucleic acid binding fraction from a cytoskeletal-enriched rice seed extract was isolated by Poly(U)-Sepharose affinity chromatography and analyzed using 2D gel electrophoresis. Analysis of 162 excised protein spots using mass spectrometry led to the identification of 148 distinct proteins, in addition to the highly abundant globulin and glutelin seed storage proteins. Identified proteins include those involved in RNA processing, translation, protein modification, cell signaling, and metabolism, as well as a number of hypothetical proteins. Proteins of particular interest with roles in RNA metabolism are discussed. These results have been deposited within the Rice RNA Binding Protein Database as part of an integrated study of plant cytoskeletal-associated RBPs using developing rice seed as a model.

  17. Access of viral proteins to mitochondria via mitochondria-associated membranes.

    PubMed

    Williamson, Chad D; Colberg-Poley, Anamaris M

    2009-05-01

    By exploiting host cell machineries, viruses provide powerful tools for gaining insight into cellular pathways. Proteins from two unrelated viruses, human CMV (HCMV) and HCV, are documented to traffic sequentially from the ER into mitochondria, probably through the mitochondria-associated membrane (MAM) compartment. The MAM are sites of ER-mitochondrial contact enabling the direct transfer of membrane bound lipids and the generation of high calcium (Ca2+) microdomains for mitochondria signalling and responses to cellular stress. Both HCV core protein and HCMV UL37 proteins are associated with Ca2+ regulation and apoptotic signals. Trafficking of viral proteins to the MAM may allow viruses to manipulate a variety of fundamental cellular processes, which converge at the MAM, including Ca2+ signalling, lipid synthesis and transfer, bioenergetics, metabolic flow, and apoptosis. Because of their distinct topologies and targeted MAM sub-domains, mitochondrial trafficking (albeit it through the MAM) of the HCMV and HCV proteins predictably involves alternative pathways and, hence, distinct targeting signals. Indeed, we found that multiple cellular and viral proteins, which target the MAM, showed no apparent consensus primary targeting sequences. Nonetheless, these viral proteins provide us with valuable tools to access the poorly characterised MAM compartment, to define its cellular constituents and describe how virus infection alters these to its own end. Furthermore, because proper trafficking of viral proteins is necessary for their function, discovering the requirements for MAM to mitochondrial trafficking of essential viral proteins may provide novel targets for the rational design of anti-viral drugs.

  18. The hypoparathyroidism-associated mutation in Drosophila Gcm compromises protein stability and glial cell formation

    PubMed Central

    Xi, Xiao; Lu, Lu; Zhuge, Chun-Chun; Chen, Xuebing; Zhai, Yuanfen; Cheng, Jingjing; Mao, Haian; Yang, Chang-Ching; Tan, Bertrand Chin-Ming; Lee, Yi-Nan; Chien, Cheng-Ting; Ho, Margaret S.

    2017-01-01

    Differentiated neurons and glia are acquired from immature precursors via transcriptional controls exerted by factors such as proteins in the family of Glial Cells Missing (Gcm). Mammalian Gcm proteins mediate neural stem cell induction, placenta and parathyroid development, whereas Drosophila Gcm proteins act as a key switch to determine neuronal and glial cell fates and regulate hemocyte development. The present study reports a hypoparathyroidism-associated mutation R59L that alters Drosophila Gcm (Gcm) protein stability, rendering it unstable, and hyperubiquitinated via the ubiquitin-proteasome system (UPS). GcmR59L interacts with the Slimb-based SCF complex and Protein Kinase C (PKC), which possibly plays a role in its phosphorylation, hence altering ubiquitination. Additionally, R59L causes reduced Gcm protein levels in a manner independent of the PEST domain signaling protein turnover. GcmR59L proteins bind DNA, functionally activate transcription, and induce glial cells, yet at a less efficient level. Finally, overexpression of either wild-type human Gcmb (hGcmb) or hGcmb carrying the conserved hypoparathyroidism mutation only slightly affects gliogenesis, indicating differential regulatory mechanisms in human and flies. Taken together, these findings demonstrate the significance of this disease-associated mutation in controlling Gcm protein stability via UPS, hence advance our understanding on how glial formation is regulated. PMID:28051179

  19. Emerging roles for centromere-associated proteins in DNA repair and genetic recombination.

    PubMed

    Osman, Fekret; Whitby, Matthew C

    2013-12-01

    Centromere proteins CENP-S and CENP-X are members of the constitutive centromere-associated network, which is a conserved group of proteins that are needed for the assembly and function of kinetochores at centromeres. Intriguingly CENP-S and CENP-X have alter egos going by the names of MHF1 (FANCM-associated histone-fold protein 1) and MHF2 respectively. In this guise they function with a DNA translocase called FANCM (Fanconi's anemia complementation group M) to promote DNA repair and homologous recombination. In the present review we discuss current knowledge of the biological roles of CENP-S and CENP-X and how their dual existence may be a common feature of CCAN (constitutive centromere-associated network) proteins.

  20. Automatic Extraction of Protein Point Mutations Using a Graph Bigram Association

    PubMed Central

    Lee, Lawrence C; Horn, Florence; Cohen, Fred E

    2007-01-01

    Protein point mutations are an essential component of the evolutionary and experimental analysis of protein structure and function. While many manually curated databases attempt to index point mutations, most experimentally generated point mutations and the biological impacts of the changes are described in the peer-reviewed published literature. We describe an application, Mutation GraB (Graph Bigram), that identifies, extracts, and verifies point mutations from biomedical literature. The principal problem of point mutation extraction is to link the point mutation with its associated protein and organism of origin. Our algorithm uses a graph-based bigram traversal to identify these relevant associations and exploits the Swiss-Prot protein database to verify this information. The graph bigram method is different from other models for point mutation extraction in that it incorporates frequency and positional data of all terms in an article to drive the point mutation–protein association. Our method was tested on 589 articles describing point mutations from the G protein–coupled receptor (GPCR), tyrosine kinase, and ion channel protein families. We evaluated our graph bigram metric against a word-proximity metric for term association on datasets of full-text literature in these three different protein families. Our testing shows that the graph bigram metric achieves a higher F-measure for the GPCRs (0.79 versus 0.76), protein tyrosine kinases (0.72 versus 0.69), and ion channel transporters (0.76 versus 0.74). Importantly, in situations where more than one protein can be assigned to a point mutation and disambiguation is required, the graph bigram metric achieves a precision of 0.84 compared with the word distance metric precision of 0.73. We believe the graph bigram search metric to be a significant improvement over previous search metrics for point mutation extraction and to be applicable to text-mining application requiring the association of words. PMID

  1. Protein-energy malnutrition alters hippocampal plasticity-associated protein expression following global ischemia in the gerbil.

    PubMed

    Prosser-Loose, Erin J; Verge, Valerie M K; Cayabyab, Francisco S; Paterson, Phyllis G

    2010-11-01

    Previously it has been demonstrated that protein-energy malnutrition (PEM) impairs habituation in the open field test following global ischemia. The present study examined the hypothesis that PEM exerts some of its deleterious effects on functional outcome by altering the post-ischemic expression of the plasticity-associated genes brain-derived neurotrophic factor (BDNF), its receptor tropomyosin-related kinase B (trkB), and growth-associated protein-43 (GAP-43). Male, Mongolian gerbils (11-12 wk) were randomized to either control diet (12.5% protein) or PEM (2% protein) for 4 wk, and then underwent 5 min bilateral common carotid artery occlusion or sham surgery. Tympanic temperature was maintained at 36.5 ± 0.5°C during surgery. Brains collected at 1, 3 and 7 d post-surgery were processed by in-situ hybridization or immunofluorescence. BDNF and trkB mRNA expression was increased in hippocampal CA1 neurons after ischemia at all time points and was not significantly influenced by diet. However, increased trkB protein expression after ischemia was exacerbated by PEM at 7 d in the CA1 region. Post-ischemic GAP-43 protein increased at 3 and 7 d in the CA1 region, and PEM intensified this response and extended it to the CA3 and hilar regions. PEM exerted these effects without exacerbating CA1 neuron loss caused by global ischemia. The findings suggest that PEM increases the stress response and/or hyper-excitability in the hippocampus after global ischemia. Nutritional care appears to have robust effects on plasticity mechanisms important to recovery after brain ischemia.

  2. Huntingtin-associated protein 1 (HAP1) is a cGMP-dependent kinase anchoring protein (GKAP) specific for the cGMP-dependent protein kinase Iβ isoform.

    PubMed

    Corradini, Eleonora; Burgers, Pepijn P; Plank, Michael; Heck, Albert J R; Scholten, Arjen

    2015-03-20

    Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform*

    PubMed Central

    Corradini, Eleonora; Burgers, Pepijn P.; Plank, Michael; Heck, Albert J. R.; Scholten, Arjen

    2015-01-01

    Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins. PMID:25653285

  4. Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

    PubMed Central

    Dick, Robert A.; Kamynina, Elena

    2013-01-01

    In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV. PMID:24109216

  5. Evaluation of differential protein expression in Haliclona aquarius and sponge-associated microorganisms under cadmium stress.

    PubMed

    Wanick, Rodrigo Cunha; de Sousa Barbosa, Herbert; Frazão, Leonardo Revoredo; Santelli, Ricardo Erthal; Arruda, Marco Aurélio Zezzi; Coutinho, Cristiano Carvalho

    2013-09-01

    A comparative proteomic approach was used to assess differentially expressed proteins in marine sponges after 36 h of exposure to cadmium (Cd). After separation performed by 2-D polyacrylamide gel electrophoresis, 46 protein spots indicated differential expression, and 17 of these proteins were identified by electrospray ionization quadrupole time-of-flight mass spectrometry. From the proteins identified, 76% were attributed to sponge-associated microorganisms (fungi and bacteria), and 24% were attributed to Haliclona aquarius. Some of the proteins that were identified may be related to cell proliferation and differentiation or processes of oxidative stress repair and energy procurement. An integrated evaluation based on spot expression levels and the postulated functions of these proteins allowed a more accurate evaluation of the stress caused to the sponge holobiont system by cadmium exposure. This study could provide new insights into the use of a proteomic approach in the marine sponge to assess the effects of Cd pollution in a marine environment.

  6. Shark myelin basic protein: amino acid sequence, secondary structure, and self-association.

    PubMed

    Milne, T J; Atkins, A R; Warren, J A; Auton, W P; Smith, R

    1990-09-01

    Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.

  7. J 16: An apex protein associated with juvenility of Sequoiadendron giganteum.

    PubMed

    Bon, M C

    1988-12-01

    Shoot apex proteins from 11 seedlings, two juvenile clones and three mature grafted clones of Sequoiadendron giganteum (Lindl.) Buchh. were examined throughout the year by SDS-polyacrylamide gel electrophoresis. A membrane-associated protein known as J 16, with an approximate molecular weight of 16 kD, was always detected in juvenile tissue, but was absent in tissue of the mature clones. The J 16 protein was also present in extracts of a mature clone that had been morphologically rejuvenated by micrografting. Protein extracts from the rejuvenated clone, were separated by electrophoresis and assayed for J 16 using an immunoblotting procedure with anti-J 16 serum. This test revealed two bands, J 16 and another protein with a lower molecular weight. The small molecular weight protein was not detected in juvenile clones.

  8. Echinoderm microtubule-associated protein -like protein 5 in anterior temporal neocortex of patients with intractable epilepsy

    PubMed Central

    Sun, Ji-jun; Huang, Min; Xiao, Fei; Xi, Zhi-qin

    2015-01-01

    Objective(s): EMAP-like Protein 5 (EML5) is a new echinoderm microtubule-associated protein that is expressed in the central nervous system. The aim of this study was to investigate the expression profile of EML5 in the anterior temporal neocortex of patients presenting with intractable epilepsy (IE). Materials and Methods: Western blot assays were performed to determine EML5 expression in 36 surgically resected anterior temporal neocortices of patients with IE and eight control tissues. Immunohistochemistry and immunofluorescence were employed to explore protein expression in IE. Results: EML5 was highly expressed in both neurons and glial cells of the anterior temporal neocortex of IE patients, whereas only low levels of EML5 were detected in control brain tissues. Western blotting showed an enhanced expression of EML5 protein in the anterior temporal neocortex of IE (optical density (OD) = 1.8030 ± 0.1335/1.1852 ± 0.2253, P<0.05) compared with normal control tissues. Conclusion: The results demonstrate that highly expressed EML5 in the neurons and glial cells of the cortex of patients with epilepsy is associated with microtubular dysfunction after frequent and recurrent seizures. PMID:26730336

  9. Identification of genetic loci associated with crude protein and mineral concentrations in alfalfa (Medicago sativa) using association mapping.

    PubMed

    Jia, Congjun; Wu, Xinming; Chen, Min; Wang, Yunqi; Liu, Xiqiang; Gong, Pan; Xu, Qingfang; Wang, Xuemin; Gao, Hongwen; Wang, Zan

    2017-06-06

    Alfalfa (Medicago sativa) is one of the most important legume forage species in China and many other countries of the world. It provides a quality source of proteins and minerals to animals. Genetic underpinnings for these important traits, however, are elusive. An alfalfa (M. sativa) association mapping study for six traits, namely crude protein (CP), rumen undegraded protein (RUP), and four mineral elements (Ca, K, Mg and P), was conducted in three consecutive years using a large collection encompassing 336 genotypes genotyped with 85 simple sequence repeat (SSR) markers. All the traits were significantly influenced by genotype, environment, and genotype × environment interaction. Eight-five significant associations (P < 0.005) were identified. Among these, five associations with Ca were repeatedly observed and six co-localized associations were identified. The identified marker alleles significantly associated with the traits provided important information for understanding genetic controls of alfalfa quality. The markers could be used in assisting selection for the individual traits in breeding populations for developing new alfalfa cultivars.

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

    USDA-ARS?s Scientific Manuscript database

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

  11. Differential expression in Phanerochaete chrysosporium of membrane- associated proteins relevant to lignin degradation

    Treesearch

    Semarjit Shary; Alexander N. Kapich; Ellen A. Panisko; Jon K. Magnuson; Daniel Cullen; Kenneth E. Hammel

    2008-01-01

    Fungal lignin-degrading systems likely include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, production of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Little is known about the nature or regulation of these membrane-associated components. We grew...

  12. Identification of ASK and clock-associated proteins as molecular partners of LKP2 (LOV kelch protein 2) in Arabidopsis.

    PubMed

    Yasuhara, Masahiro; Mitsui, Shunya; Hirano, Hiroshi; Takanabe, Rieko; Tokioka, Yoko; Ihara, Norihisa; Komatsu, Akihiro; Seki, Motoaki; Shinozaki, Kazuo; Kiyosue, Tomohiro

    2004-09-01

    The ADO/FKF/LKP/ZTL family of proteins of Arabidopsis thaliana Heynh. have a LOV domain, an F-box motif, and a kelch repeat region. LKP2 is a member of this family and functions either within or very close to the circadian oscillator in Arabidopsis. Promoter-GUS fusion studies revealed that the LKP2 gene was highly active in rosette leaves. In CaMV 35S:LKP2-GFP plants, GFP-associated fluorescence was detected in nuclei, suggesting that LKP2 is a nuclear protein. Yeast two-hybrid analysis demonstrated that LKP2 interacted with some Arabidopsis Skp1-like proteins (ASK), as do other ADO/FKF/LKP/ZTL family proteins, suggesting that LKP2 can form an SCF (Skp1-Cullin-F-box protein) complex that functions as a ubiquitin E3 ligase. LKP2 interacted not only with itself but also with other members of the family, LKP1 and FKF1. The two-hybrid analysis also demonstrated that LKP2 interacted with TOC1, a clock component, but not with CCA1 or LHY, negative regulators of TOC1 gene expression. The LOV domain of LKP2 was shown to be necessary and sufficient for the interaction with TOC1. An interaction between LKP2 and APRR5, a paralogue of TOC1, was also observed, but LKP2 did not interact with APRR3, APRR7, or APRR9, other paralogues of TOC1.

  13. Association of atypical protein kinase C isotypes with the docker protein FRS2 in fibroblast growth factor signaling.

    PubMed

    Lim, Y P; Low, B C; Lim, J; Wong, E S; Guy, G R

    1999-07-02

    FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC lambda when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC zeta, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC lambda is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281-492), an isoform bearing 50% identity to FRS2, interacted with PKC lambda at a region (amino acids 240-562) that encompasses the catalytic domain. In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC lambda or zeta. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC lambda results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its "closed" wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC lambda mutant, suggesting that the activation of PKC lambda is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.

  14. Mechanism of oligomerisation of cyclase-associated protein from Dictyostelium discoideum in solution.

    PubMed

    Yusof, Adlina Mohd; Jaenicke, Elmar; Pedersen, Jan Skov; Noegel, Angelika A; Schleicher, Michael; Hofmann, Andreas

    2006-10-06

    Cyclase-associated protein (CAP) is a highly conserved modular protein implicated in the regulation of actin filament dynamics and a variety of developmental and morphological processes. The protein exists as a high molecular weight complex in cell extracts and purified protein possesses a high tendency to aggregate, a major obstacle for crystallisation. Using a mutagenesis approach, we show that two structural features underlie the mechanism of oligomerisation in Dictyostelium discoideum CAP. Positively charged clusters on the surface of the N-terminal helix-barrel domain are involved in inter-molecular interactions with the N or C-terminal domains. Abolishing these interactions mainly renders dimers due to a domain swap feature in the extreme C-terminal region of the protein that was previously described. Based on earlier studies with yeast CAP, we also generated constructs with mutations in the extreme N-terminal region of Dictyostelium CAP that did not show significantly altered oligomerisation behaviour. Constructs with mutations in the earlier identified protein-protein interaction interface on the N-terminal domain of CAP could not be expressed as soluble protein. Assessment of the soluble proteins indicates that the mutations did not affect their overall fold. Further studies point to the correlation between stability of full-length CAP with its multimerisation behaviour, where oligomer formation leads to a more stable protein.

  15. Multiple cellular proteins modulate the dynamics of K-ras association with the plasma membrane.

    PubMed

    Bhagatji, Pinkesh; Leventis, Rania; Rich, Rebecca; Lin, Chen-ju; Silvius, John R

    2010-11-17

    Although specific proteins have been identified that regulate the membrane association and facilitate intracellular transport of prenylated Rho- and Rab-family proteins, it is not known whether cellular proteins fulfill similar roles for other prenylated species, such as Ras-family proteins. We used a previously described method to evaluate how several cellular proteins, previously identified as potential binding partners (but not effectors) of K-ras4B, influence the dynamics of K-ras association with the plasma membrane. Overexpression of either PDEδ or PRA1 enhances, whereas knockdown of either protein reduces, the rate of dissociation of K-ras from the plasma membrane. Inhibition of calmodulin likewise reduces the rate of K-ras dissociation from the plasma membrane, in this case in a manner specific for the activated form of K-ras. By contrast, galectin-3 specifically reduces the rate of plasma membrane dissociation of activated K-ras, an effect that is blocked by the K-ras antagonist farnesylthiosalicylic acid (salirasib). Multiple cellular proteins thus control the dynamics of membrane association and intercompartmental movement of K-ras to an important degree even under basal cellular conditions. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Effect of free or protein-associated soy isoflavones on the antioxidant status in rats.

    PubMed

    Barbosa, Ana Cl; Lajolo, Franco M; Genovese, Maria I

    2011-03-15

    The objective of this study was to investigate the effect of chronic ingestion of free and protein-associated soy isoflavones on the antioxidant status in male Wistar rats. Free isoflavone (iso), protein-associated soy isoflavone (iso + prot) and soy protein (prot) extracts were administered for 30 days by gavage to the rats at a dosage of 1 mg aglycone isoflavones per 200 g body weight, adjusted daily, and the prot group was given the same concentration of soy protein received by the iso + prot group. Antioxidant capacity of plasma, thiobarbituric acid-reactive substance (TBARS) and glutathione (GSH) levels and catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities in plasma, erythrocytes and tissues and gene expression levels in liver and kidney were evaluated. Chronic ingestion of free but not of protein-associated soy isoflavones nor of solely soy protein increased plasma antioxidant capacity and GPx activity in erythrocytes. Soy protein increased CAT activity and gene expression in liver. SOD activity in erythrocytes was increased by all treatments. The overall results confirm that dietary soy isoflavones have a positive effect on antioxidant status, enhancing antioxidant capacity of plasma and antioxidant enzymes in various tissues, but the effects are dependent on the form of administration and on a complex mechanism of antioxidant status balance on the organism. Copyright © 2010 Society of Chemical Industry.

  17. Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

    SciTech Connect

    Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun; Chun, ChangJu; Im, Young Jun

    2014-09-12

    Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.

  18. A novel 58-kDa protein associates with the Golgi apparatus and microtubules.

    PubMed

    Bloom, G S; Brashear, T A

    1989-09-25

    With the aim of identifying proteins involved in linking microtubules to other cytoplasmic structures, microtubule-binding proteins were isolated from rat liver extracts by a taxol-dependent procedure. The major non-tubulin component, a 58-kDa protein (designated 58K), was purified to homogeneity by gel filtration chromatography. To aid further characterization of 58K, purified preparations of the protein were used as immunogen for the production of monoclonal antibodies. Five different monoclonals were obtained, and each of these reacted on immunoblots of liver homogenates with a single band that comigrated with 58K. Based on the results of immunochemical, peptide mapping, and microsequencing experiments, 58K was found to be unrelated structurally to similarly sized cytoskeleton-associated proteins, such as tubulin, tau, vimentin, or keratin, and to represent a new protein species. Several in vitro properties of 58K were found to be characteristic of microtubule-associated proteins. For instance, 58K cosedimented quantitatively with microtubules out of liver extracts, stimulated polymerization of tubulin, and bound to microtubules in a saturable manner. In contrast to traditional microtubule-associated proteins, however, 58K was not found to be distributed uniformly along microtubules in cells. Immunofluorescence microscopy of cultured hepatoma cells revealed, instead, that 58K is associated principally with the Golgi apparatus. Moreover, Golgi membranes isolated from rat liver were observed by immunoblotting to contain significant levels of 58K, which, upon subfractionation of the membranes, partitioned as if it were a peripheral membrane protein exposed to the cytoplasmic side of the Golgi. These collective results have been evaluated in terms of earlier evidence that the intracellular position and structural integrity of the Golgi relies on the presence and organization of microtubules. In that context, the observations reported here suggest that the in vivo

  19. Nodavirus RNA Replication Protein A Induces Membrane Association of Genomic RNA▿

    PubMed Central

    Van Wynsberghe, Priscilla M.; Chen, Hau-Ren; Ahlquist, Paul

    2007-01-01

    Positive-strand RNA virus genome replication occurs in membrane-associated RNA replication complexes, whose assembly remains poorly understood. Here we show that prior to RNA replication, the multifunctional, transmembrane RNA replication protein A of the nodavirus flock house virus (FHV) recruits FHV genomic RNA1 to a membrane-associated state in both Drosophila melanogaster and Saccharomyces cerevisiae cells. Protein A has mitochondrial membrane-targeting, self-interaction, RNA-dependent RNA polymerase (RdRp), and RNA capping domains. In the absence of RdRp activity due to an active site mutation (AD692E), protein A stimulated RNA1 accumulation by increasing RNA1 stability. Protein AD692E stimulated RNA1 accumulation in wild-type cells and in xrn1− yeast defective in decapped RNA decay, showing that increased RNA1 stability was not due to protein A-mediated RNA1 recapping. Increased RNA1 stability was closely linked with protein A-induced membrane association of the stabilized RNA and was highly selective for RNA1. Substantial N- and C-proximal regions of protein A were dispensable for these activities. However, increased RNA1 accumulation was eliminated by deleting protein A amino acids (aa) 1 to 370 but was restored completely by adding back the transmembrane domain (aa 1 to 35) and partially by adding back peripheral membrane association sequences in aa 36 to 370. Moreover, although RNA polymerase activity was not required, even small deletions in or around the RdRp domain abolished increased RNA1 accumulation. These and other results show that prior to negative-strand RNA synthesis, multiple domains of mitochondrially targeted protein A cooperate to selectively recruit FHV genomic RNA to membranes where RNA replication complexes form. PMID:17301137

  20. Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

    PubMed

    Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

    2017-08-01

    The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes.IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many

  1. Predicting Cell Association of Surface-Modified Nanoparticles Using Protein Corona Structure - Activity Relationships (PCSAR).

    PubMed

    Kamath, Padmaja; Fernandez, Alberto; Giralt, Francesc; Rallo, Robert

    2015-01-01

    Nanoparticles are likely to interact in real-case application scenarios with mixtures of proteins and biomolecules that will absorb onto their surface forming the so-called protein corona. Information related to the composition of the protein corona and net cell association was collected from literature for a library of surface-modified gold and silver nanoparticles. For each protein in the corona, sequence information was extracted and used to calculate physicochemical properties and statistical descriptors. Data cleaning and preprocessing techniques including statistical analysis and feature selection methods were applied to remove highly correlated, redundant and non-significant features. A weighting technique was applied to construct specific signatures that represent the corona composition for each nanoparticle. Using this basic set of protein descriptors, a new Protein Corona Structure-Activity Relationship (PCSAR) that relates net cell association with the physicochemical descriptors of the proteins that form the corona was developed and validated. The features that resulted from the feature selection were in line with already published literature, and the computational model constructed on these features had a good accuracy (R(2)LOO=0.76 and R(2)LMO(25%)=0.72) and stability, with the advantage that the fingerprints based on physicochemical descriptors were independent of the specific proteins that form the corona.

  2. Evolutionary conservation of mammalian sperm proteins associates with overall, not tyrosine, phosphorylation in human spermatozoa.

    PubMed

    Schumacher, Julia; Ramljak, Sanja; Asif, Abdul R; Schaffrath, Michael; Zischler, Hans; Herlyn, Holger

    2013-12-06

    We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein-protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility.

  3. Identification of an RNA Polymerase III Regulator Linked to Disease-Associated Protein Aggregation.

    PubMed

    Sin, Olga; de Jong, Tristan; Mata-Cabana, Alejandro; Kudron, Michelle; Zaini, Mohamad Amr; Aprile, Francesco A; Seinstra, Renée I; Stroo, Esther; Prins, Roméo Willinge; Martineau, Céline N; Wang, Hai Hui; Hogewerf, Wytse; Steinhof, Anne; Wanker, Erich E; Vendruscolo, Michele; Calkhoven, Cornelis F; Reinke, Valerie; Guryev, Victor; Nollen, Ellen A A

    2017-03-16

    Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer's and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases.

  4. Physicochemical principles that regulate the competition between functional and dysfunctional association of proteins.

    PubMed

    Pechmann, Sebastian; Levy, Emmanuel D; Tartaglia, Gian Gaetano; Vendruscolo, Michele

    2009-06-23

    To maintain protein homeostasis, a variety of quality control mechanisms, such as the unfolded protein response and the heat shock response, enable proteins to fold and to assemble into functional complexes while avoiding the formation of aberrant and potentially harmful aggregates. We show here that a complementary contribution to the regulation of the interactions between proteins is provided by the physicochemical properties of their amino acid sequences. The results of a systematic analysis of the protein-protein complexes in the Protein Data Bank (PDB) show that interface regions are more prone to aggregate than other surface regions, indicating that many of the interactions that promote the formation of functional complexes, including hydrophobic and electrostatic forces, can potentially also cause abnormal intermolecular association. We also show, however, that aggregation-prone interfaces are prevented from triggering uncontrolled assembly by being stabilized into their functional conformations by disulfide bonds and salt bridges. These results indicate that functional and dysfunctional association of proteins are promoted by similar forces but also that they are closely regulated by the presence of specific interactions that stabilize native states.

  5. Insights into disease-associated mutations in the human proteome through protein structural analysis

    PubMed Central

    Gao, Mu; Zhou, Hongyi; Skolnick, Jeffrey

    2015-01-01

    Summary Most known disease-associated mutations are missense mutations involving changes of amino acids of proteins encoded by their genes. Given the plethora of genetic studies, sequenced exomes, and new protein structures determined each year, it is appropriate to revisit the role that structure plays in providing insights into the molecular basis of disease associated mutations. In that regard, a large-scale structural analysis on 6,025 disease-associated mutations as well as 4,536 neutral variations for comparison was performed. While buried amino acids are common among the disease-associated mutations as reported previously, more are statistically significantly enriched at observed or predicted functional sites. Interesting findings are that ligand-binding sites adjacent to protein-protein interfaces and residues involved in enzymatic function are especially vulnerable to disease-associated mutations. Finally, a compositional analysis of disease-associated mutations in comparison to variants identified in the 1000 Genomes Project provides a structural rationalization of the most disease-associated residue types. PMID:26027735

  6. Association mapping of seed oil and protein content in Sesamum indicum L. using SSR markers.

    PubMed

    Li, Chun; Miao, Hongmei; Wei, Libin; Zhang, Tide; Han, Xiuhua; Zhang, Haiyang

    2014-01-01

    Sesame is an important oil crop for the high oil content and quality. The seed oil and protein contents are two important traits in sesame. To identify the molecular markers associated with the seed oil and protein contents in sesame, we systematically performed the association mapping among 369 worldwide germplasm accessions under 5 environments using 112 polymorphic SSR markers. The general linear model (GLM) was applied with the criteria of logP ≥ 3.0 and high stability under all 5 environments. Among the 369 sesame accessions, the oil content ranged from 27.89%-58.73% and the protein content ranged from 16.72%-27.79%. A significant negative correlation of the oil content with the protein content was found in the population. A total of 19 markers for oil content were detected with a R2 value range from 4% to 29%; 24 markers for protein content were detected with a R2 value range from 3% to 29%, of which 19 markers were associated with both traits. Moreover, partial markers were confirmed using mixed linear model (MLM) method, which suggested that the oil and protein contents are controlled mostly by major genes. Allele effect analysis showed that the allele associated with high oil content was always associated with low protein content, and vice versa. Of the 19 markers associated with oil content, 17 presented near the locations of the plant lipid pathway genes and 2 were located just next to a fatty acid elongation gene and a gene encoding Stearoyl-ACP Desaturase, respectively. The findings provided a valuable foundation for oil synthesis gene identification and molecular marker assistant selection (MAS) breeding in sesame.

  7. Aberrant expression of DNA damage response proteins is associated with breast cancer subtype and clinical features

    PubMed Central

    Guler, Gulnur; Himmetoglu, Cigdem; Jimenez, Rafael E.; Geyer, Susan M.; Wang, Wenle P.; Costinean, Stefan; Pilarski, Robert T.; Morrison, Carl; Suren, Dinc; Liu, Jianhua; Chen, Jingchun; Kamal, Jyoti; Shapiro, Charles L.

    2013-01-01

    Landmark studies of the status of DNA damage checkpoints and associated repair functions in preneoplastic and neoplastic cells has focused attention on importance of these pathways in cancer development, and inhibitors of repair pathways are in clinical trials for treatment of triple negative breast cancer. Cancer heterogeneity suggests that specific cancer subtypes will have distinct mechanisms of DNA damage survival, dependent on biological context. In this study, status of DNA damage response (DDR)-associated proteins was examined in breast cancer subtypes in association with clinical features; 479 breast cancers were examined for expression of DDR proteins γH2AX, BRCA1, pChk2, and p53, DNA damage-sensitive tumor suppressors Fhit and Wwox, and Wwox-interacting proteins Ap2α, Ap2γ, ErbB4, and correlations among proteins, tumor subtypes, and clinical features were assessed. In a multivariable model, triple negative cancers showed significantly reduced Fhit and Wwox, increased p53 and Ap2γ protein expression, and were significantly more likely than other subtype tumors to exhibit aberrant expression of two or more DDR-associated proteins. Disease-free survival was associated with subtype, Fhit and membrane ErbB4 expression level and aberrant expression of multiple DDR-associated proteins. These results suggest that definition of specific DNA repair and checkpoint defects in subgroups of triple negative cancer might identify new treatment targets. Expression of Wwox and its interactor, ErbB4, was highly significantly reduced in metastatic tissues vs. matched primary tissues, suggesting that Wwox signal pathway loss contributes to lymph node metastasis, perhaps by allowing survival of tumor cells that have detached from basement membranes, as proposed for the role of Wwox in ovarian cancer spread. PMID:21069451

  8. Association Mapping of Seed Oil and Protein Content in Sesamum indicum L. Using SSR Markers

    PubMed Central

    Li, Chun; Miao, Hongmei; Wei, Libin; Zhang, Tide; Han, Xiuhua; Zhang, Haiyang

    2014-01-01

    Sesame is an important oil crop for the high oil content and quality. The seed oil and protein contents are two important traits in sesame. To identify the molecular markers associated with the seed oil and protein contents in sesame, we systematically performed the association mapping among 369 worldwide germplasm accessions under 5 environments using 112 polymorphic SSR markers. The general linear model (GLM) was applied with the criteria of logP≥3.0 and high stability under all 5 environments. Among the 369 sesame accessions, the oil content ranged from 27.89%–58.73% and the protein content ranged from 16.72%–27.79%. A significant negative correlation of the oil content with the protein content was found in the population. A total of 19 markers for oil content were detected with a R2 value range from 4% to 29%; 24 markers for protein content were detected with a R2 value range from 3% to 29%, of which 19 markers were associated with both traits. Moreover, partial markers were confirmed using mixed linear model (MLM) method, which suggested that the oil and protein contents are controlled mostly by major genes. Allele effect analysis showed that the allele associated with high oil content was always associated with low protein content, and vice versa. Of the 19 markers associated with oil content, 17 presented near the locations of the plant lipid pathway genes and 2 were located just next to a fatty acid elongation gene and a gene encoding Stearoyl-ACP Desaturase, respectively. The findings provided a valuable foundation for oil synthesis gene identification and molecular marker assistant selection (MAS) breeding in sesame. PMID:25153139

  9. Levels of the growth-associated protein GAP-43 are selectively increased in association cortices in schizophrenia

    PubMed Central

    Perrone-Bizzozero, Nora I.; Sower, Angela C.; Bird, Edward D.; Benowitz, Larry I.; Ivins, Kathryn J.; Neve, Rachael L.

    1996-01-01

    The pathophysiology of schizophrenia may involve perturbations of synaptic organization during development. The presence of cytoarchitectural abnormalities that may reflect such perturbations in the brains of patients with this disorder has been well-documented. Yet the mechanistic basis for these features of the disorder is still unknown. We hypothesized that altered regulation of the neuronal growth-associated protein GAP-43, a membrane phosphoprotein found at high levels in the developing brain, may play a role in the alterations in brain structure and function observed in schizophrenia. In the mature human brain, GAP-43 remains enriched primarily in association cortices and in the hippocampus, and it has been suggested that this protein marks circuits involved in the acquisition, processing, and/or storage of new information. Because these processes are known to be altered in schizophrenia, we proposed that GAP-43 levels might be altered in this disorder. Quantitative immunoblots revealed that the expression of GAP-43 is increased preferentially in the visual association and frontal cortices of schizophrenic patients, and that these changes are not present in other neuropsychiatric conditions requiring similar treatments. Examination of the levels of additional markers in the brain revealed that the levels of the synaptic vesicle protein synaptophysin are reduced in the same areas, but that the abundance of the astrocytic marker of neurodegeneration, the glial fibrillary acidic protein, is unchanged. In situ hybridization histochemistry was used to show that the laminar pattern of GAP-43 expression appears unaltered in schizophrenia. We propose that schizophrenia is associated with a perturbed organization of synaptic connections in distinct cortical associative areas of the human brain, and that increased levels of GAP-43 are one manifestation of this dysfunctional organization. PMID:8943081

  10. Associations between proteins and heavy metals in urine at low environmental exposures: evidence of reverse causality.

    PubMed

    Chaumont, Agnès; Nickmilder, Marc; Dumont, Xavier; Lundh, Thomas; Skerfving, Staffan; Bernard, Alfred

    2012-05-05

    Heavy metals can cause renal effects on vulnerable populations but it is uncertain whether these metals still pose health risks at the low exposure levels now prevailing in most industrialized countries. In a cross-sectional study performed on 736 adolescents, we assessed the associations between the concentrations of cadmium and lead in blood and urine and the urinary concentrations of albumin and of low-molecular-weight (LMW) proteins, retinol-binding protein (RBP) and β(2)-microglobulin. Multiple regression analyses were tested using urinary markers normalized to urinary creatinine or specific gravity. Median metal concentrations were in blood (μg/L): lead, 15.1, cadmium, 0.18 and in urine (μg/g creatinine): cadmium, 0.09 and lead, 0.82. Multivariate analyses revealed significant associations in urine between RBP and cadmium as well as between β(2)-microglobulin and lead whereas no associations were seen with metals in blood. These associations were completely abolished in subjects with increased urinary albumin, which may be explained by the competitive inhibition of LMW protein reabsorption by albumin. Given the evidence that cadmium and lead circulate mainly bound to LMW proteins, these associations observed at low exposure might simply reflect the interindividual variations in the renal uptake of proteins sharing the same affinity for tubular binding sites.

  11. The small myelin-associated glycoprotein is a zinc-binding protein.

    PubMed

    Kursula, P; Meriläinen, G; Lehto, V P; Heape, A M

    1999-11-01

    The myelin-associated glycoprotein is a transmembrane cell adhesion molecule expressed specifically by myelinating glial cells of the nervous system. Its two isoforms, whose amino acid sequences differ only by their respective cytoplasmic carboxy-terminal domains, are important for the formation and maintenance of a normal functional myelin sheath. In this study, by using recombinant proteins, we identify the cytoplasmic domain of the small isoform of the myelin-associated glycoprotein as a zinc-binding protein. The observed dissociation constant lies in the low micromolar range (K(D) = 6-7 microM). The binding of zinc by the small myelin-associated glycoprotein induces a conformational change that enables the protein to reversibly bind to a hydrophobic phenyl-Sepharose matrix. Our results also suggest that zinc may induce dimerization of the small myelin-associated glycoprotein. We suggest roles for zinc in the stabilization of the structure of the cytoplasmic domain of the small myelin-associated glycoprotein and in protein-protein interactions that involve this short domain.

  12. The Ribosomal Biogenesis Protein Utp21 Interacts with Hsp90 and Has Differing Requirements for Hsp90-Associated Proteins

    PubMed Central

    Tenge, Victoria R.; Knowles, Jared; Johnson, Jill L.

    2014-01-01

    The molecular chaperone Hsp90 buffers the effects of genetic variation by assisting the stabilization and folding of multiple clients critical for cell signaling and growth. We identified an interaction of Hsp90 and associated proteins with the essential nucleolar protein, Utp21, part of a large complex required for biogenesis of the small ribosomal subunit. The utp21-S602F mutation, which causes minor defects in otherwise wild-type yeast, exhibited severe or lethal growth defects when combined with mutations in Hsp90 or co-chaperones. WT Utp21 and Utp21-S602F exhibited similar interactions with Hsp90, and steady-state levels of WT Utp21 were reduced upon Hsp90 mutation or inhibition. Mutations in the human homolog of UTP21, WDR36, have been associated with adult-onset primary open-angle glaucoma, a leading cause of blindness worldwide. Three different mutant forms of Utp21 analogous to glaucoma-associated WDR36 mutations exhibit reduced levels in yeast cells expressing mutations in Hsp90 or associated chaperones, suggesting that Hsp90 and co-chaperones buffer the effects of those mutations. PMID:24647762

  13. Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein.

    PubMed

    Miwa, Naofumi

    2015-05-22

    Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP), which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

  14. Identification of potential molecular associations between chikungunya virus non-structural protein 2 and human host proteins.

    PubMed

    Rana, J; Gulati, S; Rajasekharan, S; Gupta, A; Chaudhary, V; Gupta, S

    2017-01-01

    Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is considered to be the master regulator of viral RNA replication and host responses generated during viral infection. This protein has two main functional domains: an N-terminal domain which exhibits NTPase, RNA triphosphatase and helicase activities and a C-terminal protease domain. Understanding how CHIKV nsP2 interacts with its host proteins is essential for elucidating all the required processes for viral replication and pathogenesis along with the identification of potential targets for antiviral therapy. In current study yeast two-hybrid (Y2H) screening of a human fetal brain cDNA library was performed using nsP2 protein as bait. The analysis identified seven host proteins (CCDC130, CPNE6, POLR2C, MAPK9, EIF4A2, EEF1A1 and EIF3I) as putative interactors of CHIKV nsP2 which were selected for further analysis based on their roles in host cellular machinery. The gene ontology analysis indicates that these proteins are mainly involved in apoptosis, transcription and translational mechanism of host cell. Domain mapping of nsP2 revealed that these associations are not random connections but instead they have functional significance. Further studies to identify the amino acid residues and their chemical interactions that may help in opening new possibilities for preventing these interactions, thus reducing chances of chikungunya infection were performed. This study expands the understanding of CHIKV-host interactions and is important for rational approaches of discovering new antiviral agents.

  15. Protein phosphorylation associated with epipodophyllotoxin-induced apoptosis of lymphoid cells: role of a serine/threonine protein kinase.

    PubMed

    Ye, X; Mody, N S; Hingley, S T; Coffman, F D; Cohen, S; Fresa, K L

    1998-11-01

    We have previously shown that apoptosis induced in thymocytes by dexamethasone or teniposide (VM-26) could be inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and sangivamycin, both relatively specific inhibitors for protein kinase C, but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor for cAMP-dependent protein kinases. Apoptosis in this model system was not blocked by EGTA and no increase in cytosolic Ca2+ was observed during apoptosis induced by either dexamethasone or VM-26, suggesting that this kinase was Ca2+-independent. In the present study, we demonstrate that addition of 10 microM sangivamycin to thymocyte cultures up to 2 h after addition of either inducer resulted in virtually complete inhibition of apoptosis. Addition of 10 microM sangivamycin at 3 or 4 h after addition of inducer resulted in partial inhibition of apoptosis. Computerized image analysis of two-dimensional PAGE analyses of whole-cell lysates demonstrated that treatment of mouse thymocytes with VM-26 resulted in a limited number of de novo phosphorylation events within 1 h of treatment. The most prominent phosphorylation events associated with VM-26-induced apoptosis were that two intracellular protein species (Protein 1: m.w. = 22.9 kDa, pI, 5.11; and Protein 2: m.w. = 22.9 kDa, pI, 4.98). Similar phosphorylation events were seen in cells treated with dexamethasone. Finally, Western blot analysis suggests that de novo protein phosphorylation induced by VM-26 is on serine/threonine residues. These results provide further evidence that the mechanism of VM-26-induced apoptosis of murine thymocytes involves the action of one or more serine/threonine kinases. Copyright 1998 Academic Press.

  16. Wool Keratin-Associated Protein Genes in Sheep-A Review.

    PubMed

    Gong, Hua; Zhou, Huitong; Forrest, Rachel H J; Li, Shaobin; Wang, Jiqing; Dyer, Jolon M; Luo, Yuzhu; Hickford, Jon G H

    2016-05-28

    The importance of sheep's wool in making textiles has inspired extensive research into its structure and the underlying genetics since the 1960s. Wool keratin-associated proteins (KAPs) are a key structural component of the wool fibre. The characterisation of the genes encoding these proteins has progressed rapidly with advances in the nucleotide and protein sequencing. This review describes our knowledge of ovine KAPs, their categorisation into families, polymorphism in the proteins and genes, the clustering and chromosomal location of the genes, some characteristics of gene expression and some potential effects of the KAPs on wool traits. The extent and nature of genetic variation in wool KAP genes and its association with fibre characteristics, provides an opportunity for the development of gene-markers for selective breeding of sheep to produce better wool with properties highly matched to specific end-uses.

  17. Wool Keratin-Associated Protein Genes in Sheep—A Review

    PubMed Central

    Gong, Hua; Zhou, Huitong; Forrest, Rachel H. J.; Li, Shaobin; Wang, Jiqing; Dyer, Jolon M.; Luo, Yuzhu; Hickford, Jon G. H.

    2016-01-01

    The importance of sheep’s wool in making textiles has inspired extensive research into its structure and the underlying genetics since the 1960s. Wool keratin-associated proteins (KAPs) are a key structural component of the wool fibre. The characterisation of the genes encoding these proteins has progressed rapidly with advances in the nucleotide and protein sequencing. This review describes our knowledge of ovine KAPs, their categorisation into families, polymorphism in the proteins and genes, the clustering and chromosomal location of the genes, some characteristics of gene expression and some potential effects of the KAPs on wool traits. The extent and nature of genetic variation in wool KAP genes and its association with fibre characteristics, provides an opportunity for the development of gene-markers for selective breeding of sheep to produce better wool with properties highly matched to specific end-uses. PMID:27240405

  18. The CCN Family Proteins: Modulators of Bone Development and Novel Targets in Bone-Associated Tumors

    PubMed Central

    Chen, Po-Chun; Cheng, Hsu-Chen; Yang, Shun-Fa; Tang, Chih-Hsin

    2014-01-01

    The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors. PMID:24551846

  19. Postranslational modifications significantly alter the binding-folding pathways of proteins associating with DNA

    NASA Astrophysics Data System (ADS)

    Papoian, Garegin

    2012-02-01

    Many important regulators of gene activity are natively disordered, but fully or partially order when they bind to their targets on DNA. Interestingly, the ensembles of disordered states for such free proteins are not structurally featureless, but can qualitatively differ from protein to protein. In particular, in random coil like states the chains are swollen, making relatively few contacts, while in molten globule like states a significant collapse occurs, with ensuing high density of intra-protein interactions. Furthermore, since many DNA binding proteins are positively charged polyelectrolytes, the electrostatic self-repulsion also influences the degree of collapse of the chain and its conformational preferences in the free state and upon binding to DNA. In our work, we have found that the nature of the natively disordered ensemble significantly affects the way the protein folds upon binding to DNA. In particular, we showed that posttranslational modifications of amino acid residues, such as lysine acetylation, can alter the degree of collapse and conformational preferences for a free protein, and also profoundly impact the binding affinity and pathways for the protein DNA association. These trends will be discussed in the context of DNA interacting with various histone tails and the p53 protein.

  20. Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements.

    PubMed

    Kirchner, Liselotte; Weitzdoerfer, Rachel; Hoeger, Harald; Url, Angelika; Schmidt, Peter; Engelmann, Mario; Villar, Santiago Rosell; Fountoulakis, Michael; Lubec, Gert; Lubec, Barbara

    2004-12-01

    Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.

  1. The CCN family proteins: modulators of bone development and novel targets in bone-associated tumors.

    PubMed

    Chen, Po-Chun; Cheng, Hsu-Chen; Yang, Shun-Fa; Lin, Chiao-Wen; Tang, Chih-Hsin

    2014-01-01

    The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors.

  2. Binding of lysozyme to phospholipid bilayers: evidence for protein aggregation upon membrane association.

    PubMed

    Gorbenko, Galyna P; Ioffe, Valeriya M; Kinnunen, Paavo K J

    2007-07-01

    Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5'-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated

  3. Binding of Lysozyme to Phospholipid Bilayers: Evidence for Protein Aggregation upon Membrane Association

    PubMed Central

    Gorbenko, Galyna P.; Ioffe, Valeriya M.; Kinnunen, Paavo K. J.

    2007-01-01

    Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5′-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated

  4. Oct3/4-associating proteins from embryonal carcinoma and spermatogenic cells of mouse.

    PubMed

    Tomilin, A; Vorob'ev, V; Drosdowsky, M; Séralini, G E

    1998-03-01

    The octamer-binding protein Oct3/4 was postulated to active transcription through protein-protein interactions with hypothetical cellular coactivator(s). We have used a bacterially produced Oct3/4, as a protein-binding probe, to detect by far-Western assay the Oct3/4-associating proteins (OTAPs) from the embryonal carcinoma (EC) cells F9 and pachytene spermatocytes. Both common and cell-specific OTAPs were shown to interact directly with Oct3/4. Differentiation of the EC cells results in disappearance of most of OTAPs, supporting their coactivator nature. Several OTAPs detected in pachytene spermatocyte may represent germ cell-specific Oct3/4 coactivators.

  5. Wnt family proteins are secreted and associated with the cell surface.

    PubMed Central

    Smolich, B D; McMahon, J A; McMahon, A P; Papkoff, J

    1993-01-01

    Members of the Wnt gene family are proposed to function in both normal development and differentiation as well as in mammary tumorigenesis. To understand the function of Wnt proteins in these two processes, we present here a biochemical characterization of seven Wnt family members. For these studies, AtT-20 cells, a neuroendocrine cell line previously shown to efficiently process and secrete Wnt-1, was transfected with expression vectors encoding Wnt family members. All of the newly characterized Wnt proteins are glycosylated, secreted proteins that are tightly associated with the cell surface or extracellular matrix. We have also identified native Wnt proteins in retinoic acid-treated P19 embryonal carcinoma cells, and they exhibit the same biochemical characteristics as the recombinant proteins. These data suggest that Wnt family members function in cell to cell signaling in a fashion similar to Wnt-1. Images PMID:8167409

  6. Autoadaptive ER-associated degradation defines a preemptive unfolded protein response pathway.

    PubMed

    Bernasconi, Riccardo; Galli, Carmela; Kokame, Koichi; Molinari, Maurizio

    2013-12-26

    Folding-defective proteins must be cleared efficiently from the endoplasmic reticulum (ER) to prevent perturbation of the folding environment and to maintain cellular proteostasis. Misfolded proteins engage dislocation machineries (dislocons) built around E3 ubiquitin ligases that promote their transport across the ER membrane, their polyubiquitylation, and their proteasomal degradation. Here, we report on the intrinsic instability of the HRD1 dislocon and the constitutive, rapid turnover of the scaffold protein HERP. We show that HRD1 dislocon integrity relies on the presence of HRD1 clients that interrupt, in a dose-dependent manner, the UBC6e/RNF5/p97/proteasome-controlled relay that controls HERP turnover. We propose that ER-associated degradation (ERAD) deploys autoadaptive regulatory pathways, collectively defined as ERAD tuning, to rapidly adapt degradation activity to misfolded protein load and to preempt the unfolded protein response (UPR) activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Arabinogalactan protein and wall-associated kinase in a plasmalemmal reticulum with specialized vertices

    NASA Technical Reports Server (NTRS)

    Gens, J. S.; Fujiki, M.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Arabinogalactan protein and wall-associated kinase (WAK) are suspected to be regulatory players at the interface between cytoplasm and cell wall. Both WAK(s) and arabinogalactan shown likely to represent arabinogalactan protein(s) have been visualized there with computational optical-sectioning microscopy. The arabinogalactan occurs in a polyhedral array at the external face of the cell membrane. WAK, and other proteins as yet unidentified, appear to fasten the membrane to the wall at vertices of the array. Evidence is presented that the array bears an important part of the mechanical stress experienced by the membrane, and it is speculated that the architectural organization of arabinogalactan protein, WAK, and other components of the array is critical for coordination of endomembrane activities, growth, and differentiation. The array has been named the plasmalemmal reticulum.

  8. Arabinogalactan protein and wall-associated kinase in a plasmalemmal reticulum with specialized vertices

    NASA Technical Reports Server (NTRS)

    Gens, J. S.; Fujiki, M.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Arabinogalactan protein and wall-associated kinase (WAK) are suspected to be regulatory players at the interface between cytoplasm and cell wall. Both WAK(s) and arabinogalactan shown likely to represent arabinogalactan protein(s) have been visualized there with computational optical-sectioning microscopy. The arabinogalactan occurs in a polyhedral array at the external face of the cell membrane. WAK, and other proteins as yet unidentified, appear to fasten the membrane to the wall at vertices of the array. Evidence is presented that the array bears an important part of the mechanical stress experienced by the membrane, and it is speculated that the architectural organization of arabinogalactan protein, WAK, and other components of the array is critical for coordination of endomembrane activities, growth, and differentiation. The array has been named the plasmalemmal reticulum.

  9. ESCRTs and associated proteins in lysosomal fusion with endosomes and autophagosomes.

    PubMed

    Majumder, Priyanka; Chakrabarti, Oishee

    2016-07-22

    Endolysosomal and autophagosomal degradation pathways are highly connected at various levels, sharing multiple molecular effectors that modulate them individually or simultaneously. These two lysosomal degradative pathways are primarily involved in the disposal of cargo internalized from the cell surface or long-lived proteins or aggregates and aged organelles present in the cytosol. Both of these pathways involve a number of carefully regulated vesicular fusion events that are dependent on ESCRT proteins. The ESCRT proteins especially ESCRT-I and III participate in the regulation of fusion events between autophagosome/amphisome and lysosome. Along with these, a number of functionally diverse ESCRT associated and regulatory proteins such as, endosomal PtdIns (3) P 5-kinase Fab1, ALIX, mahogunin ring finger 1, atrogin 1, syntaxin 17, ATG12-ATG3 complex, and protein kinase CK2α are involved in fusion events in either or both the lysosomal degradative pathways.

  10. Dissecting the molecular organization of the translocon-associated protein complex

    PubMed Central

    Pfeffer, Stefan; Dudek, Johanna; Schaffer, Miroslava; Ng, Bobby G.; Albert, Sahradha; Plitzko, Jürgen M.; Baumeister, Wolfgang; Zimmermann, Richard; Freeze, Hudson H.; Engel, Benjamin D.; Förster, Friedrich

    2017-01-01

    In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. The translocon-associated protein (TRAP) complex is an integral component of the translocon, assisting the Sec61 protein-conducting channel by regulating signal sequence and transmembrane helix insertion in a substrate-dependent manner. Here we use cryo-electron tomography (CET) to study the structure of the native translocon in evolutionarily divergent organisms and disease-linked TRAP mutant fibroblasts from human patients. The structural differences detected by subtomogram analysis form a basis for dissecting the molecular organization of the TRAP complex. We assign positions to the four TRAP subunits within the complex, providing insights into their individual functions. The revealed molecular architecture of a central translocon component advances our understanding of membrane protein biogenesis and sheds light on the role of TRAP in human congenital disorders of glycosylation. PMID:28218252

  11. Identifying the Association Between Alzheimer's Disease and Parkinson's Disease Using Genome-Wide Association Studies and Protein-Protein Interaction Network.

    PubMed

    Liu, Guiyou; Bao, Xinjie; Jiang, Yongshuai; Liao, Mingzhi; Jiang, Qinghua; Feng, Rennan; Zhang, Liangcai; Ma, Guoda; Chen, Zugen; Wang, Guangyu; Wang, Renzhi; Zhao, Bin; Li, Keshen

    2015-12-01

    Alzheimer's disease (AD) and Parkinson's disease (PD) are the first and second most common neurodegenerative diseases in the elderly. Shared clinical and pathological features have been reported. Recent large-scale genome-wide association studies (GWAS) have been conducted and reported a number of AD and PD variants. Until now, the underlying genetic mechanisms for all these newly identified PD variants as well as the association between AD and PD are still unclear exactly. We think that PD variants may contribute to AD and PD by influence on brain gene expression. Here, we conducted a systems analysis using (1) AD and PD variants (P < 5.00E-08) identified by the published GWAS; (2) four brain expression GWAS datasets using expression quantitative trait loci from the cerebellum and temporal cortex; (3) large-scale AD GWAS from the Alzheimer Disease Genetics Consortium (ADGC); (4) a protein-protein interaction network. Our results indicated that PD variants around the 17q21 were associated with gene expression and suggestive AD risk. We also identified significant interaction among AD and PD susceptibility genes. We believe that our findings may explain the underlying genetic mechanisms for newly identified PD variants in PD and AD, as well as the association between AD and PD, which may be very useful for future genetic studies for both diseases.

  12. Identification of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein-protein interactions.

    PubMed Central

    Peles, E; Nativ, M; Lustig, M; Grumet, M; Schilling, J; Martinez, R; Plowman, G D; Schlessinger, J

    1997-01-01

    Receptor protein tyrosine phosphatase beta (RPTPbeta) expressed on the surface of glial cells binds to the glycosylphosphatidylinositol (GPI)-anchored recognition molecule contactin on neuronal cells leading to neurite outgrowth. We describe the cloning of a novel contactin-associated transmembrane receptor (p190/Caspr) containing a mosaic of domains implicated in protein-protein interactions. The extracellular domain of Caspr contains a neurophilin/coagulation factor homology domain, a region related to fibrinogen beta/gamma, epidermal growth factor-like repeats, neurexin motifs as well as unique PGY repeats found in a molluscan adhesive protein. The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains of signaling molecules. Caspr and contactin exist as a complex in rat brain and are bound to each other by means of lateral (cis) interactions in the plasma membrane. We propose that Caspr may function as a signaling component of contactin, enabling recruitment and activation of intracellular signaling pathways in neurons. The binding of RPTPbeta to the contactin-Caspr complex could provide a mechanism for cell-cell communication between glial cells and neurons during development. PMID:9118959

  13. Isolation and Analysis of Keratins and Keratin-Associated Proteins from Hair and Wool.

    PubMed

    Deb-Choudhury, Santanu; Plowman, Jeffrey E; Harland, Duane P

    2016-01-01

    The presence of highly cross-linked protein networks in hair and wool makes them very difficult substrates for protein extraction, a prerequisite for further protein analysis and characterization. It is therefore imperative that these cross-links formed by disulfide bridges are first disrupted for the efficient extraction of proteins. Chaotropes such as urea are commonly used as efficient extractants. However, a combination of urea and thiourea not only improves recovery of proteins but also results in improved resolution of the keratins in 2DE gels. Reductants also play an important role in protein dissolution. Dithiothreitol effectively removes keratinous material from the cortex, whereas phosphines, like Tris(2-carboxyethyl)phosphine, remove material from the exocuticle. The relative extractability of the keratins and keratin-associated proteins is also dependent on the concentration of chaotropes, reductants, and pH, thus providing a means to preferentially extract these proteins. Ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIM(+)[Cl](-)) are known to solubilize wool by disrupting noncovalent interactions, specifically intermolecular hydrogen bonds. BMIM(+)[Cl](-) proved to be an effective extractant of wool proteins and complementary in nature to chaotropes such as urea and thiourea for identifying unique peptides of wool proteins using mass spectrometry (MS). Successful identification of proteins resolved by one- or two-dimensional electrophoresis and MS is highly dependent on the optimal recovery of its protease-digested peptides with an efficient removal of interfering substances. The detergent sodium deoxycholate used in conjunction with Empore™ disks improved identification of proteins by mass spectrometry leading to higher percentage sequence coverage, identification of unique peptides and higher score. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes.

    PubMed

    Giauque, Christopher C; Bickel, Sharon E

    2016-05-01

    Accurate segregation of homologous chromosomes during meiosis depends on their ability to remain physically connected throughout prophase I. For homologs that achieve a crossover, sister chromatid cohesion distal to the chiasma keeps them attached until anaphase I. However, in Drosophila melanogaster wild-type oocytes, chromosome 4 never recombines, and the X chromosome fails to cross over in 6-10% of oocytes. Proper segregation of these achiasmate homologs relies on their pericentric heterochromatin-mediated association, but the mechanism(s) underlying this attachment remains poorly understood. Using an inducible RNA interference (RNAi) strategy combined with fluorescence in situ hybridization (FISH) to monitor centromere proximal association of the achiasmate FM7a/X homolog pair, we asked whether specific heterochromatin-associated proteins are required for the association and proper segregation of achiasmate homologs in Drosophila oocytes. When we knock down HP1a, H3K9 methytransferases, or the HP1a binding partner Piwi during mid-prophase, we observe significant disruption of pericentric heterochromatin-mediated association of FM7a/X homologs. Furthermore, for both HP1a and Piwi knockdown oocytes, transgenic coexpression of the corresponding wild-type protein is able to rescue RNAi-induced defects, but expression of a mutant protein with a single amino acid change that disrupts the HP1a-Piwi interaction is unable to do so. We show that Piwi is stably bound to numerous sites along the meiotic chromosomes, including centromere proximal regions. In addition, reduction of HP1a or Piwi during meiotic prophase induces a significant increase in FM7a/X segregation errors. We present a speculative model outlining how HP1a and Piwi could collaborate to keep achiasmate chromosomes associated in a homology-dependent manner.

  15. Stimulation of Vesicular Stomatitis Virus in vitro RNA Synthesis by Microtubule-Associated Proteins

    NASA Astrophysics Data System (ADS)

    Hill, Virginia M.; Harmon, Shirley A.; Summers, Donald F.

    1986-08-01

    Microtubule-associated proteins purified from bovine brains stimulated the in vitro transcription and replication reactions of vesicular stomatitis virus. The products of these reactions were intact messenger or genome-sized RNA species. A preparation from HeLa cells containing tubulin and microtubule-associated proteins also stimulated vesicular stomatitis virus transcription in vitro. This observation is in accord with previous studies, which suggested that a host cell factor was involved with the function of the vesicular stomatitis virus RNA polymerase, and others that indicated that several animal viruses displayed an association with host cell cytoskeletal elements during their replication cycles. We show evidence in this report of a host cell protein that seems to have a functional role in interacting with the virion polymerase.

  16. Identification of messenger RNAs and microRNAs associated with fragile X mental retardation protein.

    PubMed

    Duan, Ranhui; Jin, Peng

    2006-01-01

    Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of the Fragile X mental retardation protein (FMRP). FMRP, which may regulate translation in neurons, not only associates with specific messenger RNAs (mRNAs) and with microRNAs (miRNAs), but also associates with the components of the miRNA pathway, including the Dicer and Argonaute proteins. It has been proposed that FMRP regulates the translation of its mRNA targets through miRNAs. In this chapter, we describe the protocol to identify the mRNAs and miRNAs associated with FMRP in vivo. The same method could also be applied to other RNA-binding proteins interacting with specific mRNAs or miRNAs.

  17. Protein-like fully reversible tetramerisation and super-association of an aminocellulose.

    PubMed

    Nikolajski, Melanie; Adams, Gary G; Gillis, Richard B; Besong, David Tabot; Rowe, Arthur J; Heinze, Thomas; Harding, Stephen E

    2014-01-24

    Unusual protein-like, partially reversible associative behaviour has recently been observed in solutions of the water soluble carbohydrates known as 6-deoxy-6-(ω-aminoalkyl)aminocelluloses, which produce controllable self-assembling films for enzyme immobilisation and other biotechnological applications. Now, for the first time, we have found a fully reversible self-association (tetramerisation) within this family of polysaccharides. Remarkably these carbohydrate tetramers are then seen to associate further in a regular way into supra-molecular complexes. Fully reversible oligomerisation has been hitherto completely unknown for carbohydrates and instead resembles in some respects the assembly of polypeptides and proteins like haemoglobin and its sickle cell mutation. Our traditional perceptions as to what might be considered "protein-like" and what might be considered as "carbohydrate-like" behaviour may need to be rendered more flexible, at least as far as interaction phenomena are concerned.

  18. Protein-like fully reversible tetramerisation and super-association of an aminocellulose

    NASA Astrophysics Data System (ADS)

    Nikolajski, Melanie; Adams, Gary G.; Gillis, Richard B.; Besong, David Tabot; Rowe, Arthur J.; Heinze, Thomas; Harding, Stephen E.

    2014-01-01

    Unusual protein-like, partially reversible associative behaviour has recently been observed in solutions of the water soluble carbohydrates known as 6-deoxy-6-(ω-aminoalkyl)aminocelluloses, which produce controllable self-assembling films for enzyme immobilisation and other biotechnological applications. Now, for the first time, we have found a fully reversible self-association (tetramerisation) within this family of polysaccharides. Remarkably these carbohydrate tetramers are then seen to associate further in a regular way into supra-molecular complexes. Fully reversible oligomerisation has been hitherto completely unknown for carbohydrates and instead resembles in some respects the assembly of polypeptides and proteins like haemoglobin and its sickle cell mutation. Our traditional perceptions as to what might be considered ``protein-like'' and what might be considered as ``carbohydrate-like'' behaviour may need to be rendered more flexible, at least as far as interaction phenomena are concerned.

  19. Surface-associated proteins of wheat starch granules: suitability of wheat starch for celiac patients.

    PubMed

    Kasarda, Donald D; Dupont, Frances M; Vensel, William H; Altenbach, Susan B; Lopez, Rocio; Tanaka, Charlene K; Hurkman, William J

    2008-11-12

    Wheat starch is used to make baked products for celiac patients in several European countries but is avoided in the United States because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye, and barley proteins and products that contain them. These proteins are capable of initiating damage to the absorptive lining of the small intestine in CD patients, apparently as a consequence of undesirable interactions with the innate and adaptive immune systems. In this study, starch surface-associated proteins were extracted from four commercial wheat starches, fractionated by high-performance liquid chromatography and gel electrophoresis, and identified by tandem mass spectrometry analysis. More than 150 proteins were identified, many of which (for example, histones, purothionins, and glutenins) had not been recognized previously as starch-associated. The commercial starches were analyzed by the R-5 enzyme-linked immunosorbent assay method to estimate the amount of harmful gluten protein present. One of these starches had a low gluten content of 7 ppm and actually fell within the range proposed as a new Codex Alimentarius Standard for naturally gluten-free foods (maximum 20 ppm). This low level of gluten indicates that the starch should be especially suitable for use by celiac patients, although wheat starches with levels up to 100 ppm are deemed safe in the proposed Codex standards.

  20. Protein–Protein and Protein–Membrane Associations in the Lignin Pathway[W][OA

    PubMed Central

    Bassard, Jean-Etienne; Richert, Ludovic; Geerinck, Jan; Renault, Hugues; Duval, Frédéric; Ullmann, Pascaline; Schmitt, Martine; Meyer, Etienne; Mutterer, Jerôme; Boerjan, Wout; De Jaeger, Geert; Mely, Yves; Goossens, Alain; Werck-Reichhart, Danièle

    2012-01-01

    Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association. PMID:23175744

  1. Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins.

    PubMed

    Yu, G; Swiston, J; Young, D

    1994-06-01

    We previously reported the identification of human CAP, a protein that is related to the Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylyl cyclase-associated CAP proteins. The two yeast CAP proteins have similar functions: the N-terminal domains are required for the normal function of adenylyl cyclase, while loss of the C-terminal domains result in morphological and nutritional defects that are unrelated to the cAMP pathways. We have amplified and cloned cDNAs from a human glioblastoma library that encode a second CAP-related protein, CAP2. The human CAP and CAP2 proteins are 64% identical. Expression of either human CAP or CAP2 in S. cerevisiae cap- strains suppresses phenotypes associated with deletion of the C-terminal domain of CAP, but does not restore hyper-activation of adenylyl cyclase by RAS2val19. Similarly, expression of either human CAP or CAP2 in S. pombe cap- strains suppresses the morphological and temperature-sensitive phenotypes associated with deletion of the C-terminal domain of CAP in this yeast. In addition, expression of human CAP, but not CAP2, suppresses the propensity to sporulate due to deletion of the N-terminal domain of CAP in S. pombe. This latter observation suggests that human CAP restores normal adenylyl cyclase activity in S. pombe cap- cells. Thus, functional properties of both N-terminal and C-terminal domains are conserved between the human and S. pombe CAP proteins.

  2. V-myc- and c-myc-encoded proteins are associated with the nuclear matrix.

    PubMed Central

    Eisenman, R N; Tachibana, C Y; Abrams, H D; Hann, S R

    1985-01-01

    A series of extraction procedures were applied to avian nuclei which allowed us to define three types of association of v-myc- and c-myc-encoded proteins with nuclei: (i) a major fraction (60 to 90%) which is retained in DNA- and RNA-depleted nuclei after low- and high-salt extraction, (ii) a small fraction (1%) released during nuclease digestion of DNA in intact nuclei in the presence of low-salt buffer, and (iii) a fraction of myc protein (less than 10%) extractable with salt or detergents and found to have affinity for both single- and double-stranded DNA. Immunofluorescence analysis with anti-myc peptide sera on cells extracted sequentially with nucleases and salts confirmed the idea that myc proteins were associated with a complex residual nuclear structure (matrix-lamin fraction) which also contained avian nuclear lamin protein. Dispersal of myc proteins into the cytoplasm was found to occur during mitosis. Both c-myc and v-myc proteins were associated with the matrix-lamin, suggesting that the function of myc may relate to nuclear structural organization. Images PMID:3872410

  3. Identification and Characterization of a Novel Heme-Associated Cell Surface Protein Made by Streptococcus pyogenes

    PubMed Central

    Lei, Benfang; Smoot, Laura M.; Menning, Heather M.; Voyich, Jovanka M.; Kala, Subbarao V.; Deleo, Frank R.; Reid, Sean D.; Musser, James M.

    2002-01-01

    Analysis of the genome sequence of a serotype M1 group A Streptococcus (GAS) strain identified a gene encoding a previously undescribed putative cell surface protein. The gene was cloned from a serotype M1 strain, and the recombinant protein was overexpressed in Escherichia coli and purified to homogeneity. The purified protein was associated with heme in a 1:1 stoichiometry. This streptococcal heme-associated protein, designated Shp, was produced in vitro by GAS, located on the bacterial cell surface, and accessible to specific antibody raised against the purified recombinant protein. Mice inoculated subcutaneously with GAS and humans with invasive infections and pharyngitis caused by GAS seroconverted to Shp, indicating that Shp was produced in vivo. The blood of mice actively immunized with Shp had significantly higher bactericidal activity than the blood of unimmunized mice. The shp gene was cotranscribed with eight contiguous genes, including homologues of an ABC transporter involved in iron uptake in gram-negative bacteria. Our results indicate that Shp is a novel cell surface heme-associated protein. PMID:12117961

  4. Interaction with the Yes-associated protein (YAP) allows TEAD1 to positively regulate NAIP expression.

    PubMed

    Landin Malt, André; Georges, Adrien; Silber, Joël; Zider, Alain; Flagiello, Domenico

    2013-10-01

    Although the expression of the neuronal apoptosis inhibitory protein (NAIP) gene is considered involved in apoptosis suppression as well as in inflammatory response, the molecular basis of the NAIP gene expression is poorly understood. Here we show that the TEA domain protein 1 (TEAD1) is able to positively activate the transcription of NAIP. We further demonstrate that this regulation is mediated by the presence of the endogenous Yes associated protein (YAP) cofactor, and requires the interaction with YAP. We finally identified an intronic region of the NAIP gene responding to TEAD1/YAP activity, suggesting that regulation of NAIP by TEAD1/YAP is at the transcriptional level.

  5. Identification of a thioredoxin-related protein associated with plasma membranes

    SciTech Connect

    Martin, H.; Dean, M. )

    1991-02-28

    A low molecular weight membrane associated sulphydryl protein was detected on a wide range of nucleated cells when ({sup 14}C)-iodoacetamide was used as a probe. This protein was extracted from THP-1 monocytes, purified to homogeneity and its isoelectric point, Mr and N-terminal amino acid sequence determined. These were shown to be almost identical to the corresponding values for both human thioredoxin and a Tac interleukin-2 receptor activator, indicating that the protein may be a member of this family and function as an essential growth factor.

  6. Human telomerase and Cajal body ribonucleoproteins share a unique specificity of Sm protein association.

    PubMed

    Fu, Dragony; Collins, Kathleen

    2006-03-01

    Cajal bodies are nuclear structures that host RNA modification and assembly reactions. Some RNAs transit Cajal bodies, while others must concentrate in Cajal bodies to function. Here we report that at least a subfraction of human telomerase RNA and individual resident Cajal body RNAs is associated with Sm proteins. Surprisingly, of seven Sm proteins assembled into a heteroheptameric ring, only a subset copurifies telomerase and Cajal body ribonucleoproteins. We show that a Cajal body RNA localization motif determines this specificity. These discoveries expand the cellular repertoire of Sm protein assemblies and their involvement in ribonucleoprotein localization and function.

  7. Association of oxidative status and semen characteristics with seminal plasma proteins of buffalo semen.

    PubMed

    Sharma, L; Pandey, V; Nigam, R; Saxena, A; Swain, D K; Yadav, B

    2016-01-01

    To study the influence of season on oxidative status of buffalo semen and their association with semen characteristics and seminal plasma proteins, ejaculates were collected twice a week in winter, summer and rainy seasons from six adult Bhadawari buffalo bulls. The neat semen was analyzed for semen characteristics immediately after collection and oxidative status viz. lipid peroxidation (LPO), catalase (CAT), super oxide dismutase (SOD) activity, and total protein (TP) were estimated in seminal plasma. The protein profiling was carried out by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The significant effect of season was observed on TP, SOD activity and % protein fractions of seminal plasma proteins of buffalo bulls. The TP values showed positive correlation with ejaculate volume (EV), sperm concentration (SC), and % live-dead (LD) and negative correlation with progressive motility (PM), and hypo-osmotic swelling test (HOST). The SOD activity showed positive correlation with PM, LD, HOST and % acrosoamal integrity (AI). Besides that, results showed correlation of TP with 6.5, 38 and 66 kDa proteins, LPO with 70, 72, 84 and 86 kDa proteins, CAT with 70 kDa and 86 kDa proteins, and SOD with 6.5, 24.5, 44.5, 70 and 72 kDa proteins. In conclusion, this study indicated that TP and SOD activity of seminal plasma of buffalo bulls were influenced by season and were found to be associated with some of the semen characteristics and expression of seminal plasma proteins.

  8. Association of oxidative status and semen characteristics with seminal plasma proteins of buffalo semen

    PubMed Central

    Sharma, L.; Pandey, V.; Nigam, R.; Saxena, A.; Swain, D. K.; Yadav, B.

    2016-01-01

    To study the influence of season on oxidative status of buffalo semen and their association with semen characteristics and seminal plasma proteins, ejaculates were collected twice a week in winter, summer and rainy seasons from six adult Bhadawari buffalo bulls. The neat semen was analyzed for semen characteristics immediately after collection and oxidative status viz. lipid peroxidation (LPO), catalase (CAT), super oxide dismutase (SOD) activity, and total protein (TP) were estimated in seminal plasma. The protein profiling was carried out by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The significant effect of season was observed on TP, SOD activity and % protein fractions of seminal plasma proteins of buffalo bulls. The TP values showed positive correlation with ejaculate volume (EV), sperm concentration (SC), and % live-dead (LD) and negative correlation with progressive motility (PM), and hypo-osmotic swelling test (HOST). The SOD activity showed positive correlation with PM, LD, HOST and % acrosoamal integrity (AI). Besides that, results showed correlation of TP with 6.5, 38 and 66 kDa proteins, LPO with 70, 72, 84 and 86 kDa proteins, CAT with 70 kDa and 86 kDa proteins, and SOD with 6.5, 24.5, 44.5, 70 and 72 kDa proteins. In conclusion, this study indicated that TP and SOD activity of seminal plasma of buffalo bulls were influenced by season and were found to be associated with some of the semen characteristics and expression of seminal plasma proteins. PMID:28224004

  9. Serum Proteins Alteration in Association with Body Mass Index in Human Volunteers.

    PubMed

    Madhuvanthi, M; Lathadevi, G V

    2016-06-01

    Serum proteins are an important indicator of the nutritional status in an individual. There is a worldwide prevalence of both undernourishment and obesity. It has been suggested that low Body Mass Index (BMI) is associated with a decrease in serum protein levels predisposing them to other illnesses. Overweight and obese individuals carry risk for various other non-communicable diseases. To compare the serum protein levels in underweight, overweight and obese individuals with that of normal body mass index individuals. This prospective study was conducted in subjects who attended the master health checkup clinic of PSG hospitals. Subjects in the age group of 20-50 years were selected. Their serum proteins and BMI was measured. Twenty subjects each of underweight, normal, overweight and obese individuals were selected, categorized and compared. The serum protein level of normal individuals (Group I) was compared with underweight (Group II), overweight (Group III) and obese subjects (Group IV) by one-way ANOVA analysis. The mean serum total proteins in gm/dl in group I controls was 7.555±0.37 compared to Group II (underweight) which was 7.295±0.419. Low BMI was found to be associated with a decrease in serum protein level which was not statistically significant. Elevated BMI as in overweight and obese subjects showed no significant alterations in serum protein levels with p >0.05 and the changes were found to be independent of the body mass index. Underweight individuals showed a decrease in serum protein levels whereas there were no significant changes in the serum protein levels in overweight and obese individuals.

  10. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

    SciTech Connect

    Jiao, Yongqin; D'Haeseleer, Patrik M; Dill, Brian; Shah, Manesh B; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; Banfield, Jillian F.; Thelen, Michael P.

    2011-01-01

    In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by 2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as -N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.

  11. Dynamics of putative raft-associated proteins at the cell surface

    PubMed Central

    Kenworthy, Anne K.; Nichols, Benjamin J.; Remmert, Catha L.; Hendrix, Glenn M.; Kumar, Mukesh; Zimmerberg, Joshua; Lippincott-Schwartz, Jennifer

    2004-01-01

    Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (>4 μm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface. PMID:15173190

  12. Dynamics of putative raft-associated proteins at the cell surface.

    PubMed

    Kenworthy, Anne K; Nichols, Benjamin J; Remmert, Catha L; Hendrix, Glenn M; Kumar, Mukesh; Zimmerberg, Joshua; Lippincott-Schwartz, Jennifer

    2004-06-07

    Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (> 4 microm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.

  13. EB1 protein alteration characterizes sporadic but not ulcerative colitis associated colorectal cancer

    PubMed Central

    Karstens, Karl F.; Hò, Gia G.; Hartwig, Sonja; Strohkamp, Sarah; Schillo, Katharina; Thorns, Christoph; Oberländer, Martina; Kalies, Kathrin; Lehr, Stefan; Habermann, Jens K.

    2017-01-01

    Background While carcinogenesis in Sporadic Colorectal Cancer (SCC) has been thoroughly studied, less is known about Ulcerative Colitis associated Colorectal Cancer (UCC). This study aimed to identify and validate differentially expressed proteins between clinical samples of SCC and UCC to elucidate new insights of UCC/SCC carcinogenesis and progression. Results Multiplex-fluorescence two-dimensional gel electrophoresis (2-D DIGE) and mass spectrometry identified 67 proteoforms representing 43 distinct proteins. After analysis by Ingenuity Pathway Analysis® (IPA), subsequent Western blot validation proofed the differential expression of Heat shock 27 kDA protein 1 (HSPB1) and Microtubule-associated protein R/EB family, member 1 (EB1) while the latter one showed also expression differences by immunohistochemistry. Materials and Methods Fresh frozen tissue of UCC (n = 10) matched with SCC (n = 10) was investigated. Proteins of cancerous intestinal mucosal cells were obtained by Laser Capture Microdissection (LCM) and compared by 2-D DIGE. Significant spots were identified by mass spectrometry. After IPA, three proteins [EB1, HSPB1, and Annexin 5 (ANXA5)] were chosen for further validation by Western blotting and tissue microarray-based immunohistochemistry. Conclusions This study identified significant differences in protein expression of colorectal carcinoma cells from UCC patients compared to patients with SCC. Particularly, EB1 was validated in an independent clinical cohort. PMID:28903393

  14. Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations*

    PubMed Central

    Wilkie, Gavin S.; Korfali, Nadia; Swanson, Selene K.; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G.; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R. W.; Florens, Laurence; Schirmer, Eric C.

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  15. Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein.

    PubMed

    Halperin, R; Hadas, E; Fleminger, G; Ovadia, Y; Kraicer, P F

    1990-01-01

    A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.

  16. Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis.

    PubMed

    Nam, Sang-Chul

    2016-11-01

    Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Here, Tau, a microtubule associated protein, was analyzed to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins' localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. It is found that Tau has a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation.

  17. The Evolutionary History of MAPL (Mitochondria-Associated Protein Ligase) and Other Eukaryotic BAM/GIDE Domain Proteins.

    PubMed

    Wideman, Jeremy G; Moore, Blake P

    2015-01-01

    MAPL (mitochondria-associated protein ligase, also called MULAN/GIDE/MUL1) is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane) domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists Bigelowiella natans and Ectocarpus siliculosis. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings. Our work paves

  18. Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi.

    PubMed

    Whittingham, Jean L; Blagova, Elena V; Finn, Ciaran E; Luo, Haixia; Miranda-CasoLuengo, Raúl; Turkenburg, Johan P; Leech, Andrew P; Walton, Paul H; Murzin, Alexey G; Meijer, Wim G; Wilkinson, Anthony J

    2014-08-01

    Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vap proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-D-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.

  19. Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals

    PubMed Central

    Ghezzi, Alfredo; Liebeskind, Benjamin J.; Thompson, Ammon; Atkinson, Nigel S.; Zakon, Harold H.

    2014-01-01

    Neuronal resting potential can tune the excitability of neural networks, affecting downstream behavior. Sodium leak channels (NALCN) play a key role in rhythmic behaviors by helping set, or subtly changing neuronal resting potential. The full complexity of these newly described channels is just beginning to be appreciated, however. NALCN channels can associate with numerous subunits in different tissues and can be activated by several different peptides and second messengers. We recently showed that NALCN channels are closely related to fungal calcium channels, which they functionally resemble. Here, we use this relationship to predict a family of NALCN-associated proteins in animals on the basis of homology with the yeast protein Mid1, the subunit of the yeast calcium channel. These proteins all share a cysteine-rich region that is necessary for Mid1 function in yeast. We validate this predicted association by showing that the Mid1 homolog in Drosophila, encoded by the CG33988 gene, is coordinately expressed with NALCN, and that knockdown of either protein creates identical phenotypes in several behaviors associated with NALCN function. The relationship between Mid1 and leak channels has therefore persisted over a billion years of evolution, despite drastic changes to both proteins and the organisms in which they exist. PMID:24639627

  20. Protein association of the neurotoxin and non-protein amino acid BMAA (β-N-methylamino-L-alanine) in the liver and brain following neonatal administration in rats.

    PubMed

    Karlsson, Oskar; Jiang, Liying; Andersson, Marie; Ilag, Leopold L; Brittebo, Eva B

    2014-04-07

    The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) is not an amino acid that is normally found in proteins. Our previous autoradiographic study of (3)H-labeled BMAA in adult mice unexpectedly revealed a tissue distribution similar to that of protein amino acids. The aim of this study was to characterize the distribution of free and protein-bound BMAA in neonatal rat tissues following a short exposure using autoradiographic imaging and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The autoradiographic imaging of (14)C-L-BMAA demonstrated a distinct uptake of radioactivity that was retained following acid extraction in tissues with a high rate of cell turnover and/or protein synthesis. The UHPLC-MS/MS analysis conclusively demonstrated a dose-dependent increase of protein-associated BMAA in neonatal rat tissues. The level of protein-associated BMAA in the liver was more than 10 times higher than that in brain regions not fully protected by the blood-brain barrier which may be due to the higher rate of protein synthesis in the liver. In conclusion, this study demonstrated that BMAA was associated with rat proteins suggesting that BMAA may be misincorporated into proteins. However, protein-associated BMAA seemed to be cleared over time, as none of the samples from adult rats had any detectable free or protein-associated BMAA.

  1. Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine, Arginine, and Lysine Residues

    PubMed Central

    Richarme, Gilbert; Mihoub, Mouadh; Dairou, Julien; Bui, Linh Chi; Leger, Thibaut; Lamouri, Aazdine

    2015-01-01

    Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein. PMID:25416785

  2. Symmetry-mismatch reconstruction of genomes and associated proteins within icosahedral viruses using cryo-EM.

    PubMed

    Li, Xiaowu; Liu, Hongrong; Cheng, Lingpeng

    2016-01-01

    Although near-atomic resolutions have been routinely achieved for structural determination of many icosahedral viral capsids, structures of genomes and associated proteins within the capsids are still less characterized because the genome information is overlapped by the highly symmetric capsid information in the virus particle images. We recently developed a software package for symmetry-mismatch structural reconstruction and determined the structures of the genome and RNA polymerases within an icosahedral virus for the first time. Here, we describe the protocol used for this structural determination, which may facilitate structural biologists in investigating the structures of viral genome and associated proteins.

  3. Identification of a mitochondrial protein associated with cytoplasmic male sterility in petunia.

    PubMed Central

    Nivison, H T; Hanson, M R

    1989-01-01

    The petunia fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxII and urfS portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXII but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines. The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility. PMID:2562768

  4. Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide.

    PubMed Central

    Ilan, O; Bloch, Y; Frankel, G; Ullrich, H; Geider, K; Rosenshine, I

    1999-01-01

    In eukaryotes, tyrosine protein phosphorylation has been studied extensively, while in bacteria, it is considered rare and is poorly defined. We demonstrate that Escherichia coli possesses a gene, etk, encoding an inner membrane protein that catalyses tyrosine autophosphorylation and phosphorylation of a synthetic co-polymer poly(Glu:Tyr). This protein tyrosine kinase (PTK) was termed Ep85 or Etk. All the E.coli strains examined possessed etk; however, only a subset of pathogenic strains expressed it. Etk is homologous to several bacterial proteins including the Ptk protein of Acinetobacter johnsonii, which is the only other known prokaryotic PTK. Other Etk homologues are AmsA of the plant pathogen Erwinia amylovora and Orf6 of the human pathogen Klebsiella pneumoniae. These proteins are involved in the production of exopolysaccharide (EPS) required for virulence. We demonstrated that like Etk, AmsA and probably also Orf6 are PTKs. Taken together, these findings suggest that tyrosine protein phosphorylation in prokaryotes is more common than was appreciated previously, and that Etk and its homologues define a distinct protein family of prokaryotic membrane-associated PTKs involved in EPS production and virulence. These prokaryotic PTKs may serve as a new target for the development of new antibiotics. PMID:10369665

  5. Proteins associated with cork formation in Quercus suber L. stem tissues.

    PubMed

    Ricardo, Cândido P P; Martins, Isabel; Francisco, Rita; Sergeant, Kjell; Pinheiro, Carla; Campos, Alexandre; Renaut, Jenny; Fevereiro, Pedro

    2011-08-12

    Cork (phellem) formation in Quercus suber stem was studied by proteomic analysis of young shoots of increasing age (Y0, Y1 and Y4) and recently-formed phellem (Y8Ph) and xylem (Y8X) from an 8-year-old branch. In this study 99 proteins were identified, 45 excised from Y8X and 54 from Y8Ph. These ones, specifically associated with phellem, are of "carbohydrate metabolism" (28%), "defence" (22%), "protein folding, stability and degradation" (19%), "regulation/signalling" (11%), "secondary metabolism" (9%), "energy metabolism" (6%), and "membrane transport" (2%). The identification in phellem of galactosidases, xylosidases, apiose/xylose synthase, laccases and diphenol oxidases suggests intense cell wall reorganization, possibly with participation of hemicellulose/pectin biosynthesis and phenol oxidation. The identification of proteasome subunits, heat shock proteins, cyclophylin, subtilisin-like proteases, 14-3-3 proteins, Rab2 protein and enzymes interacting with nucleosides/nucleic acids gives additional evidence for cellular reorganization, involving cellular secretion, protein turnover regulation and active control processes. The high involvement in phellem of defence proteins (thioredoxin-dependent peroxidase, glutathione-S-transferase, SGT1 protein, cystatin, and chitinases) suggests a strong need for cell protection from the intense stressful events occurring in active phellem, namely, desiccation, pests/disease protection, detoxification and cell death. Identically, highly enhanced defence functions were previously reported for potato periderm formation.

  6. Properties of a microtubule-associated cofactor-independent protein kinase from pig brain.

    PubMed Central

    Scott, C W; Caputo, C B; Salama, A I

    1989-01-01

    A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures. Images Fig. 1. Fig. 6. Fig. 7. PMID:2557823

  7. Fragile X Mental Retardation protein determinants required for its association with polyribosomal mRNPs.

    PubMed

    Mazroui, Rachid; Huot, Marc-Etienne; Tremblay, Sandra; Boilard, Nathalie; Labelle, Yves; Khandjian, Edouard W

    2003-12-01

    Fragile X Mental Retardation protein (FMRP) is an RNA-binding protein that contains multiple domains with apparently differential affinity to mRNA and to the ribonucleotide homopolymer poly(G). Attempts have been made to map the RNA-binding sites along the protein sequence with a view to determining which of the KH1, KH2 and RGG domains are required to recognize and bind to RNA. While these studies have greatly contributed to the delineation of domains that bind homopolymers or mRNA in vitro, little is known concerning their implications in FMRP function(s) in vivo. To address this question, we have prepared a series of FMRP versions, in which each known in vitro functional domain has been individually deleted, leaving the rest of the protein intact. Constructs with deletions in the protein-protein interaction and RNA-binding as well as in the phosphorylation domains were expressed in STEK-KO cells lacking FMRP and their recruitment into polyribosomal mRNPs and their intra-cellular localization were determined. Our results indicate that the KH RNA-binding domains and the Protein-Protein Interacting domain are essential for FMRP to associate with polyribosomal mRNPs, while the RGG box and the phosphorylated domains are dispensable.

  8. Histone deacetylase 6 associates with ribosomes and regulates de novo protein translation during arsenite stress.

    PubMed

    Kappeler, Kyle V; Zhang, Jack; Dinh, Thai Nho; Strom, Joshua G; Chen, Qin M

    2012-05-01

    Histone deacetylase 6 (HDAC6) is known as a cytoplasmic enzyme that regulates cell migration, cell adhesion, and degradation of misfolded proteins by deacetylating substrates such as α-tubulin and Hsp90. When HaCaT keratinocytes were exposed to 1-200μM sodium arsenite, we observed perinuclear localization of HDAC6 within 30 min. Although the overall level of HDAC6 protein did not change, sodium arsenite caused an increase of HDAC6 in ribosomal fractions. Separation of ribosomal subunits versus intact ribosomes or polysomes indicated that HDAC6 was mainly detected in 40/43S fractions containing the small ribosomal subunit in untreated cells but was associated with 40/43S and 60/80S ribosomal fractions in arsenite-treated cells. Immunocytochemistry studies revealed that arsenite caused colocalization of HDAC6 with the ribosomal large and small subunit protein L36a and S6. Both L36a and S6 were detected in the immunocomplex of HDAC6 isolated from arsenite-treated cells. The observed physical interaction of HDAC6 with ribosomes pointed to a role of HDAC6 in stress-induced protein translation. Among arsenite stress-induced proteins, de novo Nrf2 protein translation was inhibited by Tubastatin A. These data demonstrate that HDAC6 was recruited to ribosomes, physically interacted with ribosomal proteins, and regulated de novo protein translation in keratinocytes responding to arsenite stress.

  9. Localization of Drosophila retinal degeneration B, a membrane- associated phosphatidylinositol transfer protein

    PubMed Central

    1993-01-01

    The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160- kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium. PMID:8354691

  10. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control.

    PubMed

    Bengtson, Mario H; Joazeiro, Claudio A P

    2010-09-23

    Messenger RNA lacking stop codons ('non-stop mRNA') can arise from errors in gene expression, and encode aberrant proteins whose accumulation could be deleterious to cellular function. In bacteria, these 'non-stop proteins' become co-translationally tagged with a peptide encoded by ssrA/tmRNA (transfer-messenger RNA), which signals their degradation by energy-dependent proteases. How eukaryotic cells eliminate non-stop proteins has remained unknown. Here we show that the Saccharomyces cerevisiae Ltn1 RING-domain-type E3 ubiquitin ligase acts in the quality control of non-stop proteins, in a process that is mechanistically distinct but conceptually analogous to that performed by ssrA: Ltn1 is predominantly associated with ribosomes, and it marks nascent non-stop proteins with ubiquitin to signal their proteasomal degradation. Ltn1-mediated ubiquitylation of non-stop proteins seems to be triggered by their stalling in ribosomes on translation through the poly(A) tail. The biological relevance of this process is underscored by the finding that loss of Ltn1 function confers sensitivity to stress caused by increased non-stop protein production. We speculate that defective protein quality control may underlie the neurodegenerative phenotype that results from mutation of the mouse Ltn1 homologue Listerin.

  11. Salivary proteins associated with periodontitis in patients with Type 2 diabetes mellitus.

    PubMed

    Chan, Hang Haw; Rahim, Zubaidah H A; Jessie, Kala; Hashim, Onn H; Taiyeb-Ali, Tara B

    2012-01-01

    The objective of this study was to investigate the salivary proteins that are associated with periodontitis in patients with Type 2 diabetes mellitus (T2DM). Volunteers for the study were patients from the Diabetic Unit, University of Malaya Medical Centre, whose periodontal status was determined. The diabetic volunteers were divided into two groups, i.e., patients with periodontitis and those who were periodontally healthy. Saliva samples were collected and treated with 10% TCA/acetone/20 mM DTT to precipitate the proteins, which were then separated using two-dimensional polyacrylamide gel electrophoresis. Gel images were scanned using the GS-800(TM) Calibrated Densitometer. The protein spots were analyzed and expressed in percentage volumes. The percentage volume of each protein spot was subjected to Mann-Whitney statistical analysis using SPSS software and false discovery rate correction. When the expression of the salivary proteins was compared between the T2DM patients with periodontitis with those who were periodontally healthy, seven proteins, including polymeric immunoglobulin receptor, plastin-2, actin related protein 3, leukocyte elastase inhibitor, carbonic anhydrases 6, immunoglobulin J and interleukin-1 receptor antagonist, were found to be differentially expressed (p < 0.01304). This implies that the proteins may have the potential to be used as biomarkers for the prediction of T2DM patients who may be prone to periodontitis.

  12. Salivary Proteins Associated with Periodontitis in Patients with Type 2 Diabetes Mellitus

    PubMed Central

    Chan, Hang Haw; Rahim, Zubaidah H. A.; Jessie, Kala; Hashim, Onn H.; Taiyeb-Ali, Tara B.

    2012-01-01

    The objective of this study was to investigate the salivary proteins that are associated with periodontitis in patients with Type 2 diabetes mellitus (T2DM). Volunteers for the study were patients from the Diabetic Unit, University of Malaya Medical Centre, whose periodontal status was determined. The diabetic volunteers were divided into two groups, i.e., patients with periodontitis and those who were periodontally healthy. Saliva samples were collected and treated with 10% TCA/acetone/20 mM DTT to precipitate the proteins, which were then separated using two-dimensional polyacrylamide gel electrophoresis. Gel images were scanned using the GS-800TM Calibrated Densitometer. The protein spots were analyzed and expressed in percentage volumes. The percentage volume of each protein spot was subjected to Mann-Whitney statistical analysis using SPSS software and false discovery rate correction. When the expression of the salivary proteins was compared between the T2DM patients with periodontitis with those who were periodontally healthy, seven proteins, including polymeric immunoglobulin receptor, plastin-2, actin related protein 3, leukocyte elastase inhibitor, carbonic anhydrases 6, immunoglobulin J and interleukin-1 receptor antagonist, were found to be differentially expressed (p < 0.01304). This implies that the proteins may have the potential to be used as biomarkers for the prediction of T2DM patients who may be prone to periodontitis. PMID:22606001