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Sample records for functional proteins involved

  1. Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation

    PubMed Central

    Karpowicz, Steven J.; Heinnickel, Mark; Dewez, David; Hamel, Blaise; Dent, Rachel; Niyogi, Krishna K.; Johnson, Xenie; Alric, Jean; Wollman, Francis-André; Li, Huiying; Merchant, Sabeeha S.

    2010-01-01

    Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus. PMID:20490922

  2. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map.

    PubMed

    Collins, Sean R; Miller, Kyle M; Maas, Nancy L; Roguev, Assen; Fillingham, Jeffrey; Chu, Clement S; Schuldiner, Maya; Gebbia, Marinella; Recht, Judith; Shales, Michael; Ding, Huiming; Xu, Hong; Han, Junhong; Ingvarsdottir, Kristin; Cheng, Benjamin; Andrews, Brenda; Boone, Charles; Berger, Shelley L; Hieter, Phil; Zhang, Zhiguo; Brown, Grant W; Ingles, C James; Emili, Andrew; Allis, C David; Toczyski, David P; Weissman, Jonathan S; Greenblatt, Jack F; Krogan, Nevan J

    2007-04-12

    Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal; however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.

  3. Nuclear pore proteins are involved in the biogenesis of functional tRNA.

    PubMed Central

    Simos, G; Tekotte, H; Grosjean, H; Segref, A; Sharma, K; Tollervey, D; Hurt, E C

    1996-01-01

    Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA. Images PMID:8641292

  4. Protein-protein interactions involving voltage-gated sodium channels: Post-translational regulation, intracellular trafficking and functional expression.

    PubMed

    Shao, Dongmin; Okuse, Kenji; Djamgoz, Mustafa B A

    2009-07-01

    Voltage-gated sodium channels (VGSCs), classically known to play a central role in excitability and signalling in nerves and muscles, have also been found to be expressed in a range of 'non-excitable' cells, including lymphocytes, fibroblasts and endothelia. VGSC abnormalities are associated with various diseases including epilepsy, long-QT syndrome 3, Brugada syndrome, sudden infant death syndrome and, more recently, various human cancers. Given their pivotal role in a wide range of physiological and pathophysiological processes, regulation of functional VGSC expression has been the subject of intense study. An emerging theme is post-translational regulation and macro-molecular complexing by protein-protein interactions and intracellular trafficking, leading to changes in functional VGSC expression in plasma membrane. This partially involves endoplasmic reticulum associated degradation and ubiquitin-proteasome system. Several proteins have been shown to associate with VGSCs. Here, we review the interactions involving VGSCs and the following proteins: p11, ankyrin, syntrophin, beta-subunit of VGSC, papin, ERM and Nedd4 proteins. Protein kinases A and C, as well as Ca(2+)-calmodulin dependent kinase II that have also been shown to regulate intracellular trafficking of VGSCs by changing the balance of externalization vs. internalization, and an effort is made to separate these effects from the short-term phosphorylation of mature proteins in plasma membrane. Two further modulatory mechanisms are reciprocal interactions with the cytoskeleton and, late-stage, activity-dependent regulation. Thus, the review gives an updated account of the range of post-translational molecular mechanisms regulating functional VGSC expression. However, many details of VGSC subtype-specific regulation and pathophysiological aspects remain unknown and these are highlighted throughout for completeness.

  5. Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles.

    PubMed

    Neuman, Benjamin W

    2016-11-01

    Replication of eukaryotic positive-stranded RNA viruses is usually linked to the presence of membrane-associated replicative organelles. The purpose of this review is to discuss the function of proteins responsible for formation of the coronavirus replicative organelle. This will be done by identifying domains that are conserved across the order Nidovirales, and by summarizing what is known about function and structure at the level of protein domains.

  6. Evolution of an ancient protein function involved in organized multicellularity in animals

    PubMed Central

    Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

    2016-01-01

    To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which – the evolution of GKPID’s capacity to bind the cortical marker protein – can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals. DOI: http://dx.doi.org/10.7554/eLife.10147.001 PMID:26740169

  7. Cell-to-cell movement of potato virus X involves distinct functions of the coat protein.

    PubMed

    Fedorkin, O; Solovyev, A; Yelina, N; Zamyatnin, A; Zinovkin, R; Mäkinen, K; Schiemann, J; Yu Morozov, S

    2001-02-01

    Complementation of movement-deficient potato virus X (PVX) coat protein (CP) mutants, namely PVX.CP-Xho lacking the 18 C-terminal amino acid residues and PVX.DeltaCP lacking the entire CP gene, was studied by transient co-expression with heterologous proteins. These data demonstrated that the potyvirus CPs and both the major and minor CPs of beet yellows closterovirus could complement cell-to-cell movement of PVX.CP-Xho but not PVX.DeltaCP. These data also indicated that the C-terminally truncated PVX CP lacked a movement function which could be provided in trans by the CPs of other filamentous viruses, whereas another movement determinant specified by some region outside the most C-terminal part of the PVX CP could not be complemented either by potyvirus or closterovirus CPs. Surprisingly, the CP of spherical cocksfoot mottle sobemovirus rescued all of the PVX CP movement functions, complementing the spread of PVX.CP-Xho and, to a lesser extent, PVX.DeltaCP. Both these mutants were also rescued by the tobacco mosaic virus (TMV) movement protein (MP). To shed light on the movement function of PVX CP, attempts were made to complement PVX.CP-Xho by a series of TMV MP mutants. An internal deletion abolished complementation, suggesting that the internal region of TMV MP, which includes a number of overlapping functional domains important for cell-to-cell transport, provides an activity complementing movement determinant(s) specified by the C-terminal region of PVX CP.

  8. Involvement of Hevea latex organelle membrane proteins in the rubber biosynthesis activity and regulatory function.

    PubMed

    Wititsuwaannakul, Dhirayos; Rattanapittayaporn, Atiya; Koyama, Tanetoshi; Wititsuwaannakul, Rapepun

    2004-03-15

    Centrifugation of fresh Hevea rubber latex yields three distinct fractions. The sediment bottom fraction (BF) content of membrane-bound organelles is ca. 20 vol.-% of latex. Prolonged storage or delayed use of fresh latex will result in disintegration and loss of the bottom fraction. This is due to the osmotically sensitive BF rupture and its membrane debris being tightly bound to the top rubber particles (RP) phase. The BF membrane was found to be highly active for rubber biosynthesis (RB), in contrast to previous reports that describe RB only occurring on the RP surface. It was clearly shown that washed BF membrane (WBM) was much more active than fresh RP for RB activity. WBM was highly activated by SDS for RB in a biphasic manner, but SDS strongly inhibited the RP. Probably WBM micelle formation resulted in a highly increased active surface area for RB. C55-PP (UPP) was a very active allylic for WBM in RB function, but inactive for RP. Serial acetone extraction of WBM proteins showed a distinct profile of the fractions with different RB activity. WBM isolated proteins suspended in 2% sodium dodecyl sulfate (SDS) with an RB activity equal to that of intact WBM was with the 20% acetone protein fraction. The 60 and 80% fractions were inactive. Combining the 20 with 80% fractions showed a complete inhibition of RB activity. Complete RB loss was also found when WBM was mixed with the 80% fraction, indicating that WBM has both an enzyme system and a factor for regulation of the RB activity in a well controlled metabolic function for the latex RB process.

  9. Functional Characterization of Bacterial Oligosaccharyltransferases Involved in O-Linked Protein Glycosylation▿

    PubMed Central

    Faridmoayer, Amirreza; Fentabil, Messele A.; Mills, Dominic C.; Klassen, John S.; Feldman, Mario F.

    2007-01-01

    Protein glycosylation is an important posttranslational modification that occurs in all domains of life. Pilins, the structural components of type IV pili, are O glycosylated in Neisseria meningitidis, Neisseria gonorrhoeae, and some strains of Pseudomonas aeruginosa. In this work, we characterized the P. aeruginosa 1244 and N. meningitidis MC58 O glycosylation systems in Escherichia coli. In both cases, sugars are transferred en bloc by an oligosaccharyltransferase (OTase) named PglL in N. meningitidis and PilO in P. aeruginosa. We show that, like PilO, PglL has relaxed glycan specificity. Both OTases are sufficient for glycosylation, but they require translocation of the undecaprenol-pyrophosphate-linked oligosaccharide substrates into the periplasm for activity. Whereas PilO activity is restricted to short oligosaccharides, PglL is able to transfer diverse oligo- and polysaccharides. This functional characterization supports the concept that despite their low sequence similarity, PilO and PglL belong to a new family of “O-OTases” that transfer oligosaccharides from lipid carriers to hydroxylated amino acids in proteins. To date, such activity has not been identified for eukaryotes. To our knowledge, this is the first report describing recombinant O glycoproteins synthesized in E. coli. PMID:17890310

  10. Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling.

    PubMed Central

    Leberer, E; Dignard, D; Hougan, L; Thomas, D Y; Whiteway, M

    1992-01-01

    The STE4 gene, which encodes the beta subunit of the mating response G-protein in the yeast Saccharomyces cerevisiae, was subjected to a saturation mutagenesis using 'doped' oligodeoxynucleotides. We employed a genetic screen to select dominant-negative STE4 mutants, which when overexpressed from the GAL1 promoter, interfered with the signalling function of the wild type protein. The identified inhibitory amino acid alterations define two small regions that are crucially involved in transmitting the mating signal from G beta to downstream components of the signalling pathway. These results underline the positive signalling role of yeast G beta and assign for the first time the positive signalling function of a G-protein beta subunit to specific structural features. Images PMID:1464310

  11. Functional dissection of protein domains involved in the immunomodulatory properties of PE_PGRS33 of Mycobacterium tuberculosis.

    PubMed

    Zumbo, Antonella; Palucci, Ivana; Cascioferro, Alessandro; Sali, Michela; Ventura, Marcello; D'Alfonso, Pamela; Iantomasi, Raffaella; Di Sante, Gabriele; Ria, Francesco; Sanguinetti, Maurizio; Fadda, Giovanni; Manganelli, Riccardo; Delogu, Giovanni

    2013-12-01

    PE_PGRSs are a large family of proteins identified in Mycobacterium tuberculosis complex and in few other pathogenic mycobacteria. The PE domain of PE_PGRS33 mediates localization of the protein on the mycobacterial cell surface, where the PGRS domain is available to interact with host components. In this study, PE_PGRS33 and its functional deletion mutants were expressed in M. smegmatis, and in vitro and in vivo assays were used to dissect the protein domains involved in the immunomodulatory properties of the protein. We demonstrate that PE_PGRS33-mediated secretion of TNF-α by macrophages occurs by extracellular interaction with TLR2. Our results also show that while the PGRS domain of the protein is required for triggering TNF-α secretion, mutation in the PE domain affects the pro-inflammatory properties of the protein. These results indicate that PE_PGRS33 is a protein with immunomodulatory activity and that protein stability and localization on the mycobacterial surface can affect these properties.

  12. Alterations in left ventricular function during intermittent hypoxia: Possible involvement of O-GlcNAc protein and MAPK signaling.

    PubMed

    Guo, Xueling; Shang, Jin; Deng, Yan; Yuan, Xiao; Zhu, Die; Liu, Huiguo

    2015-07-01

    Obstructive sleep apnea, characterized by recurrent episodes of hypoxia [intermittent hypoxia (IH)], has been identified as a risk factor for cardiovascular diseases. The O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) of proteins has important regulatory implications on the pathophysiology of cardiovascular disorders. In this study, we examined the role of O-GlcNAcylation in cardiac architecture and left ventricular function following IH. Rats were randomly assigned to a normoxia and IH group (2 min 21% O2; 2 min 6-8% O2). Left ventricular function, myocardial morphology and the levels of signaling molecules were then measured. IH induced a significant increase in blood pressure, associated with a gradually abnormal myocardial architecture. The rats exposed to 2 or 3 weeks of IH presented with augmented left ventricular systolic and diastolic function, which declined at week 4. Consistently, the O-GlcNAc protein and O-GlcNAcase (OGA) levels in the left ventricular tissues steadily increased following IH, reaching peak levels at week 3. The O-GlcNAc transferase (OGT), extracellular signal-regulated kinase 1/2 (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation levels were affected in an opposite manner. The phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) remained unaltered. In parallel, compared with exposure to normoxia, 4 weeks of IH augmented the O-GlcNAc protein, OGT, phosphorylated ERK1/2 and p38 MAPK levels, accompanied by a decrease in OGA levels and an increase in the levels of myocardial nuclear factor-κB (NF-κB), inflammatory cytokines, caspase-3 and cardiomyocyte apoptosis. Taken together, our suggest a possible involvement of O-GlcNAc protein and MAPK signaling in the alterations of left ventricular function and cardiac injury following IH.

  13. Structural and Functional Study of Yer067w, a New Protein Involved in Yeast Metabolism Control and Drug Resistance

    PubMed Central

    Domitrovic, Tatiana; Kozlov, Guennadi; Freire, João Claudio Gonçalves; Masuda, Claudio Akio; da Silva Almeida, Marcius; Montero-Lomeli, Mónica; Atella, Georgia Correa; Matta-Camacho, Edna; Gehring, Kalle; Kurtenbach, Eleonora

    2010-01-01

    The genome of Saccharomyces cerevisiae is arguably the best studied eukaryotic genome, and yet, it contains approximately 1000 genes that are still relatively uncharacterized. As the majority of these ORFs have no homologs with characterized sequence or protein structure, traditional sequence-based approaches cannot be applied to deduce their biological function. Here, we characterize YER067W, a conserved gene of unknown function that is strongly induced in response to many stress conditions and repressed in drug resistant yeast strains. Gene expression patterns of YER067W and its paralog YIL057C suggest an involvement in energy metabolism. We show that yeast lacking YER067W display altered levels of reserve carbohydrates and a growth deficiency in media that requires aerobic metabolism. Impaired mitochondrial function and overall reduction of ergosterol content in the YER067W deleted strain explained the observed 2- and 4-fold increase in resistance to the drugs fluconazole and amphotericin B, respectively. Cell fractionation and immunofluorescence microscopy revealed that Yer067w is associated with cellular membranes despite the absence of a transmembrane domain in the protein. Finally, the 1.7 Å resolution crystal structure of Yer067w shows an alpha-beta fold with low similarity to known structures and a putative functional site. YER067W's involvement with aerobic energetic metabolism suggests the assignment of the gene name RGI1, standing for respiratory growth induced 1. Altogether, the results shed light on a previously uncharacterized protein family and provide basis for further studies of its apparent role in energy metabolism control and drug resistance. PMID:20567505

  14. Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi.

    PubMed

    Pavani, Raphael Souza; da Silva, Marcelo Santos; Fernandes, Carlos Alexandre Henrique; Morini, Flavia Souza; Araujo, Christiane Bezerra; Fontes, Marcos Roberto de Mattos; Sant'Anna, Osvaldo Augusto; Machado, Carlos Renato; Cano, Maria Isabel; Fragoso, Stenio Perdigão; Elias, Maria Carolina

    2016-12-01

    Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi.

  15. Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi

    PubMed Central

    Pavani, Raphael Souza; da Silva, Marcelo Santos; Fernandes, Carlos Alexandre Henrique; Morini, Flavia Souza; Araujo, Christiane Bezerra; Fontes, Marcos Roberto de Mattos; Sant’Anna, Osvaldo Augusto; Machado, Carlos Renato; Cano, Maria Isabel; Fragoso, Stenio Perdigão; Elias, Maria Carolina

    2016-01-01

    Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi. PMID:27984589

  16. Functional Characterizations of Chemosensory Proteins of the Alfalfa Plant Bug Adelphocoris lineolatus Indicate Their Involvement in Host Recognition

    PubMed Central

    Zhang, Xue-Ying; Ji, Ping; Liu, Jing-Tao; Wang, Gui-Rong; Wu, Kong-Ming; Guo, Yu-Yuan; Zhou, Jing-Jiang; Zhang, Yong-Jun

    2012-01-01

    Insect chemosensory proteins (CSPs) have been proposed to capture and transport hydrophobic chemicals from air to olfactory receptors in the lymph of antennal chemosensilla. They may represent a new class of soluble carrier protein involved in insect chemoreception. However, their specific functional roles in insect chemoreception have not been fully elucidated. In this study, we report for the first time three novel CSP genes (AlinCSP1-3) of the alfalfa plant bug Adelphocoris lineolatus (Goeze) by screening the antennal cDNA library. The qRT-PCR examinations of the transcript levels revealed that all three genes (AlinCSP1-3) are mainly expressed in the antennae. Interestingly, these CSP genes AlinCSP1-3 are also highly expressed in the 5th instar nymphs, suggesting a proposed function of these CSP proteins (AlinCSP1-3) in the olfactory reception and in maintaining particular life activities into the adult stage. Using bacterial expression system, the three CSP proteins were expressed and purified. For the first time we characterized the types of sensilla in the antennae of the plant bug using scanning electron microscopy (SEM). Immunocytochemistry analysis indicated that the CSP proteins were expressed in the pheromone-sensitive sensilla trichodea and general odorant-sensitive sensilla basiconica, providing further evidence of their involvement in chemoreception. The antennal activity of 55 host-related semiochemicals and sex pheromone compounds in the host location and mate selection behavior of A. lineolatus was investigated using electroantennogram (EAG), and the binding affinities of these chemicals to the three CSPs (AlinCSP1-3) were measured using fluorescent binding assays. The results showed several host-related semiochemicals, (Z)-3-hexen-1-ol, (E)-2-hexen-1-al and valeraldehyde, have a high binding affinity with AlinCSP1-3 and can elicit significant high EAG responses of A. lineolatus antennae. Our studies indicate the three antennae-biased CSPs may

  17. Protein kinase C overexpression suppresses calcineurin-associated defects in Aspergillus nidulans and is involved in mitochondrial function.

    PubMed

    Colabardini, Ana Cristina; Ries, Laure Nicolas Annick; Brown, Neil Andrew; Savoldi, Marcela; Dinamarco, Taísa Magnani; von Zeska Kress, Marcia Regina; von Zeska, Marcia Regina; Goldman, Maria Helena S; Goldman, Gustavo Henrique

    2014-01-01

    In filamentous fungi, intracellular signaling pathways which are mediated by changing calcium levels and/or by activated protein kinase C (Pkc), control fungal adaptation to external stimuli. A rise in intracellular Ca2+ levels activates calcineurin subunit A (CnaA), which regulates cellular calcium homeostasis among other processes. Pkc is primarily involved in maintaining cell wall integrity (CWI) in response to different environmental stresses. Cross-talk between the Ca2+ and Pkc-mediated pathways has mainly been described in Saccharomyces cerevisiae and in a few other filamentous fungi. The presented study describes a genetic interaction between CnaA and PkcA in the filamentous fungus Aspergillus nidulans. Overexpression of pkcA partially rescues the phenotypes caused by a cnaA deletion. Furthermore, CnaA appears to affect the regulation of a mitogen-activated kinase, MpkA, involved in the CWI pathway. Reversely, PkcA is involved in controlling intracellular calcium homeostasis, as was confirmed by microarray analysis. Furthermore, overexpression of pkcA in a cnaA deletion background restores mitochondrial number and function. In conclusion, PkcA and CnaA-mediated signaling appear to share common targets, one of which appears to be MpkA of the CWI pathway. Both pathways also regulate components involved in mitochondrial biogenesis and function. This study describes targets for PkcA and CnaA-signaling pathways in an A. nidulans and identifies a novel interaction of both pathways in the regulation of cellular respiration.

  18. Protein Kinase C Overexpression Suppresses Calcineurin-Associated Defects in Aspergillus nidulans and Is Involved in Mitochondrial Function

    PubMed Central

    Brown, Neil Andrew; Savoldi, Marcela; Dinamarco, Taísa Magnani; von Zeska, Marcia Regina; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2014-01-01

    In filamentous fungi, intracellular signaling pathways which are mediated by changing calcium levels and/or by activated protein kinase C (Pkc), control fungal adaptation to external stimuli. A rise in intracellular Ca2+ levels activates calcineurin subunit A (CnaA), which regulates cellular calcium homeostasis among other processes. Pkc is primarily involved in maintaining cell wall integrity (CWI) in response to different environmental stresses. Cross-talk between the Ca2+ and Pkc-mediated pathways has mainly been described in Saccharomyces cerevisiae and in a few other filamentous fungi. The presented study describes a genetic interaction between CnaA and PkcA in the filamentous fungus Aspergillus nidulans. Overexpression of pkcA partially rescues the phenotypes caused by a cnaA deletion. Furthermore, CnaA appears to affect the regulation of a mitogen-activated kinase, MpkA, involved in the CWI pathway. Reversely, PkcA is involved in controlling intracellular calcium homeostasis, as was confirmed by microarray analysis. Furthermore, overexpression of pkcA in a cnaA deletion background restores mitochondrial number and function. In conclusion, PkcA and CnaA-mediated signaling appear to share common targets, one of which appears to be MpkA of the CWI pathway. Both pathways also regulate components involved in mitochondrial biogenesis and function. This study describes targets for PkcA and CnaA-signaling pathways in an A. nidulans and identifies a novel interaction of both pathways in the regulation of cellular respiration. PMID:25153325

  19. Functional Analysis of Adenovirus Protein IX Identifies Domains Involved in Capsid Stability, Transcriptional Activity, and Nuclear Reorganization

    PubMed Central

    Rosa-Calatrava, Manuel; Grave, Linda; Puvion-Dutilleul, Francine; Chatton, Bruno; Kedinger, Claude

    2001-01-01

    The product of adenovirus (Ad) type 5 gene IX (pIX) is known to actively participate in the stability of the viral icosahedron, acting as a capsid cement. We have previously demonstrated that pIX is also a transcriptional activator of several viral and cellular TATA-containing promoters, likely contributing to the transactivation of the Ad expression program. By extensive mutagenesis, we have now delineated the functional domains involved in each of the pIX properties: residues 22 to 26 of the highly conserved N-terminal domain are crucial for incorporation of the protein into the virion; specific residues of the C-terminal leucine repeat are responsible for pIX interactions with itself and possibly other proteins, a property that is critical for pIX transcriptional activity. We also show that pIX takes part in the virus-induced nuclear reorganization of late infected cells: the protein induces, most likely through self-assembly, the formation of specific nuclear structures which appear as dispersed nuclear globules by immunofluorescence staining and as clear amorphous spherical inclusions by electron microscopy. The integrity of the leucine repeat appears to be essential for the formation and nuclear retention of these inclusions. Together, our results demonstrate the multifunctional nature of pIX and provide new insights into Ad biology. PMID:11435594

  20. A previously uncharacterized tetratricopeptide-repeat-containing protein is involved in cell envelope function in Rhizobium leguminosarum.

    PubMed

    Neudorf, Kara D; Vanderlinde, Elizabeth M; Tambalo, Dinah D; Yost, Christopher K

    2015-01-01

    Rhizobium leguminosarum is a soil bacterium that is an intracellular symbiont of leguminous plants through the formation of nitrogen-fixing root nodules. Due to the changing environments that rhizobia encounter, the cell is often faced with a variety of cell altering stressors that can compromise the cell envelope integrity. A previously uncharacterized operon (RL3499-RL3502) has been linked to proper cell envelope function, and mutants display pleiotropic phenotypes including an inability to grow on peptide-rich media. In order to identify functional partners to the operon, suppressor mutants capable of growth on complex, peptide-rich media were isolated. A suppressor mutant of a non-polar mutation to RL3500 was chosen for further characterization. Transposon mutagenesis, screening for loss of the suppressor phenotype, led to the identification of a Tn5 insertion in an uncharacterized tetratricopeptide-repeat-containing protein RL0936. Furthermore, RL0936 had a 3.5-fold increase in gene expression in the suppressor strain when compared with the WT and a 1.5-fold increase in the original RL3500 mutant. Mutation of RL0936 decreased desiccation tolerance and lowered the ability to form biofilms when compared with the WT strain. This work has identified a potential interaction between RL0936 and the RL3499-RL3502 operon that is involved in cell envelope development in R. leguminosarum, and has described phenotypic activities to a previously uncharacterized conserved hypothetical gene.

  1. RBP-Var: a database of functional variants involved in regulation mediated by RNA-binding proteins

    PubMed Central

    Mao, Fengbiao; Xiao, Luoyuan; Li, Xianfeng; Liang, Jialong; Teng, Huajing; Cai, Wanshi; Sun, Zhong Sheng

    2016-01-01

    Transcription factors bind to the genome by forming specific contacts with the primary DNA sequence; however, RNA-binding proteins (RBPs) have greater scope to achieve binding specificity through the RNA secondary structure. It has been revealed that single nucleotide variants (SNVs) that alter RNA structure, also known as RiboSNitches, exhibit 3-fold greater local structure changes than replicates of the same DNA sequence, demonstrated by the fact that depletion of RiboSNitches could result in the alteration of specific RNA shapes at thousands of sites, including 3′ UTRs, binding sites of microRNAs and RBPs. However, the network between SNVs and post-transcriptional regulation remains unclear. Here, we developed RBP-Var, a database freely available at http://www.rbp-var.biols.ac.cn/, which provides annotation of functional variants involved in post-transcriptional interaction and regulation. RBP-Var provides an easy-to-use web interface that allows users to rapidly find whether SNVs of interest can transform the secondary structure of RNA and identify RBPs whose binding may be subsequently disrupted. RBP-Var integrates DNA and RNA biology to understand how various genetic variants and post-transcriptional mechanisms cooperate to orchestrate gene expression. In summary, RBP-Var is useful in selecting candidate SNVs for further functional studies and exploring causal SNVs underlying human diseases. PMID:26635394

  2. An Interactome-Centered Protein Discovery Approach Reveals Novel Components Involved in Mitosome Function and Homeostasis in Giardia lamblia.

    PubMed

    Rout, Samuel; Zumthor, Jon Paulin; Schraner, Elisabeth M; Faso, Carmen; Hehl, Adrian B

    2016-12-01

    Protozoan parasites of the genus Giardia are highly prevalent globally, and infect a wide range of vertebrate hosts including humans, with proliferation and pathology restricted to the small intestine. This narrow ecological specialization entailed extensive structural and functional adaptations during host-parasite co-evolution. An example is the streamlined mitosomal proteome with iron-sulphur protein maturation as the only biochemical pathway clearly associated with this organelle. Here, we applied techniques in microscopy and protein biochemistry to investigate the mitosomal membrane proteome in association to mitosome homeostasis. Live cell imaging revealed a highly immobilized array of 30-40 physically distinct mitosome organelles in trophozoites. We provide direct evidence for the single giardial dynamin-related protein as a contributor to mitosomal morphogenesis and homeostasis. To overcome inherent limitations that have hitherto severely hampered the characterization of these unique organelles we applied a novel interaction-based proteome discovery strategy using forward and reverse protein co-immunoprecipitation. This allowed generation of organelle proteome data strictly in a protein-protein interaction context. We built an initial Tom40-centered outer membrane interactome by co-immunoprecipitation experiments, identifying small GTPases, factors with dual mitosome and endoplasmic reticulum (ER) distribution, as well as novel matrix proteins. Through iterative expansion of this protein-protein interaction network, we were able to i) significantly extend this interaction-based mitosomal proteome to include other membrane-associated proteins with possible roles in mitosome morphogenesis and connection to other subcellular compartments, and ii) identify novel matrix proteins which may shed light on mitosome-associated metabolic functions other than Fe-S cluster biogenesis. Functional analysis also revealed conceptual conservation of protein translocation

  3. An Interactome-Centered Protein Discovery Approach Reveals Novel Components Involved in Mitosome Function and Homeostasis in Giardia lamblia

    PubMed Central

    Rout, Samuel; Zumthor, Jon Paulin; Schraner, Elisabeth M.

    2016-01-01

    Protozoan parasites of the genus Giardia are highly prevalent globally, and infect a wide range of vertebrate hosts including humans, with proliferation and pathology restricted to the small intestine. This narrow ecological specialization entailed extensive structural and functional adaptations during host-parasite co-evolution. An example is the streamlined mitosomal proteome with iron-sulphur protein maturation as the only biochemical pathway clearly associated with this organelle. Here, we applied techniques in microscopy and protein biochemistry to investigate the mitosomal membrane proteome in association to mitosome homeostasis. Live cell imaging revealed a highly immobilized array of 30–40 physically distinct mitosome organelles in trophozoites. We provide direct evidence for the single giardial dynamin-related protein as a contributor to mitosomal morphogenesis and homeostasis. To overcome inherent limitations that have hitherto severely hampered the characterization of these unique organelles we applied a novel interaction-based proteome discovery strategy using forward and reverse protein co-immunoprecipitation. This allowed generation of organelle proteome data strictly in a protein-protein interaction context. We built an initial Tom40-centered outer membrane interactome by co-immunoprecipitation experiments, identifying small GTPases, factors with dual mitosome and endoplasmic reticulum (ER) distribution, as well as novel matrix proteins. Through iterative expansion of this protein-protein interaction network, we were able to i) significantly extend this interaction-based mitosomal proteome to include other membrane-associated proteins with possible roles in mitosome morphogenesis and connection to other subcellular compartments, and ii) identify novel matrix proteins which may shed light on mitosome-associated metabolic functions other than Fe-S cluster biogenesis. Functional analysis also revealed conceptual conservation of protein translocation

  4. Crustacean oxi-reductases protein sequences derived from a functional genomic project potentially involved in ecdysteroid hormones metabolism - a starting point for function examination.

    PubMed

    Tom, Moshe; Manfrin, Chiara; Giulianini, Piero G; Pallavicini, Alberto

    2013-12-01

    A transcriptomic assembly originated from hypodermis and Y organ of the crustacean Pontastacus leptodactylus is used here for in silico characterization of oxi-reductase enzymes potentially involved in the metabolism of ecdysteroid molting hormones. RNA samples were extracted from male Y organ and its neighboring hypodermis in all stages of the molt cycle. An equimolar RNA mix from all stages was sequenced using next generation sequencing technologies and de novo assembled, resulting with 74,877 unique contigs. These transcript sequences were annotated by examining their resemblance to all GenBank translated transcripts, determining their Gene Ontology terms and their characterizing domains. Based on the present knowledge of arthropod ecdysteroid metabolism and more generally on steroid metabolism in other taxa, transcripts potentially related to ecdysteroid metabolism were identified and their longest possible conceptual protein sequences were constructed in two stages, correct reading frame was deduced from BLASTX resemblances, followed by elongation of the protein sequence by identifying the correct translation frame of the original transcript. The analyzed genes belonged to several oxi-reductase superfamilies including the Rieske non heme iron oxygenases, cytochrome P450s, short-chained hydroxysteroid oxi-reductases, aldo/keto oxireductases, lamin B receptor/sterol reductases and glucose-methanol-cholin oxi-reductatses. A total of 68 proteins were characterized and the most probable participants in the ecdysteroid metabolism where indicated. The study provides transcript and protein structural information, a starting point for further functional studies, using a variety of gene-specific methods to demonstrate or disprove the roles of these proteins in relation to ecdysteroid metabolism in P. leptodactylus.

  5. Genome-wide analysis and functional characterization of candidate effector proteins potentially involved in Fusarium graminearum-wheat interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal pathogens often produce certain small secreted cysteine-rich proteins (SSCPs) during pathogenesis that may function in triggering resistance or susceptibility in specific host plants. We have identified a total of 190 SSCPs encoded in the genome of the wheat scab fungus Fusarium graminearum a...

  6. GAS, a new glutamate-rich protein, interacts differentially with SRCs and is involved in oestrogen receptor function

    PubMed Central

    Liang, Jing; Zhang, Hua; Zhang, Yu; Zhang, Ying; Shang, Yongfeng

    2009-01-01

    Steroid receptor coactivators (SRCs) exert profound effects on animal development and physiology. Genetic ablation experiments indicate that various SRC proteins might have differential physiological roles; however, clear evidence of functional specificity has not yet been shown at the molecular level. Here we report the identification of a new SRC1 interacting protein, glutamate-rich coactivator interacting with SRC1 (GAS), which contains a central glutamate-rich region and has transactivation activity. Interestingly, GAS interacts only with SRC1, and not with glucocorticoid receptor interacting protein 1 (GRIP1) or amplified in breast cancer 1 (AIB1), the other two members of the SRC family. It interacts with oestrogen receptor-α (ERα) and participates in both oestrogen receptor-regulated gene transcription and oestrogen-stimulated G1/S cell-cycle transition. Our data thus indicate that GAS is a new transcription cofactor and that different SRCs are associated with distinct secondary cofactors. PMID:19039327

  7. Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: Involvement of the effector proteins in the regulatory mechanism

    SciTech Connect

    Saxena, Sunil K. . E-mail: ssaxena@stevens.edu; Kaur, Simarna

    2006-07-21

    Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.

  8. Structural organization and function of mouse photoreceptor ribbon synapses involve the immunoglobulin protein synaptic cell adhesion molecule 1.

    PubMed

    Ribic, Adema; Liu, Xinran; Crair, Michael C; Biederer, Thomas

    2014-03-01

    Adhesive interactions in the retina instruct the developmental specification of inner retinal layers. However, potential roles of adhesion in the development and function of photoreceptor synapses remain incompletely understood. This contrasts with our understanding of synapse development in the CNS, which can be guided by select adhesion molecules such as the Synaptic Cell Adhesion Molecule 1 (SynCAM 1/CADM1/nectin-like 2 protein). This immunoglobulin superfamily protein modulates the development and plasticity of classical excitatory synapses. We show here by immunoelectron microscopy and immunoblotting that SynCAM 1 is expressed on mouse rod photoreceptors and their terminals in the outer nuclear and plexiform layers in a developmentally regulated manner. Expression of SynCAM 1 on rods is low in early postnatal stages (P3-P7) but increases after eye opening (P14). In support of functional roles in the photoreceptors, electroretinogram recordings demonstrate impaired responses to light stimulation in SynCAM 1 knockout (KO) mice. In addition, the structural integrity of synapses in the OPL requires SynCAM 1. Quantitative ultrastructural analysis of SynCAM 1 KO retina measured fewer fully assembled, triadic rod ribbon synapses. Furthermore, rod synapse ribbons are shortened in KO mice, and protein levels of Ribeye, a major structural component of ribbons, are reduced in SynCAM 1 KO retina. Together, our results implicate SynCAM 1 in the synaptic organization of the rod visual pathway and provide evidence for novel roles of synaptic adhesion in the structural and functional integrity of ribbon synapses.

  9. Possible involvement of Helios in controlling the immature B cell functions via transcriptional regulation of protein kinase Cs.

    PubMed

    Kikuchi, Hidehiko; Nakayama, Masami; Takami, Yasunari; Kuribayashi, Futoshi; Nakayama, Tatsuo

    2011-01-01

    The transcription factor Ikaros family consists of five zinc-finger proteins: Ikaros, Aiolos, Helios, Eos and Pegasus; these proteins except Pegasus are essential for development and differentiation of lymphocytes. However, in B lymphocytes, the physiological role of Helios remains to be elucidated yet, because its expression level is very low. Here, we generated the Helios-deficient DT40 cells, Helios (-/-), and showed that the Helios-deficiency caused significant increases in transcriptions of four protein kinase Cs (PKCs); PKC-δ, PKC-ε, PKC-η and PKC-ζ, whereas their expressions were drastically down-regulated in the Aiolos-deficient DT40 cells, Aiolos (-/-). In addition, Helios (-/-) was remarkably resistant against phorbol 12-myristate 13-acetate (PMA)/ionomycin treatment, which mimics the B cell receptor (BCR)-mediated stimulation. In the presence of PMA/ionomycin, their viability was remarkably higher than that of DT40, and their DNA fragmentation was less severe than that of DT40 in the opposite manner for the Aiolos-deficiency. The resistance against the PMA/ionomycin-induced apoptosis of Helios (-/-) was sensitive to Rottlerin but not to Go6976. In addition, the Helios-deficiency caused remarkable up-regulation of the Rottlerin-sensitive superoxide (O2 (-))-generating activity. These data suggest that Helios may contribute to the regulation of the BCR-mediated apoptosis and O2 (-)-generating activity, via transcriptional regulation of these four PKCs (especially PKC-δ) in immature B lymphocytes. Together with previous data, our findings may significantly help in the understanding of the B lymphocyte-specific expressions of PKC genes and molecular mechanisms of both the BCR-mediated apoptosis involved in negative selection and the O2 (-)-generating system in immature B lymphocytes.

  10. [Annexins--proteins involved in organization and function of biological membranes--from Arabidopsis thaliana to Homo sapiens].

    PubMed

    Bandorowicz-Pikuła, Joanna

    2007-01-01

    The mini-review series presented in this issue of Postepy Biochemii is focussed on some aspects of biology of calcium- and membrane-binding proteins, annexins, ubiquitous in all eucaryotic organisms (excluding yeasts), from Arabidopsis thaliana to Homo sapiens. Annexins are encoded by twelve genes in verterbrates and by eight in higher plants. Their physiological significance is underlined by two facts: the numer of the annexin genes seems to grow during evolution and in some cell types they comprise up to 2% of total protein. In the present review the hypothesis is discussed suggesting that multiplication of annexin genes in evolution represents mechanism of organism adaptation to changes in environment. In addition, the experimental data are presented suggestive of annexins playing a crucial role in functioning of plasma membrane, such as signal transduction, ion and vesicular transport and membrane repair. The review is then followed by articlesdealing in details with participation of annexins in plant response to abiotic stress (Arabidopsis thaliana), in tissue mineralization (Gallus gallus), in exocytosis of catecholamines by PC12 cells (mammals) and in Niemann-Pick type C disease related to abnormal transport and intracellular storage of cholesterol (Homo sapiens).

  11. IL1-receptor accessory protein-like 1 (IL1RAPL1), a protein involved in cognitive functions, regulates N-type Ca2+-channel and neurite elongation.

    PubMed

    Gambino, Frédéric; Pavlowsky, Alice; Béglé, Aurélie; Dupont, Jean-Luc; Bahi, Nadia; Courjaret, Raphael; Gardette, Robert; Hadjkacem, Hassen; Skala, Henriette; Poulain, Bernard; Chelly, Jamel; Vitale, Nicolas; Humeau, Yann

    2007-05-22

    Null mutations in the IL1-receptor accessory protein-like 1 gene (IL1RAPL1) are responsible for an inherited X-linked form of cognitive impairment. IL1RAPL1 protein physically interacts with neuronal calcium sensor-1 (NCS-1), but the functional impact of the IL1RAPL1/NCS-1 interaction remains unknown. Here, we demonstrate that stable expression of IL1RAPL1 in PC12 cells induces a specific silencing of N-type voltage-gated calcium channels (N-VGCC) activity that explains a secretion deficit observed in these IL1RAPL1 cells. Importantly, this modulation of VGCC activity is mediated by NCS-1. Indeed, a specific loss-of-function of N-VGCC was observed in PC12 cells overexpressing NCS-1, and a total recovery of N-VGCC activity was obtained by a down-regulation of NCS-1 in IL1RAPL1 cells. The functional relevance of the interaction between IL1RAPL1 and NCS-1 was also suggested by the reduction of neurite elongation observed in nerve growth factor (NGF)-treated IL1RAPL1 cells, a phenotype rescued by NCS-1 inactivation. Because both proteins are highly expressed in neurons, these results suggest that IL1RAPL1-related mental retardation could result from a disruption of N-VGCC and/or NCS-1-dependent synaptic and neuronal activities.

  12. CtBP/BARS: a dual-function protein involved in transcription co-repression and Golgi membrane fission.

    PubMed

    Nardini, Marco; Spanò, Stefania; Cericola, Claudia; Pesce, Alessandra; Massaro, Anna; Millo, Enrico; Luini, Alberto; Corda, Daniela; Bolognesi, Martino

    2003-06-16

    C-terminal-binding protein/brefeldin A-ADP ribosylated substrate (CtBP/BARS) plays key roles in development and oncogenesis as a transcription co-repressor, and in intracellular traffic as a promoter of Golgi membrane fission. Co-repressor activity is regulated by NAD(H) binding to CtBP/BARS, while membrane fission is associated with its acyl-CoA-dependent acyltransferase activity. Here, we report the crystal structures of rat CtBP/BARS in a binary complex with NAD(H), and in a ternary complex with a PIDLSKK peptide mimicking the consensus motif (PXDLS) recognized in CtBP/BARS cellular partners. The structural data show CtBP/BARS in a NAD(H)-bound dimeric form; the peptide binding maps the recognition site for DNA-binding proteins and histone deacetylases to an N-terminal region of the protein. The crystal structure together with the site-directed mutagenesis data and binding experiments suggest a rationale for the molecular mechanisms underlying the two fundamental co-existing, but diverse, activities supported by CtBP/BARS in the nucleus and in Golgi membranes.

  13. Mitogen-activated protein kinase activity is involved in effector functions triggered by the CD94/NKG2-C NK receptor specific for HLA-E.

    PubMed

    Carretero, M; Llano, M; Navarro, F; Bellón, T; López-Botet, M

    2000-10-01

    The CD94/NKG2C heterodimer constitutes an activating receptor involved in NK cell-mediated recognition of the class lb molecule HLA-E. It transduces the triggering signal through an ITAM-bearing molecule, DAP12/KARAP, coupled non-covalently to the receptor. Here we show that specific engagement of the receptor complex expressed on the surface of an NK clone induced the phosphorylation of mitogen-activated protein kinase (MAPK). By the use of the MEK inhibitor PD098059 we demonstrate that the MAPK pathway participates in the CD94-dependent TNF-alpha production and cytotoxicity. Moreover, we transferred the activating function by transfection of the heterologous RBL cell line with CD94/NKG2-C/DAP12. In this system, cross-linking of the receptor induced calcium mobilization, serotonin release and phosphorylation of MAPK.

  14. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  15. Proteins involved in meiotic recombination: a role in male infertility?

    PubMed

    Sanderson, Matthew L; Hassold, Terry J; Carrell, Douglas T

    2008-01-01

    Meiotic recombination results in the formation of crossovers, by which genetic information is exchanged between homologous chromosomes during prophase I of meiosis. Recombination is a complex process involving many proteins. Alterations in the genes involved in recombination may result in infertility. Molecular studies have improved our understanding of the roles and mechanisms of the proteins and protein complexes involved in recombination, some of which have function in mitotic cells as well as meiotic cells. Human gene sequencing studies have been performed for some of these genes and have provided further information on the phenotypes observed in some infertile individuals. However, further studies are needed to help elucidate the particular role(s) of a given protein and to increase our understanding of these protein systems. This review will focus on our current understanding of proteins involved in meiotic recombination from a genomic perspective, summarizing our current understanding of known mutations and single nucleotide polymorphisms that may affect male fertility by altering meiotic recombination.

  16. Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility

    PubMed Central

    Aparicio, I. M.; Espino, J.; Bejarano, I.; Gallardo-Soler, A.; Campo, M. L.; Salido, G. M.; Pariente, J. A.; Peña, F. J.; Tapia, J. A.

    2016-01-01

    Macroautophagy (hereafter autophagy) is an evolutionarily highly conserved cellular process that participates in the maintenance of intracellular homeostasis through the degradation of most long-lived proteins and entire organelles. Autophagy participates in some reproductive events; however, there are not reports regarding the role of autophagy in the regulation of sperm physiology. Hence, the aim of this study was to investigate whether autophagy-related proteins are present and functionally active in human spermatozoa. Proteins related to autophagy/mitophagy process (LC3, Atg5, Atg16, Beclin 1, p62, m-TOR, AMPKα 1/2, and PINK1) were present in human spermatozoa. LC3 colocalized with p62 in the middle piece of the spermatozoa. Autophagy activation induced a significant increase in motility and a decrease in PINK1, TOM20 expression and caspase 3/7 activation. In contrast, autophagy inhibition resulted in decreased motility, viability, ATP and intracellular calcium concentration whereas PINK1, TOM20 expression, AMPK phosphorylation and caspase 3/7 activation were significantly increased. In conclusion our results show that autophagy related proteins and upstream regulators are present and functional in human spermatozoa. Modification of mitochondrial proteins expression after autophagy activation/inhibition may be indicating that a specialized form of autophagy named mitophagy may be regulating sperm function such as motility and viability and may be cooperating with apoptosis. PMID:27633131

  17. Effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: involvement of MAP kinases and protein kinase C.

    PubMed

    Canesi, Laura; Lorusso, Lucia Cecilia; Ciacci, Caterina; Betti, Michele; Gallo, Gabriella

    2005-11-10

    -) production. The results demonstrate that TBBPA in vitro activates the immune function of mussel hemocytes through kinase-mediated cell signaling and that common transduction pathways are involved in mediating the effects of this BFR in mammalian and aquatic invertebrate cells.

  18. Involvement of PCH family proteins in cytokinesis and actin distribution.

    PubMed

    Lippincott, J; Li, R

    2000-04-15

    Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.

  19. Novel protein-protein interaction family proteins involved in chloroplast movement response.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Wada, Masamitsu

    2011-04-01

    To optimize photosynthetic activity, chloroplasts change their intracellular location in response to ambient light conditions; chloroplasts move toward low intensity light to maximize light capture, and away from high intensity light to avoid photodamage. Although several proteins have been reported to be involved in the chloroplast photorelocation movement response, any physical interaction among them was not found so far. We recently found a physical interaction between two plant-specific coiled-coil proteins, WEB1 (Weak Chloroplast Movement under Blue Light 1) and PMI2 (Plastid Movement Impaired 2), that were identified to regulate chloroplast movement velocity. Since the both coiled-coil regions of WEB1 and PMI2 were classified into an uncharacterized protein family having DUF827 (DUF: Domain of Unknown Function) domain, it was the first report that DUF827 proteins could mediate protein-protein interaction. In this mini-review article, we discuss regarding molecular function of WEB1 and PMI2, and also define a novel protein family composed of WEB1, PMI2 and WEB1/PMI2-like proteins for protein-protein interaction in land plants.

  20. DsrR, a novel IscA-like protein lacking iron- and Fe-S-binding functions, involved in the regulation of sulfur oxidation in Allochromatium vinosum.

    PubMed

    Grimm, Frauke; Cort, John R; Dahl, Christiane

    2010-03-01

    In the purple sulfur bacterium Allochromatium vinosum, the reverse-acting dissimilatory sulfite reductase (DsrAB) is the key enzyme responsible for the oxidation of intracellular sulfur globules. The genes dsrAB are the first and the gene dsrR is the penultimate of the 15 genes of the dsr operon in A. vinosum. Genes homologous to dsrR occur in a number of other environmentally important sulfur-oxidizing bacteria utilizing Dsr proteins. DsrR exhibits sequence similarities to A-type scaffolds, like IscA, that partake in the maturation of protein-bound iron-sulfur clusters. We used nuclear magnetic resonance (NMR) spectroscopy to solve the solution structure of DsrR and to show that the protein is indeed structurally highly similar to A-type scaffolds. However, DsrR does not retain the Fe-S- or the iron-binding ability of these proteins, which is due to the lack of all three highly conserved cysteine residues of IscA-like scaffolds. Taken together, these findings suggest a common function for DsrR and IscA-like proteins different from direct participation in iron-sulfur cluster maturation. An A. vinosum DeltadsrR deletion strain showed a significantly reduced sulfur oxidation rate that was fully restored upon complementation with dsrR in trans. Immunoblot analyses revealed a reduced level of DsrE and DsrL in the DeltadsrR strain. These proteins are absolutely essential for sulfur oxidation. Transcriptional and translational gene fusion experiments suggested the participation of DsrR in the posttranscriptional control of the dsr operon, similar to the alternative function of cyanobacterial IscA as part of the sense and/or response cascade set into action upon iron limitation.

  1. Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis.

    PubMed

    Turowski, Valeria R; Aknin, Cindy; Maliandi, Maria V; Buchensky, Celeste; Leaden, Laura; Peralta, Diego A; Busi, Maria V; Araya, Alejandro; Gomez-Casati, Diego F

    2015-01-01

    Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S) cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR) and ferredoxin (Fd), two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.

  2. Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis

    PubMed Central

    Turowski, Valeria R.; Aknin, Cindy; Maliandi, Maria V.; Buchensky, Celeste; Leaden, Laura; Peralta, Diego A.; Busi, Maria V.; Araya, Alejandro; Gomez-Casati, Diego F.

    2015-01-01

    Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S) cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR) and ferredoxin (Fd), two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle. PMID:26517126

  3. Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction

    PubMed Central

    Sterner, David E.; Grant, Patrick A.; Roberts, Shannon M.; Duggan, Laura J.; Belotserkovskaya, Rimma; Pacella, Lisa A.; Winston, Fred; Workman, Jerry L.; Berger, Shelley L.

    1999-01-01

    SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Δ spt3Δ and gcn5Δ spt8Δ) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7Δ, spt20Δ, or ada1Δ mutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription. PMID:9858534

  4. Key proteins involved in insulin vesicle exocytosis and secretion

    PubMed Central

    Xiong, Qian-Yin; Yu, Cui; Zhang, Yao; Ling, Liefeng; Wang, Lizhuo; Gao, Jia-Lin

    2017-01-01

    In vivo insulin secretion is predominantly affected by blood glucose concentration, blood concentration of amino acids, gastrointestinal hormones and free nerve functional status, in addition to other factors. Insulin is one of the most important hormones in the body, and its secretion is precisely controlled by nutrients, neurotransmitters and hormones. The insulin exocytosis process is similar to the neurotransmitter release mechanism. There are various types of proteins and lipids that participate in the insulin secretory vesicle fusion process, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, Ras-related proteins and vacuolar-type H+-ATPase (V-ATPase). Notably, the SNARE protein is the molecular basis of exocytotic activity. In the current review, the role of the vesicle membrane proteins (synaptobrevins, vesicle associated membrane proteins and target membrane proteins) and auxiliary proteins (Rab proteins and Munc-18 proteins) in vesicle fusion activity were summarized. A summary of these key proteins involved in insulin granule secretion will facilitate understanding of the pathogenesis of diabetes. PMID:28357064

  5. Precocious leaf senescence by functional loss of PROTEIN S-ACYL TRANSFERASE14 involves the NPR1-dependent salicylic acid signaling.

    PubMed

    Zhao, Xin-Ying; Wang, Jia-Gang; Song, Shi-Jian; Wang, Qun; Kang, Hui; Zhang, Yan; Li, Sha

    2016-02-04

    We report here that Arabidopsis PROTEIN S-ACYL TRANSFERASE14 (PAT14), through its palmitate transferase activity, acts at the vacuolar trafficking route to repress salicylic acid (SA) signaling, thus mediating age-dependent but not carbon starvation-induced leaf senescence. Functional loss of PAT14 resulted in precocious leaf senescence and its transcriptomic analysis revealed that senescence was dependent on salicylic acid. Overexpressing PAT14 suppressed the expression of SA responsive genes. Introducing the SA deficient mutants, npr1-5 and NahG, but not other hormonal mutants, completely suppressed the precocious leaf senescence of PAT14 loss-of-function, further supporting the epistatic relation between PAT14 and the SA pathway. By confocal fluorescence microscopy, we showed that PAT14 is localized at the Golgi, the trans-Golg network/early endosome, and prevacuolar compartments, indicating its roles through vacuolar trafficking. By reporter analysis and real time PCRs, we showed that the expression PAT14, unlike most of the senescence associated genes, is not developmentally regulated, suggesting post-transcriptional regulatory mechanisms on its functionality. We further showed that the maize and wheat homologs of PAT14 fully rescued the precocious leaf senescence of pat14-2, demonstrating that the role of PAT14 in suppressing SA signaling during age-dependent leaf senescence is evolutionarily conserved between dicots and monocots.

  6. Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast.

    PubMed

    Yamamoto, Takaharu; Fujimura-Kamada, Konomi; Shioji, Eno; Suzuki, Risa; Tanaka, Kazuma

    2017-01-05

    Type 4 P-type ATPases (P4-ATPases) function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer, to generate and maintain asymmetric distribution of phospholipids at the plasma membrane and endosomal/Golgi membranes. The budding yeast Saccharomyces cerevisiae has four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), associated with the Cdc50p family noncatalytic subunit, and one monomeric flippase, Neo1p They have been suggested to function in vesicle formation in membrane trafficking pathways, but details of their mechanisms remain to be clarified. Here, to search for novel factors that functionally interact with flippases, we screened transposon insertional mutants for strains that suppressed the cold-sensitive growth defect in the cdc50Δ mutant. We identified a mutation of YMR010W encoding a novel conserved membrane protein that belongs to the PQ-loop family including the cystine transporter cystinosin and the SWEET sugar transporters. We named this gene CFS1 (cdc fifty suppressor 1). GFP-tagged Cfs1p was partially colocalized with Drs2p and Neo1p to endosomal/late Golgi membranes. Interestingly, the cfs1Δ mutation suppressed growth defects in all flippase mutants. Accordingly, defects in membrane trafficking in the flippase mutants were also suppressed. These results suggest that Cfs1p and flippases function antagonistically in membrane trafficking pathways. A growth assay to assess sensitivity to duramycin, a phosphatidylethanolamine (PE)-binding peptide, suggested that the cfs1Δ mutation changed PE asymmetry in the plasma membrane. Cfs1p may thus be a novel regulator of phospholipid asymmetry.

  7. Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast

    PubMed Central

    Yamamoto, Takaharu; Fujimura-Kamada, Konomi; Shioji, Eno; Suzuki, Risa; Tanaka, Kazuma

    2016-01-01

    Type 4 P-type ATPases (P4-ATPases) function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer, to generate and maintain asymmetric distribution of phospholipids at the plasma membrane and endosomal/Golgi membranes. The budding yeast Saccharomyces cerevisiae has four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), associated with the Cdc50p family noncatalytic subunit, and one monomeric flippase, Neo1p. They have been suggested to function in vesicle formation in membrane trafficking pathways, but details of their mechanisms remain to be clarified. Here, to search for novel factors that functionally interact with flippases, we screened transposon insertional mutants for strains that suppressed the cold-sensitive growth defect in the cdc50Δ mutant. We identified a mutation of YMR010W encoding a novel conserved membrane protein that belongs to the PQ-loop family including the cystine transporter cystinosin and the SWEET sugar transporters. We named this gene CFS1 (cdc fifty suppressor 1). GFP-tagged Cfs1p was partially colocalized with Drs2p and Neo1p to endosomal/late Golgi membranes. Interestingly, the cfs1Δ mutation suppressed growth defects in all flippase mutants. Accordingly, defects in membrane trafficking in the flippase mutants were also suppressed. These results suggest that Cfs1p and flippases function antagonistically in membrane trafficking pathways. A growth assay to assess sensitivity to duramycin, a phosphatidylethanolamine (PE)-binding peptide, suggested that the cfs1Δ mutation changed PE asymmetry in the plasma membrane. Cfs1p may thus be a novel regulator of phospholipid asymmetry. PMID:28057802

  8. Serotonin involvement in pituitary-adrenal function

    NASA Technical Reports Server (NTRS)

    Vernikos-Danellis, J.; Kellar, K. J.; Kent, D.; Gonzales, C.; Berger, P. A.; Barchas, J. D.

    1977-01-01

    Experiments clarifying the effects of serotonin (5-HT) in the regulation of the hypothalamic-pituitary-adrenocortical system are surveyed. Lesion experiments which seek to determine functional maps of serotonergic input to areas involved in regulation are reported. Investigations of the effects of 5-HT levels on the plasma ACTH response to stress and the diurnal variation in basal plasma corticosterone are summarized, and the question of whether serotonergic transmission is involved in the regulation of all aspects of pituitary-adrenal function is considered with attention to the stimulatory and inhibitory action of 5-HT.

  9. Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration.

    PubMed

    Del Prete, Dolores; Rice, Richard C; Rajadhyaksha, Anjali M; D'Adamio, Luciano

    2016-08-12

    The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4(CRBN), which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4(CRBN), pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys(649/650/651/676/688) Deletion of Crbn reduces ubiquitination of Lys(676) suggesting that Lys(676) is physiologically ubiquitinated by CRL4(CRBN) The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible.

  10. Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

    PubMed Central

    Breunig, K D; Kuger, P

    1987-01-01

    As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription. Images PMID:2830492

  11. Proteomic analysis of proteins involved in spermiogenesis in mouse.

    PubMed

    Guo, Xuejiang; Shen, Jian; Xia, Zhengrong; Zhang, Rui; Zhang, Ping; Zhao, Chun; Xing, Jun; Chen, Ling; Chen, Wen; Lin, Min; Huo, Ran; Su, Bing; Zhou, Zuomin; Sha, Jiahao

    2010-03-05

    Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.

  12. Some completely monotonic functions involving polygamma functions and an application

    NASA Astrophysics Data System (ADS)

    Qi, Feng; Cui, Run-Qing; Chen, Chao-Ping; Guo, Bai-Ni

    2005-10-01

    By using the first Binet's formula the strictly completely monotonic properties of functions involving the psi and polygamma functions are obtainedE As direct consequences, two inequalities are proved. As an application, the best lower and upper bounds of the nth harmonic number are established.

  13. Year 2 Report: Protein Function Prediction Platform

    SciTech Connect

    Zhou, C E

    2012-04-27

    Upon completion of our second year of development in a 3-year development cycle, we have completed a prototype protein structure-function annotation and function prediction system: Protein Function Prediction (PFP) platform (v.0.5). We have met our milestones for Years 1 and 2 and are positioned to continue development in completion of our original statement of work, or a reasonable modification thereof, in service to DTRA Programs involved in diagnostics and medical countermeasures research and development. The PFP platform is a multi-scale computational modeling system for protein structure-function annotation and function prediction. As of this writing, PFP is the only existing fully automated, high-throughput, multi-scale modeling, whole-proteome annotation platform, and represents a significant advance in the field of genome annotation (Fig. 1). PFP modules perform protein functional annotations at the sequence, systems biology, protein structure, and atomistic levels of biological complexity (Fig. 2). Because these approaches provide orthogonal means of characterizing proteins and suggesting protein function, PFP processing maximizes the protein functional information that can currently be gained by computational means. Comprehensive annotation of pathogen genomes is essential for bio-defense applications in pathogen characterization, threat assessment, and medical countermeasure design and development in that it can short-cut the time and effort required to select and characterize protein biomarkers.

  14. The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5.

    PubMed

    Haas, Thomas J; Sliwinski, Marek K; Martínez, Dana E; Preuss, Mary; Ebine, Kazuo; Ueda, Takashi; Nielsen, Erik; Odorizzi, Greg; Otegui, Marisa S

    2007-04-01

    In yeast and mammals, the AAA ATPase Vps4p/SKD1 (for Vacuolar protein sorting 4/SUPPRESSOR OF K(+) TRANSPORT GROWTH DEFECT1) is required for the endosomal sorting of secretory and endocytic cargo. We identified a VPS4/SKD1 homolog in Arabidopsis thaliana, which localizes to the cytoplasm and to multivesicular endosomes. In addition, green fluorescent protein-SKD1 colocalizes on multivesicular bodies with fluorescent fusion protein endosomal Rab GTPases, such as ARA6/RabF1, RHA1/RabF2a, and ARA7/RabF2b, and with the endocytic marker FM4-64. The expression of SKD1(E232Q), an ATPase-deficient version of SKD1, induces alterations in the endosomal system of tobacco (Nicotiana tabacum) Bright Yellow 2 cells and ultimately leads to cell death. The inducible expression of SKD1(E232Q) in Arabidopsis resulted in enlarged endosomes with a reduced number of internal vesicles. In a yeast two-hybrid screen using Arabidopsis SKD1 as bait, we isolated a putative homolog of mammalian LYST-INTERACTING PROTEIN5 (LIP5)/SKD1 BINDING PROTEIN1 and yeast Vta1p (for Vps twenty associated 1 protein). Arabidopsis LIP5 acts as a positive regulator of SKD1 by increasing fourfold to fivefold its in vitro ATPase activity. We isolated a knockout homozygous Arabidopsis mutant line with a T-DNA insertion in LIP5. lip5 plants are viable and show no phenotypic alterations under normal growth conditions, suggesting that basal SKD1 ATPase activity is sufficient for plant development and growth.

  15. Functional brain networks involved in reality monitoring.

    PubMed

    Metzak, Paul D; Lavigne, Katie M; Woodward, Todd S

    2015-08-01

    Source monitoring refers to the recollection of variables that specify the context and conditions in which a memory episode was encoded. This process involves using the qualitative and quantitative features of a memory trace to distinguish its source. One specific class of source monitoring is reality monitoring, which involves distinguishing internally generated from externally generated information, that is, memories of imagined events from real events. The purpose of the present study was to identify functional brain networks that underlie reality monitoring, using an alternative type of source monitoring as a control condition. On the basis of previous studies on self-referential thinking, it was expected that a medial prefrontal cortex (mPFC) based network would be more active during reality monitoring than the control condition, due to the requirement to focus on a comparison of internal (self) and external (other) source information. Two functional brain networks emerged from this analysis, one reflecting increasing task-related activity, and one reflecting decreasing task-related activity. The second network was mPFC based, and was characterized by task-related deactivations in areas resembling the default-mode network; namely, the mPFC, middle temporal gyri, lateral parietal regions, and the precuneus, and these deactivations were diminished during reality monitoring relative to source monitoring, resulting in higher activity during reality monitoring. This result supports previous research suggesting that self-referential thinking involves the mPFC, but extends this to a network-level interpretation of reality monitoring.

  16. Structural and functional analysis of Tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process.

    PubMed Central

    Mahillon, J; Lereclus, D

    1988-01-01

    The 4149-bp transposon Tn4430 from Bacillus thuringiensis is delineated by 38-bp inverted repeats and codes for a 113-kd protein that shares homology with the transposases (TnpA) of Tn3, Tn21 and Tn501. Through transpositional recombination, this protein generates the formation of co-integrates between both donor and target replicons, with duplication of Tn4430 molecules. These features are characteristic of transposons of the Tn3 family (class II elements). The second step of the transposition process, the co-integrate resolution, is mediated by a 32-kd protein. This protein (TnpI) displays regional similarities with site-specific recombinases of the integrase family, such as Int of bacteriophage lambda, Cre of bacteriophage P1 or TnpA and TnpB of the Tn554 transposon. Moreover, the 250-bp sequence upstream to the tnpI gene contains several structural features that are reminiscent of the attP attachment site of phage lambda. This unique association between the integrase-like TnpI recombinase and the TnpA transposase qualifies Tn4430 as a member of a new group within the class II mobile genetic elements. Images PMID:2842151

  17. Functional Differentiation of Uterine Stromal Cells Involves Cross-regulation between Bone Morphogenetic Protein 2 and Kruppel-like Factor (KLF) Family Members KLF9 and KLF13

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The inability of the uterine epithelium to enter a state of receptivity for the embryo to implant is a significant underlying cause of early pregnancy loss. We previously showed that mice null for the Progesterone Receptor (PGR)-interacting protein Kruppel-like Factor (KLF) 9 are subfertile and exhi...

  18. NEDD8 protein is involved in ubiquitinated inclusion bodies.

    PubMed

    Dil Kuazi, Afroz; Kito, Katsumi; Abe, Yasuhito; Shin, Ryong-Woon; Kamitani, Tetsu; Ueda, Norifumi

    2003-02-01

    Proteolysis by the ubiquitin-proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin-like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin-proteasome system. NEDD8 (neural precursor cell-expressed and developmentally down-regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin-proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinson's disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimer's disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin-proteasome system.

  19. Van der Waals Interactions Involving Proteins

    NASA Technical Reports Server (NTRS)

    Roth, Charles M.; Neal, Brian L.; Lenhoff, Abraham M.

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models. with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth.

  20. Van der Waals interactions involving proteins.

    PubMed Central

    Roth, C M; Neal, B L; Lenhoff, A M

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models, with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth. Images FIGURE 3 PMID:8789115

  1. Prediction of protein function from protein sequence and structure.

    PubMed

    Whisstock, James C; Lesk, Arthur M

    2003-08-01

    The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function. In particular, a number of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologues often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known. However, these inferences are tenuous. Such methods provide reasonable guesses at function, but are far from foolproof. It is therefore fortunate that the development of whole-organism approaches and comparative genomics permits other approaches to function prediction when the data are available. These include the use of protein-protein interaction patterns, and correlations between occurrences of related proteins in different organisms, as

  2. Fusion excitation functions involving transitional nuclei

    SciTech Connect

    Rehm, K.E.; Jiang, C.L.; Esbensen, H.

    1995-08-01

    Measurements of fusion excitation functions involving transitional nuclei {sup 78}Kr and {sup 100}Mo showed a different behavior at low energies, if compared to measurements with {sup 86}Kr and {sup 92}Mo. This points to a possible influence of nuclear structure on the fusion process. One way to characterize the structure of vibrational nuclei is via their restoring force parameters C{sub 2} which can be calculated from the energy of the lowest 2{sup +} state and the corresponding B(E2) value. A survey of the even-even nuclei between A = 28-150 shows strong variations in C{sub 2} values spanning two orders of magnitude. The lowest values for C{sub 2} are observed for {sup 78}Kr, {sup 104}Ru and {sup 124}Xe followed by {sup 74,76}Ge, {sup 74,76}Se, {sup 100}Mo and {sup 110}Pd. In order to learn more about the influence of {open_quotes}softness{close_quotes} on the sub-barrier fusion enhancement, we measured cross sections for evaporation residue production for the systems {sup 78}Kr + {sup 104}Ru and {sup 78}Kr + {sup 76}Ge with the gas-filled magnet technique. For both systems, fusion excitation functions involving the closed neutron shell nucleus {sup 86}Kr were measured previously. The data are presently being analyzed.

  3. [Protein phosphatases: structure and function].

    PubMed

    Bulanova, E G; Budagian, V M

    1994-01-01

    The process of protein and enzyme systems phosphorylation is necessary for cell growth, differentiation and preparation for division and mitosis. The conformation changes of protein as a result of phosphorylation lead to increased enzyme activity and enhanced affinity to substrates. A large group of enzymes--protein kinases--is responsible for phosphorylation process in cell, which are divided into tyrosine- and serine-threonine-kinases depending on their ability to phosphorylate appropriate amino acid residues. In this review has been considered the functional importance and structure of protein phosphatases--enzymes, which are functional antagonists of protein kinases.

  4. Molecular and functional characterization of peptidoglycan-recognition protein SC2 (PGRP-SC2) from Nile tilapia (Oreochromis niloticus) involved in the immune response to Streptococcus agalactiae.

    PubMed

    Gan, Zhen; Chen, Shannan; Hou, Jing; Huo, Huijun; Zhang, Xiaolin; Ruan, Baiye; Laghari, Zubair Ahmed; Li, Li; Lu, Yishan; Nie, Pin

    2016-07-01

    PGRP-SC2, the member of PGRP family, plays an important role in regulation of innate immune response. In this paper, a PGRP-SC2 gene of Nile tilapia, Oreochromis niloticus (designated as On-PGRP-SC2) was cloned and its expression pattern under the infection of Streptococcus agalactiae was investigated. Sequence analysis showed main structural features required for amidase activity were detected in the deduced amino acid sequence of On-PGRP-SC2. In healthy tilapia, the On-PGRP-SC2 transcripts could be detected in all the examined tissues, with the most abundant expression in the muscle. When infected with S. agalactiae, there was a clear time-dependent expression pattern of On-PGRP-SC2 in the spleen, head kidney and brain. The assays for the amidase activity suggested that recombinant On-PGRP-SC2 protein had a Zn(2+)-dependent PGN-degrading activity. Moreover, our works showed that recombinant On-PGRP-SC2 protein could significantly reduce bacterial load in target organs attacked by S. agalactiae. These findings indicated that On-PGRP-SC2 may play important roles in the immune response to S. agalactiae in Nile tilapia.

  5. Molecular signaling involving intrinsically disordered proteins in prostate cancer

    PubMed Central

    Russo, Anna; Manna, Sara La; Novellino, Ettore; Malfitano, Anna Maria; Marasco, Daniela

    2016-01-01

    Investigations on cellular protein interaction networks (PINs) reveal that proteins that constitute hubs in a PIN are notably enriched in Intrinsically Disordered Proteins (IDPs) compared to proteins that constitute edges, highlighting the role of IDPs in signaling pathways. Most IDPs rapidly undergo disorder-to-order transitions upon binding to their biological targets to perform their function. Conformational dynamics enables IDPs to be versatile and to interact with a broad range of interactors under normal physiological conditions where their expression is tightly modulated. IDPs are involved in many cellular processes such as cellular signaling, transcriptional regulation, and splicing; thus, their high-specificity/low-affinity interactions play crucial roles in many human diseases including cancer. Prostate cancer (PCa) is one of the leading causes of cancer-related mortality in men worldwide. Therefore, identifying molecular mechanisms of the oncogenic signaling pathways that are involved in prostate carcinogenesis is crucial. In this review, we focus on the aspects of cellular pathways leading to PCa in which IDPs exert a primary role. PMID:27212129

  6. Carbon regulation of the cuticle-degrading enzyme PR1 from Metarhizium anisopliae may involve a trans-acting DNA-binding protein CRR1, a functional equivalent of the Aspergillus nidulans CREA protein.

    PubMed

    Screen, S; Bailey, A; Charnley, K; Cooper, R; Clarkson, J

    1997-06-01

    The pr1 gene of the entomopathogenic fungus Metarhizium anisopliae encodes a serine protease that is highly active towards the insect cuticle and whose synthesis is subject to both carbon and nitrogen repression. The pr1 promoter region was sequenced revealing the presence of putative CREA- and AREA-binding sites. In vitro bandshift experiments demonstrated that an Aspergillus nidulans GST-CREA fusion protein was capable of binding to two of the three putative CREA sites. Using a PCR-based strategy the M. anisopliae crr1 gene was identified; it encodes a putative C2H2-type DNA-binding protein with significant sequence similarity to A. nidulans CREA. Complementation experiments with an A. nidulans strain carrying creA204 demonstrated that CRR1 can partially substitute for CREA function.

  7. Functional characterization of TRAP1-like protein involved in modulating fibrotic processes mediated by TGF-β/Smad signaling in hypertrophic scar fibroblasts

    SciTech Connect

    Wang, X.; Chu, J.; Wen, C.J.; Fu, S.B.; Qian, Y.L.; Wo, Y.; Wang, C.; Wang, D.R.

    2015-03-15

    The transforming growth factor-β1 (TGF-β)-mediated signaling pathway is believed to be closely associated with wound healing and scar formation, in which TRAP1-like protein (TLP) plays a role in regulating the balance of Smad2 vs. Smad3 signaling. Our previous study revealed the relation between TLP and collagen synthesis in normal human skin fibroblasts. Here, we present a detailed analysis of the effects of TLP on the process of hypertrophic scar formation and contraction. To explore and verify a contribution of TLP to the pathological mechanism of hypertrophic scar fibroblasts (HSFb), we constructed lentiviral vectors that either overexpressed TLP or encoded small hairpin RNAs (shRNAs) targeting TLP, then we transfected them into HSFb. TLP knockdown in HSFb resulted in reduced levels of cell contraction, type I and type III collagen mRNA transcripts and protein expression, and higher levels of fibronectin (FN) compared to control groups. In addition, knockdown of TLP promoted the phosphorylation of Smad3 but repressed Smad2 and Erk-1/2 phosphorylation in human hypertrophic scar fibroblasts compared to control groups. The reduction of TLP did not interfere with HSF proliferative ability, but exogenous TLP cooperated with TGF-β1 to increase cell viability. Together, our findings demonstrate evidence for a contribution of TLP expression in hypertrophic scar formation and contraction. - Highlights: • TLP acted different roles in the activating of Smad2- and Smad3-dependent signaling. • TLP may induce TGF-β1-mediated collagens expression through Smad signalings and MAPK signaling. • TLP may enhance HSFb contraction by increasing the expression of α-SMA. • Exogenous TLP can cooperate with TGF-β1 to increase cell viability.

  8. Food Protein Functionality--A New Model.

    PubMed

    Foegeding, E Allen

    2015-12-01

    Proteins in foods serve dual roles as nutrients and structural building blocks. The concept of protein functionality has historically been restricted to nonnutritive functions--such as creating emulsions, foams, and gels--but this places sole emphasis on food quality considerations and potentially overlooks modifications that may also alter nutritional quality or allergenicity. A new model is proposed that addresses the function of proteins in foods based on the length scale(s) responsible for the function. Properties such as flavor binding, color, allergenicity, and digestibility are explained based on the structure of individual molecules; placing this functionality at the nano/molecular scale. At the next higher scale, applications in foods involving gelation, emulsification, and foam formation are based on how proteins form secondary structures that are seen at the nano and microlength scales, collectively called the mesoscale. The macroscale structure represents the arrangements of molecules and mesoscale structures in a food. Macroscale properties determine overall product appearance, stability, and texture. The historical approach of comparing among proteins based on forming and stabilizing specific mesoscale structures remains valid but emphasis should be on a common means for structure formation to allow for comparisons across investigations. For applications in food products, protein functionality should start with identification of functional needs across scales. Those needs are then evaluated relative to how processing and other ingredients could alter desired molecular scale properties, or proper formation of mesoscale structures. This allows for a comprehensive approach to achieving the desired function of proteins in foods.

  9. Functional proteins involved in regulation of intracellular Ca(2+) for drug development: chronic nicotine treatment upregulates L-type high voltage-gated calcium channels.

    PubMed

    Katsura, Masashi; Ohkuma, Seitaro

    2005-03-01

    Neurochemical mechanisms underlying drug dependence and withdrawal syndrome remain unclear. In this review, we discuss how chronic nicotine exposure to neurons affects expression of diazepam binding inhibitor (DBI), an endogenous anxiogenic neuropeptide supposed to be a common substance participating drug dependence, and function of L-type high voltage-gated Ca(2+) channels (HVCCs). We also discuss the functional interaction between DBI and L-type HVCCs in nicotine dependence. Both DBI levels and [(45)Ca(2+)] influx significantly increased in the brain from mice treated with nicotine for long term, which was further enhanced after abrupt cessation of nicotine and was abolished by nicotinic acetylcholine receptor (nAChR) antagonists. Similar responses of DBI expression and L-type HVCC function were observed in cerebral cortical neurons after sustained exposure to nicotine. In addition, increased DBI expression was inhibited by antagonists of nAChR and L-type HVCCs. Sustained exposure of neurons to nicotine significantly enhanced expression of alpha(1) and alpha(2)/delta(1) subunits for L-type HVCCs and caused an increase in the B(max) value of [(3)H]verapamil binding to the particulate fractions. Therefore, it is concluded that the alterations in DBI expression is mediated via increased influx of Ca(2+) through upregulated L-type HVCCs and these neurochemical changes have a close relationship with development of nicotine dependence and/or its withdrawal syndrome.

  10. Genes and proteins involved in bacterial magnetic particle formation.

    PubMed

    Matsunaga, Tadashi; Okamura, Yoshiko

    2003-11-01

    Magnetic bacteria synthesize intracellular magnetosomes that impart a cellular swimming behaviour referred to as magnetotaxis. The magnetic structures aligned in chains are postulated to function as biological compass needles allowing the bacterium to migrate along redox gradients through the Earth's geomagnetic field lines. Despite the discovery of this unique group of microorganisms 28 years ago, the mechanisms of magnetic crystal biomineralization have yet to be fully elucidated. This review describes the current knowledge of the genes and proteins involved in magnetite formation in magnetic bacteria and the biotechnological applications of biomagnetites in the interdisciplinary fields of nanobiotechnology, medicine and environmental management.

  11. Function and structure of inherently disordered proteins.

    PubMed

    Dunker, A Keith; Silman, Israel; Uversky, Vladimir N; Sussman, Joel L

    2008-12-01

    The application of bioinformatics methodologies to proteins inherently lacking 3D structure has brought increased attention to these macromolecules. Here topics concerning these proteins are discussed, including their prediction from amino acid sequence, their enrichment in eukaryotes compared to prokaryotes, their more rapid evolution compared to structured proteins, their organization into specific groups, their structural preferences, their half-lives in cells, their contributions to signaling diversity (via high contents of multiple-partner binding sites, post-translational modifications, and alternative splicing), their distinct functional repertoire compared to that of structured proteins, and their involvement in diseases.

  12. Zinc finger protein ZPR9 functions as an activator of AMPK-related serine/threonine kinase MPK38/MELK involved in ASK1/TGF-β/p53 signaling pathways

    PubMed Central

    Seong, Hyun-A; Manoharan, Ravi; Ha, Hyunjung

    2017-01-01

    Murine protein serine-threonine kinase 38 (MPK38), an AMP‐activated protein kinase (AMPK)-related kinase, has been implicated in the induction of apoptosis signal-regulating kinase 1 (ASK1)-, transforming growth factor-β (TGF‐β)-, and p53-mediated activity involved in metabolic homeostasis. Here, zinc finger protein ZPR9 was found to be an activator of MPK38. The association of MPK38 and ZPR9 was mediated by cysteine residues present in each of these two proteins, Cys269 and Cys286 of MPK38 and Cys305 and Cys308 of ZPR9. MPK38 phosphorylated ZPR9 at Thr252. Wild‐type ZPR9, but not the ZPR9 mutant T252A, enhanced ASK1, TGF‐β, and p53 function by stabilizing MPK38. The requirement of ZPR9 Thr252 phosphorylation was validated using CRISPR/Cas9-mediated ZPR9 (T252A) knockin cell lines. The knockdown of endogenous ZPR9 showed an opposite trend, resulting in the inhibition of MPK38‐dependent ASK1, TGF‐β, and p53 function. This effect was also demonstrated in mouse embryonic fibroblast (MEF) cells that were haploinsufficient (+/−) for ZPR9, NIH 3T3 cells with inducible knockdown of ZPR9, and CRISPR/Cas9-mediated ZPR9 knockout cells. Furthermore, high-fat diet (HFD)-fed mice displayed reduced MPK38 kinase activity and ZPR9 expression compared to that in mice on control chow, suggesting that ZPR9 acts as a physiological activator of MPK38 that may participate in obesity. PMID:28195154

  13. Involvement of heat shock proteins in gluten-sensitive enteropathy.

    PubMed

    Sziksz, Erna; Pap, Domonkos; Veres, Gábor; Fekete, Andrea; Tulassay, Tivadar; Vannay, Ádám

    2014-06-07

    Gluten-sensitive enteropathy, also known as coeliac disease (CD), is an autoimmune disorder occurring in genetically susceptible individuals that damages the small intestine and interferes with the absorption of other nutrients. As it is triggered by dietary gluten and related prolamins present in wheat, rye and barley, the accepted treatment for CD is a strict gluten-free diet. However, a complete exclusion of gluten-containing cereals from the diet is often difficult, and new therapeutic strategies are urgently needed. A class of proteins that have already emerged as drug targets for other autoimmune diseases are the heat shock proteins (HSPs), which are highly conserved stress-induced chaperones that protect cells against harmful extracellular factors. HSPs are expressed in several tissues, including the gastrointestinal tract, and their levels are significantly increased under stress circumstances. HSPs exert immunomodulatory effects, and also play a crucial role in the maintenance of epithelial cell structure and function, as they are responsible for adequate protein folding, influence the degradation of proteins and cell repair processes after damage, and modulate cell signalling, cell proliferation and apoptosis. The present review discusses the involvement of HSPs in the pathophysiology of CD. Furthermore, HSPs may represent a useful therapeutic target for the treatment of CD due to the cytoprotective, immunomodulatory, and anti-apoptotic effects in the intestinal mucosal barrier.

  14. Suppression of granulocyte-macrophage colony-stimulating factor expression by glucocorticoids involves inhibition of enhancer function by the glucocorticoid receptor binding to composite NF-AT/activator protein-1 elements.

    PubMed

    Smith, P J; Cousins, D J; Jee, Y K; Staynov, D Z; Lee, T H; Lavender, P

    2001-09-01

    Increased expression of a number of cytokines including GM-CSF is associated with chronic inflammatory conditions such as bronchial asthma. Glucocorticoid therapy results in suppression of cytokine levels by a mechanism(s) not yet fully understood. We have examined regulation of GM-CSF expression by the synthetic glucocorticoid dexamethasone in human T cells. Transient transfection assays with reporter constructs revealed that dexamethasone inhibited the function of the GM-CSF enhancer, but had no effect on regulation of GM-CSF expression occurring through the proximal promoter. Activation of the GM-CSF enhancer involves cooperative interaction between the transcription factors NF-AT and AP-1. We demonstrate here that glucocorticoid-mediated inhibition of enhancer function involves glucocorticoid receptor (GR) binding to the NF-AT/AP-1 sites. These elements, which do not constitute recognizable glucocorticoid response elements, support binding of the GR, primarily as a dimer. This binding correlates with the ability of dexamethasone to inhibit enhancer activity of the NF-AT/AP-1 elements, suggesting a competition between NF-AT/AP-1 proteins and GR.

  15. Functional domains in tetraspanin proteins.

    PubMed

    Stipp, Christopher S; Kolesnikova, Tatiana V; Hemler, Martin E

    2003-02-01

    Exciting new findings have emerged about the structure, function and biochemistry of tetraspanin proteins. Five distinct tetraspanin regions have now been delineated linking structural features to specific functions. Within the large extracellular loop of tetraspanins, there is a variable region that mediates specific interactions with other proteins, as well as a more highly conserved region that has been suggested to mediate homodimerization. Within the transmembrane region, the four tetraspanin transmembrane domains are probable sites of both intra- and inter-molecular interactions that are crucial during biosynthesis and assembly of the network of tetraspanin-linked membrane proteins known as the 'tetraspanin web'. In the intracellular juxtamembrane region, palmitoylation of cysteine residues also contributes to tetraspanin web assembly, and the C-terminal cytoplasmic tail region could provide specific functional links to cytoskeletal or signaling proteins.

  16. Life under tension: Computational studies of proteins involved in mechanotransduction

    NASA Astrophysics Data System (ADS)

    Sotomayor, Marcos Manuel

    cadherins. Simulations also revealed how calcium ions control cadherin's shape and the availability of key residues involved in cell-cell adhesion, suggesting a conceptual framework for interpreting mutations in cadherin calcium binding motifs causing hereditary deafness. Overall, simulations provided a unique nanoscopic view of the dynamics and function of some of the proteins involved in mechanotransduction.

  17. Sucrose Synthase: Expanding Protein Function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. Th...

  18. Possible involvement of poly(A) in protein synthesis.

    PubMed Central

    Jacobson, A; Favreau, M

    1983-01-01

    The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect. Images PMID:6137807

  19. Tamoxifen regulation of bone growth and endocrine function in the ovariectomized rat: discrimination of responses involving estrogen receptor α/estrogen receptor β, G protein-coupled estrogen receptor, or estrogen-related receptor γ using fulvestrant (ICI 182780).

    PubMed

    Fitts, James M; Klein, Robert M; Powers, C Andrew

    2011-07-01

    Tamoxifen is a selective estrogen receptor (ER) modulator, but it is also a deactivating ligand for estrogen-related receptor-γ (ERRγ) and a full agonist for the G protein-coupled estrogen receptor (GPER). Fulvestrant is a selective ER down-regulator that lacks agonist effects on ERα/ERβ, is inactive on ERRγ, but acts as a full agonist on GPER. Fulvestrant effects on tamoxifen actions on uterine and somatic growth, bone, the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis, and pituitary prolactin were analyzed to pharmacologically discriminate tamoxifen effects that may be mediated by ERα/ERβ versus ERRγ versus GPER. Ovariectomized rats received tamoxifen (0.6 mg/kg/daily) plus fulvestrant at 0, 3, 6, or 12 mg/kg/daily for 5 weeks; controls received vehicle or 6 mg/kg fulvestrant daily. Tamoxifen effects to increase uterine weight, decrease serum IGF-I, increase pituitary prolactin, and increase bone mineral density could be fully blocked by fulvestrant, indicating mediation by ERα/ERβ. Tamoxifen effects to decrease pituitary GH, tibia length, and body weight were only partially blocked by fulvestrant, indicating involvement of mechanisms unrelated to ERα/ERβ. Fulvestrant did not inhibit tamoxifen actions to reduce total pituitary protein, again indicating effects not mediated by ERα/ERβ. Tamoxifen actions to reduce serum GH were mimicked rather than inhibited by fulvestrant, pharmacological features consistent with GPER involvement. However, fulvestrant alone increased IGF-I and also blocked tamoxifen-evoked IGF-I decreases; thus fulvestrant effects on serum GH might reflect increased IGF-I feedback inhibition. Fulvestrant alone had no effect on the other parameters. The findings indicate that mechanisms unrelated to ERα/ERβ contribute to tamoxifen effects on body weight, bone growth, and pituitary function.

  20. Nicotinic involvement in memory function in zebrafish.

    PubMed

    Levin, Edward D; Chen, Elaine

    2004-01-01

    Zebrafish are an emerging model for the study of the molecular mechanisms of brain function. To conduct studies of the neural bases of behavior in zebrafish, we must understand the behavioral function of zebrafish and how it is altered by perturbations of brain function. This study determined nicotine actions on memory function in zebrafish. With the methods that we have developed to assess memory in zebrafish using delayed spatial alternation (DSA), we determined the dose effect function of acute nicotine on memory function in zebrafish. As in rodents and primates, low nicotine doses improve memory in zebrafish, while high nicotine doses have diminished effect and can impair memory. This study shows that nicotine affects memory function in zebrafish much like in rats, mice, monkeys and humans. Now, zebrafish can be used to help understand the molecular mechanisms crucial to nicotine effects on memory.

  1. DUF581 Is Plant Specific FCS-Like Zinc Finger Involved in Protein-Protein Interaction

    PubMed Central

    K, Muhammed Jamsheer; Laxmi, Ashverya

    2014-01-01

    Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ) domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction. PMID:24901469

  2. Evolution-Based Functional Decomposition of Proteins.

    PubMed

    Rivoire, Olivier; Reynolds, Kimberly A; Ranganathan, Rama

    2016-06-01

    The essential biological properties of proteins-folding, biochemical activities, and the capacity to adapt-arise from the global pattern of interactions between amino acid residues. The statistical coupling analysis (SCA) is an approach to defining this pattern that involves the study of amino acid coevolution in an ensemble of sequences comprising a protein family. This approach indicates a functional architecture within proteins in which the basic units are coupled networks of amino acids termed sectors. This evolution-based decomposition has potential for new understandings of the structural basis for protein function. To facilitate its usage, we present here the principles and practice of the SCA and introduce new methods for sector analysis in a python-based software package (pySCA). We show that the pattern of amino acid interactions within sectors is linked to the divergence of functional lineages in a multiple sequence alignment-a model for how sector properties might be differentially tuned in members of a protein family. This work provides new tools for studying proteins and for generally testing the concept of sectors as the principal units of function and adaptive variation.

  3. Methods for Mapping of Interaction Networks Involving Membrane Proteins

    SciTech Connect

    Hooker, Brian S.; Bigelow, Diana J.; Lin, Chiann Tso

    2007-11-23

    Numerous approaches have been taken to study protein interactions, such as tagged protein complex isolation followed by mass spectrometry, yeast two-hybrid methods, fluorescence resonance energy transfer, surface plasmon resonance, site-directed mutagenesis, and crystallography. Membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is not suitable for the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.

  4. Functional limitations due to foot involvement in spondyloarthritis

    PubMed Central

    Ozaras, Nihal; Havan, Nuri; Poyraz, Emine; Rezvanı, Aylin; Aydın, Teoman

    2016-01-01

    [Purpose] Spondyloarthritis is a major inflammatory disease followed-up in the rheumatology clinics, foot involvement in spodyloarthritis is common. The functional states of patients with spondyloarthritis are usually evaluated globally. The aim of this study was to assess the foot involvement-related functional limitations in patients with spondyloarthritis. [Subjects and Methods] Patients with ankylosing spondylitis and psoriatic arthritis with foot pain more than 4 weeks who underwent anteroposterior and lateral feet radiography were enrolled into the study. A “clinical findings score” was calculated by assigning 1 point for every finding of swelling, redness, and tenderness. C-reactive protein and erythrocyte sedimentation rate were used as serum markers for disease activity. Foot radiograms were evaluated using the spondyloarthropathy tarsal radiographic index and the foot-related functional state of patients was determined by the Turkish version of the Foot and Ankle Outcome Score. [Results] There were no relationships between Foot and Ankle Outcome Score subscales and clinical findings score, serum markers, or radiologic score. Pain and symptoms subscale scores were result positively correlated with activity of daily living, sport and recreation, and quality of life subscale scores. [Conclusion] Pain and symptoms are the main determinants of foot-related functional limitations in spondyloarthritis. PMID:27512252

  5. Molecular Simulation Studies of Proteins Involved in Parkinson's Disease

    NASA Astrophysics Data System (ADS)

    Carloni, Paolo

    2007-12-01

    This contribution describes two recent computational studies related to proteins involved in Parkinson's Disease (PD). The first focuses on the interplay between dopamine and α-synuclein (AS), which plays a central role in PD (unpublished results). The second deals with the protein DJ-1, whose mutations are present in patients suffering from familiar PD [1]. Computational methods are used to investigate the relationship between such mutations and the protein oligomeric state, which may be important for the progression of the disease.

  6. Functional type 1 secretion system involved in Legionella pneumophila virulence.

    PubMed

    Fuche, Fabien; Vianney, Anne; Andrea, Claire; Doublet, Patricia; Gilbert, Christophe

    2015-02-01

    Legionella pneumophila is a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence of L. pneumophila is now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle of L. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that the repeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation of L. pneumophila, via its T1SS, in its internalization into host cells.

  7. Genome-wide protein-protein interactions and protein function exploration in cyanobacteria.

    PubMed

    Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

    2015-10-22

    Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and "interologs" in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria.

  8. Involvement of Iron-Containing Proteins in Genome Integrity in Arabidopsis Thaliana

    PubMed Central

    Zhang, Caiguo

    2015-01-01

    The Arabidopsis genome encodes numerous iron-containing proteins such as iron-sulfur (Fe-S) cluster proteins and hemoproteins. These proteins generally utilize iron as a cofactor, and they perform critical roles in photosynthesis, genome stability, electron transfer, and oxidation-reduction reactions. Plants have evolved sophisticated mechanisms to maintain iron homeostasis for the assembly of functional iron-containing proteins, thereby ensuring genome stability, cell development, and plant growth. Over the past few years, our understanding of iron-containing proteins and their functions involved in genome stability has expanded enormously. In this review, I provide the current perspectives on iron homeostasis in Arabidopsis, followed by a summary of iron-containing protein functions involved in genome stability maintenance and a discussion of their possible molecular mechanisms. PMID:27330736

  9. Investigating neuronal function with optically controllable proteins

    PubMed Central

    Zhou, Xin X.; Pan, Michael; Lin, Michael Z.

    2015-01-01

    In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain. PMID:26257603

  10. Design of membrane proteins: toward functional systems.

    PubMed

    Ghirlanda, Giovanna

    2009-12-01

    Over the years, membrane-soluble peptides have provided a convenient model system to investigate the folding and assembly of integral membrane proteins. Recent advances in experimental and computational methods are now being translated into the design of functional membrane proteins. Applications include artificial modulators of membrane protein function, inhibitors of protein-protein interactions, and redox membrane proteins.

  11. Functional Classification of Immune Regulatory Proteins

    SciTech Connect

    Rubinstein, Rotem; Ramagopal, Udupi A.; Nathenson, Stanley G.; Almo, Steven C.; Fiser, Andras

    2013-05-01

    Members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. We were guided by the Brotherhood approach and present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

  12. P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing.

    PubMed

    Jekat, Stephan B; Ernst, Antonia M; von Bohl, Andreas; Zielonka, Sascia; Twyman, Richard M; Noll, Gundula A; Prüfer, Dirk

    2013-01-01

    Structural phloem proteins (P-proteins) are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently confirmed to be encoded by the widespread sieve element occlusion (SEO) gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb) indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing.

  13. Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. III. Permeability of spectrin-depleted inside-out membrane vesicles to hydrophilic nonelectrolytes. Formation of leaks by chemical or enzymatic modification of membrane proteins.

    PubMed

    Klonk, S; Deuticke, B

    1992-04-29

    Spectrin-depleted inside-out vesicles (IOV's) prepared from human erythrocyte membranes were characterized in terms of size, ground permeability to hydrophilic nonelectrolytes and their sensitivity to modification by SH reagents, DIDS and trypsin. IOV's proved to have the same permeability of their lipid domain to erythritol as native erythrocytes, in contrast to resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)), which have a residual leak. On the other hand, IOV's have a slightly elevated permeability for mannitol and sucrose, nonelectrolytes which are almost (mannitol) or fully (sucrose) impermeant in the native membrane. These increased fluxes, which have a high activation energy and can be stimulated by phloretin, are, however, also much smaller than the corresponding leak fluxes observed in resealed ghosts. In view of these differences, formation of IOV's can be concluded to go along with partial annealing of barrier defects persisting in the erythrocyte membrane after preparation of resealed ghosts. Oxidation of SH groups of the IOV membrane by diamide produces an enhancement of permeability for hydrophilic nonelectrolytes which is much less pronounced than that induced by a similar treatment of erythrocytes or ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)). Moreover, proteolytic treatment of the vesicle membrane, although leading to a marked digestion of integral membrane proteins, only induces a minor, saturating increase of permeability, much lower than that in trypsinized resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 137-142 (Part II of this series)). Since absence of the cytoskeletal proteins, spectrin and actin, is the major difference between IOV's and resealed ghosts, these results may be taken as further evidence for a dependence of the barrier properties of the erythrocyte membrane bilayer domain

  14. Proteins with Novel Structure, Function and Dynamics

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew

    2014-01-01

    Recently, a small enzyme that ligates two RNA fragments with the rate of 10(exp 6) above background was evolved in vitro (Seelig and Szostak, Nature 448:828-831, 2007). This enzyme does not resemble any contemporary protein (Chao et al., Nature Chem. Biol. 9:81-83, 2013). It consists of a dynamic, catalytic loop, a small, rigid core containing two zinc ions coordinated by neighboring amino acids, and two highly flexible tails that might be unimportant for protein function. In contrast to other proteins, this enzyme does not contain ordered secondary structure elements, such as alpha-helix or beta-sheet. The loop is kept together by just two interactions of a charged residue and a histidine with a zinc ion, which they coordinate on the opposite side of the loop. Such structure appears to be very fragile. Surprisingly, computer simulations indicate otherwise. As the coordinating, charged residue is mutated to alanine, another, nearby charged residue takes its place, thus keeping the structure nearly intact. If this residue is also substituted by alanine a salt bridge involving two other, charged residues on the opposite sides of the loop keeps the loop in place. These adjustments are facilitated by high flexibility of the protein. Computational predictions have been confirmed experimentally, as both mutants retain full activity and overall structure. These results challenge our notions about what is required for protein activity and about the relationship between protein dynamics, stability and robustness. We hypothesize that small, highly dynamic proteins could be both active and fault tolerant in ways that many other proteins are not, i.e. they can adjust to retain their structure and activity even if subjected to mutations in structurally critical regions. This opens the doors for designing proteins with novel functions, structures and dynamics that have not been yet considered.

  15. Stably Expressed Genes Involved in Basic Cellular Functions

    PubMed Central

    Wang, Kejian; Fuscoe, James C.

    2017-01-01

    Stably Expressed Genes (SEGs) whose expression varies within a narrow range may be involved in core cellular processes necessary for basic functions. To identify such genes, we re-analyzed existing RNA-Seq gene expression profiles across 11 organs at 4 developmental stages (from immature to old age) in both sexes of F344 rats (n = 4/group; 320 samples). Expression changes (calculated as the maximum expression / minimum expression for each gene) of >19000 genes across organs, ages, and sexes ranged from 2.35 to >109-fold, with a median of 165-fold. The expression of 278 SEGs was found to vary ≤4-fold and these genes were significantly involved in protein catabolism (proteasome and ubiquitination), RNA transport, protein processing, and the spliceosome. Such stability of expression was further validated in human samples where the expression variability of the homologous human SEGs was significantly lower than that of other genes in the human genome. It was also found that the homologous human SEGs were generally less subject to non-synonymous mutation than other genes, as would be expected of stably expressed genes. We also found that knockout of SEG homologs in mouse models was more likely to cause complete preweaning lethality than non-SEG homologs, corroborating the fundamental roles played by SEGs in biological development. Such stably expressed genes and pathways across life-stages suggest that tight control of these processes is important in basic cellular functions and that perturbation by endogenous (e.g., genetics) or exogenous agents (e.g., drugs, environmental factors) may cause serious adverse effects. PMID:28125669

  16. Site-directed mutagenesis of IRX9, IRX9L and IRX14 proteins involved in xylan biosynthesis: glycosyltransferase activity is not required for IRX9 function in Arabidopsis.

    PubMed

    Ren, Yanfang; Hansen, Sara Fasmer; Ebert, Berit; Lau, Jane; Scheller, Henrik Vibe

    2014-01-01

    Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of β-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic

  17. Phospholipid liposomes functionalized by protein

    NASA Astrophysics Data System (ADS)

    Glukhova, O. E.; Savostyanov, G. V.; Grishina, O. A.

    2015-03-01

    Finding new ways to deliver neurotrophic drugs to the brain in newborns is one of the contemporary problems of medicine and pharmaceutical industry. Modern researches in this field indicate the promising prospects of supramolecular transport systems for targeted drug delivery to the brain which can overcome the blood-brain barrier (BBB). Thus, the solution of this problem is actual not only for medicine, but also for society as a whole because it determines the health of future generations. Phospholipid liposomes due to combination of lipo- and hydrophilic properties are considered as the main future objects in medicine for drug delivery through the BBB as well as increasing their bioavailability and toxicity. Liposomes functionalized by various proteins were used as transport systems for ease of liposomes use. Designing of modification oligosaccharide of liposomes surface is promising in the last decade because it enables the delivery of liposomes to specific receptor of human cells by selecting ligand and it is widely used in pharmacology for the treatment of several diseases. The purpose of this work is creation of a coarse-grained model of bilayer of phospholipid liposomes, functionalized by specific to the structural elements of the BBB proteins, as well as prediction of the most favorable orientation and position of the molecules in the generated complex by methods of molecular docking for the formation of the structure. Investigation of activity of the ligand molecule to protein receptor of human cells by the methods of molecular dynamics was carried out.

  18. Identification of the major lipoproteins in crayfish hemolymph as proteins involved in immune recognition and clotting.

    PubMed

    Hall, M; van Heusden, M C; Söderhäll, K

    1995-11-22

    Lipid-containing hemolymph proteins from males of the crayfish Pacifastacus leniusculus were isolated by density gradient ultracentrifugation. Two major lipoproteins, one high density lipoprotein (HDL) and one very high density lipoprotein (VHDL), were characterized. The HDL and the VHDL were found to be identical to two proteins previously studied for their roles in immune recognition and hemolymph clotting, namely the beta-1,3-glucan binding protein and the clotting protein. These results imply that crayfish lipoproteins have dual functions, and that they are involved in immunity, hemolymph clotting, and lipid transport in these animals. Also, the oxygen-transporting protein hemocyanin was found to have a small lipid content.

  19. Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. I. Impairment of resealing and formation of aqueous pores in the ghost membrane after modification of SH groups.

    PubMed

    Klonk, S; Deuticke, B

    1992-04-29

    Resealed human erythrocyte ghosts prepared by a two-step procedure were shown to have small residual barrier defects with the properties of aqueous pores, such as size discrimination of hydrophilic nonelectrolytes (erythritol to sucrose), indicative of an apparent pore radius of about 0.7 nm, and a low activation energy (about 12-20 kJ/mol (mannitol, sucrose)) of the leak fluxes. As in other cases (Deuticke et al. (1991) Biochim. Biophys. Acta 1067, 111-122) these leak fluxes can be inhibited by phloretin. Treatment of such resealed ghosts with the mild SH oxidizing agent, diamide, induces additional membrane leaks to the same extent and with the same properties as in native erythrocytes (Deuticke et al. (1983) Biochim. Biophys. Acta 731, 196-210), including reversibility of the leak by SH reducing agents, inhibition by phloretin and stimulation by alkanols. In contrast, resealed ghosts prepared either from diamide-treated erythrocytes or by adding diamide to the 'open' membranes prior to reconstitution of high ionic strength and raising the temperature, exhibit a state of greater leakiness. This leakiness is somewhat different in its origin from the former class of leaks, since it can also be produced by N-ethylmaleimide, which is essentially ineffective when added to the membrane in its 'tight' state. The leaks induced in the 'open' state of the membrane, which can be regarded as a consequence of an impaired resealing, are nevertheless reversible by reducing agents added after resealing and are comparable in many, but not all their characteristics to leaks induced in the 'tight' state of the membrane. Resealing in the presence of the isothiocyanostilbenes DIDS or SITS mimicks the leak forming effect of diamide by modifying a small population of SH groups, while amino groups seem not to be involved. The findings indicate and substantiate an important role of the redox state of membrane skeletal protein sulfhydryls in the maintenance and the re-establishment of the

  20. Proteins involved in vesicular transport and membrane fusion.

    PubMed

    Waters, M G; Griff, I C; Rothman, J E

    1991-08-01

    In the past year, new information about proteins involved in vesicular transport has been plentiful. Particularly noteworthy are the complementary findings that Sec17p is required for vesicle consumption in endoplasmic reticulum-to-Golgi transport in yeast and that an analogous activity in mammalian cells, termed SNAP, is required for transport from the cis to the medial cisternae of the Golgi apparatus.

  1. Functional involvement of protein kinase C, Rho-kinase and TRPC3 decreases while PLC increases with advancement of pregnancy in mediating oxytocin-induced myometrial contractions in water buffaloes (Bubalus bubalis).

    PubMed

    Sharma, Abhishek; Nakade, Udayraj P; Choudhury, Soumen; Garg, Satish Kumar

    2017-04-01

    Present study unravels the involvement of different calcium signaling pathways in oxytocin-induced contractions in myometrium of non-pregnant and pregnant buffaloes during early and mid-pregnancy stages. Uteri of pregnant animals were more sensitive than of non-pregnant buffaloes. Phasic contractions and frequency of contraction significantly increased with advancement of pregnancy, while tonic contractions non-significantly and amplitude significantly decreased from six months pregnancy onward. Oxytocin produced concentration-dependent-contraction on isolated myometrial strips of pregnant and non-pregnant buffaloes and the dose response curves (DRCs) of oxytocin were significantly (P < 0.05) shifted to right in the presence of nifedipine (1 μM), in Ca(2+)-free Ringer Locke solution (RLS), ruthenium red (30 μM), ruthenium red + nifedipine, cyclopiazonic acid (CPA; Ca(2+) free RLS as well as RLS), CPA (10 μM)+nifedipine, U-73122 (1 μM) + nifedipine and SKF96365 (25 μM) on uteri of non-pregnant and pregnant (early and mid) animals. The DRCs were also significantly shifted towards right in the presence of Y-27632 (10 μM), GF109203X (5 μM) and Pyr3 (10 μM) on uteri of non-pregnant and early pregnancy stage buffaloes while only in the presence of U-73122 (1 μM) on uteri of mid-pregnancy stage buffaloes. Our finding suggest that and L-type Ca(2+) channels, IP3-RyR-gated, and store-operated calcium channels including transient receptor potential channel (TRPC) pathways play significant role in mediating oxytocin-induced contractions in myometrium of pregnant and non-pregnant buffaloes. SERCA plays major role only during early-pregnancy while functional role of protein kinase C (PKC), Rho-kinase and TRPC3 pathways decreased and role of G-protein coupled receptor-phospholipase C (GPCR-PLC) pathway increased with advancement of pregnancy.

  2. Functional involvement of human discs large tumor suppressor in cytokinesis

    SciTech Connect

    Unno, Kenji; Hanada, Toshihiko; Chishti, Athar H.

    2008-10-15

    Cytokinesis is the final step of cell division that completes the separation of two daughter cells. We found that the human discs large (hDlg) tumor suppressor homologue is functionally involved in cytokinesis. The guanylate kinase (GUK) domain of hDlg mediates the localization of hDlg to the midbody during cytokinesis, and over-expression of the GUK domain in U2OS and HeLa cells impaired cytokinesis. Mouse embryonic fibroblasts (MEFs) derived from dlg mutant mice contained an increased number of multinucleated cells and showed reduced proliferation in culture. A kinesin-like motor protein, GAKIN, which binds directly to the GUK domain of hDlg, exhibited a similar intracellular distribution pattern with hDlg throughout mitosis and localized to the midbody during cytokinesis. However, the targeting of hDlg and GAKIN to the midbody appeared to be independent of each other. The midbody localization of GAKIN required its functional kinesin-motor domain. Treatment of cells with the siRNA specific for hDlg and GAKIN caused formation of multinucleated cells and delayed cytokinesis. Together, these results suggest that hDlg and GAKIN play functional roles in the maintenance of midbody architecture during cytokinesis.

  3. Alkylation damage by lipid electrophiles targets functional protein systems.

    PubMed

    Codreanu, Simona G; Ullery, Jody C; Zhu, Jing; Tallman, Keri A; Beavers, William N; Porter, Ned A; Marnett, Lawrence J; Zhang, Bing; Liebler, Daniel C

    2014-03-01

    Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions.

  4. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    PubMed Central

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  5. Monotonicity and Logarithmic Concavity of Two Functions Involving Exponential Function

    ERIC Educational Resources Information Center

    Liu, Ai-Qi; Li, Guo-Fu; Guo, Bai-Ni; Qi, Feng

    2008-01-01

    The function 1 divided by "x"[superscript 2] minus "e"[superscript"-x"] divided by (1 minus "e"[superscript"-x"])[superscript 2] for "x" greater than 0 is proved to be strictly decreasing. As an application of this monotonicity, the logarithmic concavity of the function "t" divided by "e"[superscript "at"] minus "e"[superscript"(a-1)""t"] for "a"…

  6. Heterogeneity in Retroviral Nucleocapsid Protein Function

    NASA Astrophysics Data System (ADS)

    Landes, Christy

    2009-03-01

    Time-resolved single-molecule fluorescence spectroscopy was used to study the human T-cell lymphotropic virus type 1 (HTLV-1) nucleocapsid protein (NC) chaperone activity as compared to that of the HIV-1 NC protein. HTLV-1 NC contains two zinc fingers with each having a CCHC binding motif similar to HIV-1 NC. HIV-1 NC is required for recognition and packaging of the viral RNA and is also a nucleic acid chaperone protein that facilitates nucleic acid restructuring during reverse transcription. Because of similarities in structures between the two retroviruses, we have used single-molecule fluorescence energy transfer to investigate the chaperoning activity of HTLV-1 NC protein. The results indicate that HTLV-1 NC protein induces structural changes by opening the transactivation response (TAR)-DNA hairpin to an even greater extent than HIV-1 NC. However, unlike HIV-1 NC, HTLV-1 NC does not chaperone the strand-transfer reaction involving TAR-DNA. These results suggest that despite its effective destabilization capability, HTLV-1 NC is not as effective at overall chaperone function as is its HIV-1 counterpart.

  7. An iterative approach of protein function prediction

    PubMed Central

    2011-01-01

    Background Current approaches of predicting protein functions from a protein-protein interaction (PPI) dataset are based on an assumption that the available functions of the proteins (a.k.a. annotated proteins) will determine the functions of the proteins whose functions are unknown yet at the moment (a.k.a. un-annotated proteins). Therefore, the protein function prediction is a mono-directed and one-off procedure, i.e. from annotated proteins to un-annotated proteins. However, the interactions between proteins are mutual rather than static and mono-directed, although functions of some proteins are unknown for some reasons at present. That means when we use the similarity-based approach to predict functions of un-annotated proteins, the un-annotated proteins, once their functions are predicted, will affect the similarities between proteins, which in turn will affect the prediction results. In other words, the function prediction is a dynamic and mutual procedure. This dynamic feature of protein interactions, however, was not considered in the existing prediction algorithms. Results In this paper, we propose a new prediction approach that predicts protein functions iteratively. This iterative approach incorporates the dynamic and mutual features of PPI interactions, as well as the local and global semantic influence of protein functions, into the prediction. To guarantee predicting functions iteratively, we propose a new protein similarity from protein functions. We adapt new evaluation metrics to evaluate the prediction quality of our algorithm and other similar algorithms. Experiments on real PPI datasets were conducted to evaluate the effectiveness of the proposed approach in predicting unknown protein functions. Conclusions The iterative approach is more likely to reflect the real biological nature between proteins when predicting functions. A proper definition of protein similarity from protein functions is the key to predicting functions iteratively. The

  8. Identifying Unstable Regions of Proteins Involved in Misfolding Diseases

    NASA Astrophysics Data System (ADS)

    Guest, Will; Cashman, Neil; Plotkin, Steven

    2009-05-01

    Protein misfolding is a necessary step in the pathogenesis of many diseases, including Creutzfeldt-Jakob disease (CJD) and familial amyotrophic lateral sclerosis (fALS). Identifying unstable structural elements in their causative proteins elucidates the early events of misfolding and presents targets for inhibition of the disease process. An algorithm was developed to calculate the Gibbs free energy of unfolding for all sequence-contiguous regions of a protein using three methods to parameterize energy changes: a modified G=o model, changes in solvent-accessible surface area, and all-atoms molecular dynamics. The entropic effects of disulfide bonds and post-translational modifications are treated analytically. It incorporates a novel method for finding local dielectric constants inside a protein to accurately handle charge effects. We have predicted the unstable parts of prion protein and superoxide dismutase 1, the proteins involved in CJD and fALS respectively, and have used these regions as epitopes to prepare antibodies that are specific to the misfolded conformation and show promise as therapeutic agents.

  9. First identification of proteins involved in motility of Mycoplasma gallisepticum.

    PubMed

    Indikova, Ivana; Vronka, Martin; Szostak, Michael P

    2014-10-17

    Mycoplasma gallisepticum, the most pathogenic mycoplasma in poultry, is able to glide over solid surfaces. Although this gliding motility was first observed in 1968, no specific protein has yet been shown to be involved in gliding. We examined M. gallisepticum strains and clonal variants for motility and found that the cytadherence proteins GapA and CrmA were required for gliding. Loss of GapA or CrmA resulted in the loss of motility and hemadsorption and led to drastic changes in the characteristic flask-shape of the cells. To identify further genes involved in motility, a transposon mutant library of M. gallisepticum was generated and screened for motility-deficient mutants, using a screening assay based on colony morphology. Motility-deficient mutants had transposon insertions in gapA and the neighbouring downstream gene crmA. In addition, insertions were seen in gene mgc2, immediately upstream of gapA, in two motility-deficient mutants. In contrast to the GapA/CrmA mutants, the mgc2 motility mutants still possessed the ability to hemadsorb. Complementation of these mutants with a mgc2-hexahistidine fusion gene restored the motile phenotype. This is the first report assigning specific M. gallisepticum proteins to involvement in gliding motility.

  10. [Extranuclear functions of protein sumoylation in the central nervous system].

    PubMed

    Martin, Stéphane

    2009-01-01

    Post-translational protein modifications play essential roles in many aspects of cellular functions and therefore in the maintenance of cell integrity. These protein modifications are involved at all stages of neuronal communication within the central nervous system. Sumoylation is a reversible post-translational protein modification that consists in the covalent labelling of a small protein called SUMO to lysine residues of selected target proteins. Sumoylation is a well characterized regulator of nuclear functions and has recently emerged as a key factor for numerous extranuclear processes. Furthermore, sumoylation has recently been shown to modulate synaptic transmission and is also implicated in a wide range of neurodegenerative diseases.

  11. The VHL short variant involves in protein quality control.

    PubMed

    Liu, Yanbin; Yang, Haixia; Zuo, Feifei; Chen, Liang

    2016-09-01

    The von Hippel-Lindau (VHL) is the most important and frequently mutated gene in human clear cell renal cell carcinoma (ccRCC). In contrast to its long counterpart, the internal translational variant of VHL protein (VHLs) is evolutionarily conserved. Herein we present evidence that VHLs associates with ribosome complex via interaction with the large subunit 6 (RPL6). Manipulation of VHLs expression significantly alters protein synthesis, cell size and mitochondrial mass. VHLs deficiency leads to remarkable sensitivity to drug treatments eliciting nascent protein mis-folding and translational errors. The ubiquitination of nascent peptides are dramatically increased upon the ectopic over-expression of VHLs, which simultaneously co-localizes with proteasome and thus may facilitate the ubiquitin-proteasome mediated degradation. In summary, VHLs contributes to protein quality control in addition to its canonical function in maintaining homeostasis of hypoxia-induced factors alpha subunit (HIFα) in response to environmental oxygen supply.

  12. Protein function annotation using protein domain family resources.

    PubMed

    Das, Sayoni; Orengo, Christine A

    2016-01-15

    As a result of the genome sequencing and structural genomics initiatives, we have a wealth of protein sequence and structural data. However, only about 1% of these proteins have experimental functional annotations. As a result, computational approaches that can predict protein functions are essential in bridging this widening annotation gap. This article reviews the current approaches of protein function prediction using structure and sequence based classification of protein domain family resources with a special focus on functional families in the CATH-Gene3D resource.

  13. Proteomic detection of proteins involved in perchlorate and chlorate metabolism.

    PubMed

    Bansal, Reema; Deobald, Lee A; Crawford, Ronald L; Paszczynski, Andrzej J

    2009-09-01

    Mass spectrometry and a time-course cell lysis method were used to study proteins involved in perchlorate and chlorate metabolism in pure bacterial cultures and environmental samples. The bacterial cultures used included Dechlorosoma sp. KJ, Dechloromonas hortensis, Pseudomonas chloritidismutans ASK-1, and Pseudomonas stutzeri. The environmental samples included an anaerobic sludge enrichment culture from a sewage treatment plant, a sample of a biomass-covered activated carbon matrix from a bioreactor used for treating perchlorate-contaminated drinking water, and a waste water effluent sample from a paper mill. The approach focused on detection of perchlorate (and chlorate) reductase and chlorite dismutase proteins, which are the two central enzymes in the perchlorate (or chlorate) reduction pathways. In addition, acetate-metabolizing enzymes in pure bacterial samples and housekeeping proteins from perchlorate (or chlorate)-reducing microorganisms in environmental samples were also identified.

  14. Bap, a Staphylococcus aureus Surface Protein Involved in Biofilm Formation

    PubMed Central

    Cucarella, Carme; Solano, Cristina; Valle, Jaione; Amorena, Beatriz; Lasa, Íñigo; Penadés, José R.

    2001-01-01

    Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection. PMID:11292810

  15. [Proteins of human milk involved in immunological processes].

    PubMed

    Lis, Jolanta; Orczyk-Pawiłowicz, Magdalena; Kątnik-Prastowska, Iwona

    2013-05-31

    Human milk contains a lot of components (i.e. proteins, carbohydrates, lipids, inorganic elements) which provide basic nutrients for infants during the first period of their lives. Qualitative composition of milk components of healthy mothers is similar, but their levels change during lactation stages. Colostrum is the fluid secreted during the first days postpartum by mammary epithelial cells. Colostrum is replaced by transitional milk during 5-15 days postpartum and from 15 days postpartum mature milk is produced. Human milk, apart from nutritional components, is a source of biologically active molecules, i.e. immunoglobulins, growth factors, cytokines, acute phase proteins, antiviral and antibacterial proteins. Such components of human milk are responsible for specific biological activities of human milk. This secretion plays an important role in growth and development of newborns. Bioactive molecules present in the milk support the immature immune system of the newborn and also protect against the development of infection. In this article we describe the pathways involved in the production and secretion of human milk, the state of knowledge on the proteome of human milk, and the contents of components of milk during lactation. Moreover, some growth factors and proteins involved in innate and specific immunity, intercellular communication, immunomodulation, and inflammatory processes have been characterized.

  16. Identification of Protein Interactions Involved in Cellular Signaling

    PubMed Central

    Westermarck, Jukka; Ivaska, Johanna; Corthals, Garry L.

    2013-01-01

    Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes. PMID:23481661

  17. Functional assignment to JEV proteins using SVM

    PubMed Central

    Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

    2008-01-01

    Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP). PMID:19052658

  18. Analysis of proteins involved in biodegradation of crop biomass

    NASA Technical Reports Server (NTRS)

    Crawford, Kamau; Trotman, Audrey

    1998-01-01

    The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

  19. Neuron membrane trafficking and protein kinases involved in autism and ADHD.

    PubMed

    Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

    2015-01-30

    A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  20. A human skeletal muscle interactome centered on proteins involved in muscular dystrophies: LGMD interactome

    PubMed Central

    2013-01-01

    Background The complexity of the skeletal muscle and the identification of numerous human disease-causing mutations in its constitutive proteins make it an interesting tissue for proteomic studies aimed at understanding functional relationships of interacting proteins in both health and diseases. Method We undertook a large-scale study using two-hybrid screens and a human skeletal-muscle cDNA library to establish a proteome-scale map of protein-protein interactions centered on proteins involved in limb-girdle muscular dystrophies (LGMD). LGMD is a group of more than 20 different neuromuscular disorders that principally affect the proximal pelvic and shoulder girdle muscles. Results and conclusion The interaction network we unraveled incorporates 1018 proteins connected by 1492 direct binary interactions and includes 1420 novel protein-protein interactions. Computational, experimental and literature-based analyses were performed to assess the overall quality of this network. Interestingly, LGMD proteins were shown to be highly interconnected, in particular indirectly through sarcomeric proteins. In-depth mining of the LGMD-centered interactome identified new candidate genes for orphan LGMDs and other neuromuscular disorders. The data also suggest the existence of functional links between LGMD2B/dysferlin and gene regulation, between LGMD2C/γ-sarcoglycan and energy control and between LGMD2G/telethonin and maintenance of genome integrity. This dataset represents a valuable resource for future functional investigations. PMID:23414517

  1. Functional Foods Containing Whey Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Whey proteins, modified whey proteins, and whey components are useful as nutrients or supplements for health maintenance. Extrusion modified whey proteins can easily fit into new products such as beverages, confectionery items (e.g., candies), convenience foods, desserts, baked goods, sauces, and in...

  2. Identification of an additional protein involved in mannan biosynthesis

    PubMed Central

    Wang, Yan; Mortimer, Jennifer C; Davis, Jonathan; Dupree, Paul; Keegstra, Kenneth

    2013-01-01

    Galactomannans comprise a β-1,4-mannan backbone substituted with α-1,6-galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side-chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum-graecum L.) seed endosperm, which accumulates large quantities of galactomannans as a reserve carbohydrate during seed development. One of the candidate genes encodes a protein that is likely to be a glycosyltransferase. Because this protein is involved in mannan biosynthesis, we named it ‘mannan synthesis-related’ (MSR). Here, we report the characterization of a fenugreek MSR gene (TfMSR) and its two Arabidopsis homologs, AtMSR1 and AtMSR2. TfMSR was highly and specifically expressed in the endosperm. TfMSR, AtMSR1 and AtMSR2 proteins were all determined to be localized to the Golgi by fluorescence confocal microscopy. The level of mannosyl residues in stem glucomannans decreased by approximately 40% for Arabidopsis msr1 single T-DNA insertion mutants and by more than 50% for msr1 msr2 double mutants, but remained unchanged for msr2 single mutants. In addition, in vitro mannan synthase activity from the stems of msr1 single and msr1 msr2 double mutants also decreased. Expression of AtMSR1 or AtMSR2 in the msr1 msr2 double mutant completely or partially restored mannosyl levels. From these results, we conclude that the MSR protein is important for mannan biosynthesis, and offer some ideas about its role. PMID:22966747

  3. The crystal structure of the thiocyanate-forming protein from Thlaspi arvense, a kelch protein involved in glucosinolate breakdown.

    PubMed

    Gumz, Frauke; Krausze, Joern; Eisenschmidt, Daniela; Backenköhler, Anita; Barleben, Leif; Brandt, Wolfgang; Wittstock, Ute

    2015-09-01

    Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade β-propeller with a wide "top" and a narrower "bottom" opening with distinct strand-connecting loops protruding far beyond the lower propeller surface. Molecular modeling and mutational analysis identified residues for glucosinolate aglucone and Fe(2+) cofactor binding within these loops. As the first experimentally determined structure of a plant kelch protein, the crystal structure of TaTFP not only enables more detailed mechanistic studies on glucosinolate breakdown product formation, but also provides a new basis for research on the diverse roles and mechanisms of other kelch proteins in plants.

  4. A new protein structure representation for efficient protein function prediction.

    PubMed

    Maghawry, Huda A; Mostafa, Mostafa G M; Gharib, Tarek F

    2014-12-01

    One of the challenging problems in bioinformatics is the prediction of protein function. Protein function is the main key that can be used to classify different proteins. Protein function can be inferred experimentally with very small throughput or computationally with very high throughput. Computational methods are sequence based or structure based. Structure-based methods produce more accurate protein function prediction. In this article, we propose a new protein structure representation for efficient protein function prediction. The representation is based on three-dimensional patterns of protein residues. In the analysis, we used protein function based on enzyme activity through six mechanistically diverse enzyme superfamilies: amidohydrolase, crotonase, haloacid dehalogenase, isoprenoid synthase type I, and vicinal oxygen chelate. We applied three different classification methods, naïve Bayes, k-nearest neighbors, and random forest, to predict the enzyme superfamily of a given protein. The prediction accuracy using the proposed representation outperforms a recently introduced representation method that is based only on the distance patterns. The results show that the proposed representation achieved prediction accuracy up to 98%, with improvement of about 10% on average.

  5. Protein Function Prediction: Problems and Pitfalls.

    PubMed

    Pearson, William R

    2015-09-03

    The characterization of new genomes based on their protein sets has been revolutionized by new sequencing technologies, but biologists seeking to exploit new sequence information are often frustrated by the challenges associated with accurately assigning biological functions to newly identified proteins. Here, we highlight some of the challenges in functional inference from sequence similarity. Investigators can improve the accuracy of function prediction by (1) being conservative about the evolutionary distance to a protein of known function; (2) considering the ambiguous meaning of "functional similarity," and (3) being aware of the limitations of annotations in functional databases. Protein function prediction does not offer "one-size-fits-all" solutions. Prediction strategies work better when the idiosyncrasies of function and functional annotation are better understood.

  6. Exploring Mouse Protein Function via Multiple Approaches

    PubMed Central

    Huang, Tao; Kong, Xiangyin; Zhang, Yunhua; Zhang, Ning

    2016-01-01

    Although the number of available protein sequences is growing exponentially, functional protein annotations lag far behind. Therefore, accurate identification of protein functions remains one of the major challenges in molecular biology. In this study, we presented a novel approach to predict mouse protein functions. The approach was a sequential combination of a similarity-based approach, an interaction-based approach and a pseudo amino acid composition-based approach. The method achieved an accuracy of about 0.8450 for the 1st-order predictions in the leave-one-out and ten-fold cross-validations. For the results yielded by the leave-one-out cross-validation, although the similarity-based approach alone achieved an accuracy of 0.8756, it was unable to predict the functions of proteins with no homologues. Comparatively, the pseudo amino acid composition-based approach alone reached an accuracy of 0.6786. Although the accuracy was lower than that of the previous approach, it could predict the functions of almost all proteins, even proteins with no homologues. Therefore, the combined method balanced the advantages and disadvantages of both approaches to achieve efficient performance. Furthermore, the results yielded by the ten-fold cross-validation indicate that the combined method is still effective and stable when there are no close homologs are available. However, the accuracy of the predicted functions can only be determined according to known protein functions based on current knowledge. Many protein functions remain unknown. By exploring the functions of proteins for which the 1st-order predicted functions are wrong but the 2nd-order predicted functions are correct, the 1st-order wrongly predicted functions were shown to be closely associated with the genes encoding the proteins. The so-called wrongly predicted functions could also potentially be correct upon future experimental verification. Therefore, the accuracy of the presented method may be much higher in

  7. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

    PubMed

    Dong, Jinlan; Bruening, Merlin L

    2015-01-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  8. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  9. Computations involving differential operators and their actions on functions

    NASA Technical Reports Server (NTRS)

    Crouch, Peter E.; Grossman, Robert; Larson, Richard

    1991-01-01

    The algorithms derived by Grossmann and Larson (1989) are further developed for rewriting expressions involving differential operators. The differential operators involved arise in the local analysis of nonlinear dynamical systems. These algorithms are extended in two different directions: the algorithms are generalized so that they apply to differential operators on groups and the data structures and algorithms are developed to compute symbolically the action of differential operators on functions. Both of these generalizations are needed for applications.

  10. FRG1P-mediated aggregation of proteins involved in pre-mRNA processing.

    PubMed

    van Koningsbruggen, Silvana; Straasheijm, Kirsten R; Sterrenburg, Ellen; de Graaf, Natascha; Dauwerse, Hans G; Frants, Rune R; van der Maarel, Silvère M

    2007-02-01

    FRG1 is considered a candidate gene for facioscapulohumeral muscular dystrophy (FSHD) based on its location at chromosome 4qter and its upregulation in FSHD muscle. The FRG1 protein (FRG1P) localizes to nucleoli, Cajal bodies (and speckles), and has been suggested to be a component of the human spliceosome but its exact function is unknown. Recently, transgenic mice overexpressing high levels of FRG1P in skeletal muscle were described to present with muscular dystrophy. Moreover, upregulation of FRG1P was demonstrated to correlate with missplicing of specific pre-mRNAs. In this study, we have combined colocalization studies with yeast two-hybrid screens to identify proteins that associate with FRG1P. We demonstrate that artificially induced nucleolar aggregates of VSV-FRG1P specifically sequester proteins involved in pre-mRNA processing. In addition, we have identified SMN, PABPN1, and FAM71B, a novel speckle and Cajal body protein, as binding partners of FRG1P. All these proteins are, or seem to be, involved in RNA biogenesis. Our data confirm the presence of FRG1P in protein complexes containing human spliceosomes and support a potential role of FRG1P in either splicing or another step in nuclear RNA biogenesis. Intriguingly, among FRG1P-associated proteins are SMN and PABPN1, both being involved in neuromuscular disorders, possibly through RNA biogenesis-related processes.

  11. Origins of Protein Functions in Cells

    NASA Technical Reports Server (NTRS)

    Seelig, Burchard; Pohorille, Andrzej

    2011-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis and in vitro evolution of very large libraries of random amino acid sequences. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions yet, important clues have been uncovered. In one example (Keefe and Szostak, 2001), novel ATP binding proteins were identified that appear to be unrelated in both sequence and structure to any known ATP binding proteins. One of these proteins was subsequently redesigned computationally to bind GTP through introducing several mutations that introduce targeted structural changes to the protein, improve its binding to guanine and prevent water from accessing the active center. This study facilitates further investigations of individual evolutionary steps that lead to a change of function in primordial proteins. In a second study (Seelig and Szostak, 2007), novel enzymes were generated that can join two pieces of RNA in a reaction for which no natural enzymes are known

  12. A Cellulose Synthase-Like Protein Involved in Hyphal Tip Growth and Morphological Differentiation in Streptomyces▿

    PubMed Central

    Xu, Hongbin; Chater, Keith F.; Deng, Zixin; Tao, Meifeng

    2008-01-01

    Cellulose synthase and cellulose synthase-like proteins, responsible for synthesizing β-glucan-containing polysaccharides, play a fundamental role in cellular architectures, such as plant cell and tissue morphogenesis, bacterial biofilm formation, and fruiting-body development. However, the roles of the proteins involved in the developmental process are not well understood. Here, we report that a cellulose synthase-like protein (CslASc) in Streptomyces has a function in hyphal tip growth and morphological differentiation. The cslASc replacement mutant showed pleiotropic defects, including the severe delay of aerial-hyphal formation and altered cell wall morphology. Calcofluor white fluorescence analysis demonstrated that polysaccharide synthesis at hyphal tips was dependent on CslASc. cslASc was constitutively transcribed, and an enhanced green fluorescent protein-CslASc fusion protein was mostly located at the hyphal tips. An extract enriched in morphogenetic chaplin proteins promoted formation of aerial hyphae by the mutant. Furthermore, a two-hybrid experiment indicated that the glycosyltransferase domain of CslASc interacted with the tropomyosin-like polarity-determining DivIVA protein, suggesting that the tip-located DivIVA governed tip recruitment of the CslASc membrane protein. These results imply that the cellulose synthase-like protein couples extracellular and cytoskeletal components functioning in tip growth and cell development. PMID:18487344

  13. 3DSwap: curated knowledgebase of proteins involved in 3D domain swapping.

    PubMed

    Shameer, Khader; Shingate, Prashant N; Manjunath, S C P; Karthika, M; Pugalenthi, Ganesan; Sowdhamini, Ramanathan

    2011-01-01

    Three-dimensional domain swapping is a unique protein structural phenomenon where two or more protein chains in a protein oligomer share a common structural segment between individual chains. This phenomenon is observed in an array of protein structures in oligomeric conformation. Protein structures in swapped conformations perform diverse functional roles and are also associated with deposition diseases in humans. We have performed in-depth literature curation and structural bioinformatics analyses to develop an integrated knowledgebase of proteins involved in 3D domain swapping. The hallmark of 3D domain swapping is the presence of distinct structural segments such as the hinge and swapped regions. We have curated the literature to delineate the boundaries of these regions. In addition, we have defined several new concepts like 'secondary major interface' to represent the interface properties arising as a result of 3D domain swapping, and a new quantitative measure for the 'extent of swapping' in structures. The catalog of proteins reported in 3DSwap knowledgebase has been generated using an integrated structural bioinformatics workflow of database searches, literature curation, by structure visualization and sequence-structure-function analyses. The current version of the 3DSwap knowledgebase reports 293 protein structures, the analysis of such a compendium of protein structures will further the understanding molecular factors driving 3D domain swapping.

  14. Detection of Functional Modes in Protein Dynamics

    PubMed Central

    Hub, Jochen S.; de Groot, Bert L.

    2009-01-01

    Proteins frequently accomplish their biological function by collective atomic motions. Yet the identification of collective motions related to a specific protein function from, e.g., a molecular dynamics trajectory is often non-trivial. Here, we propose a novel technique termed “functional mode analysis” that aims to detect the collective motion that is directly related to a particular protein function. Based on an ensemble of structures, together with an arbitrary “functional quantity” that quantifies the functional state of the protein, the technique detects the collective motion that is maximally correlated to the functional quantity. The functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. In addition, the motion that displays the largest likelihood to induce a substantial change in the functional quantity is estimated from the given protein ensemble. Two different correlation measures are applied: first, the Pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. Detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. The new approach is illustrated using a number of biomolecules, including a polyalanine-helix, T4 lysozyme, Trp-cage, and leucine-binding protein. PMID:19714202

  15. Characterization and Functionality of Corn Germ Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to evaluate the functional properties of protein extracted from wet-milled corn germ and identify potential applications of the recovered protein. Corn germ comprises 12% of the total weight of normal dent corn and about 29% of the corn protein (moisture-free and oil- free ...

  16. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis1

    PubMed Central

    Pesaresi, Paolo; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Rothbart, Maxi; Hedtke, Boris

    2016-01-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes. PMID:26823545

  17. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    PubMed

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes.

  18. The EutQ and EutP Proteins are Novel Acetate Kinases Involved in Ethanolamine Catabolism: Physiological Implications for the Function of the Ethanolamine Metabolosome in Salmonella enterica

    PubMed Central

    Moore, Theodore C.; Escalante-Semerena, Jorge C.

    2016-01-01

    Summary Salmonella enterica catabolizes ethanolamine inside a compartment known as the metabolosome. The ethanolamine utilization (eut) operon of this bacterium encodes all functions needed for the assembly and function of this structure. To date, the roles of the EutQ and EutP were not known. Herein we show that both proteins have acetate kinase activity, and that EutQ is required during anoxic growth of S. enterica on ethanolamine and tetrathionate. EutP and EutQ-dependent ATP synthesis occurred when enzymes were incubated with ADP, Mg(II) ions and acetyl-phosphate. EutQ and EutP also synthesized acetyl-phosphate from ATP and acetate. Although EutP had acetate kinase activity, ΔeutP strains lacked discernable phenotypes under the conditions where ΔeutQ strains displayed clear phenotypes. The kinetic parameters indicate that EutP is a faster enzyme than EutQ. Our evidence supports the conclusions that EutQ and EutP represent novel classes of acetate kinases. We propose that EutQ is necessary to drive flux through the pathway under physiological conditions, preventing a buildup of acetaldehyde. We also suggest that ATP generated by these enzymes may be used as a substrate for EutT, the ATP-dependent corrinoid adenosyltransferase, and for the EutA ethanolamine ammonia-lyase reactivase. PMID:26448059

  19. Children on the Autism Spectrum: Grandmother Involvement and Family Functioning

    ERIC Educational Resources Information Center

    Sullivan, Alison; Winograd, Greta; Verkuilen, Jay; Fish, Marian C.

    2012-01-01

    Background: This study investigated associations between the presence of a child with autism or Asperger's disorder in the family, family functioning and grandmother experiences with the goal of better understanding grandparent involvement in the lives of grandchildren on the autism spectrum and their families. Methods: Mothers and grandmothers of…

  20. Protein function prediction based on data fusion and functional interrelationship.

    PubMed

    Meng, Jun; Wekesa, Jael-Sanyanda; Shi, Guan-Li; Luan, Yu-Shi

    2016-04-01

    One of the challenging tasks of bioinformatics is to predict more accurate and confident protein functions from genomics and proteomics datasets. Computational approaches use a variety of high throughput experimental data, such as protein-protein interaction (PPI), protein sequences and phylogenetic profiles, to predict protein functions. This paper presents a method that uses transductive multi-label learning algorithm by integrating multiple data sources for classification. Multiple proteomics datasets are integrated to make inferences about functions of unknown proteins and use a directed bi-relational graph to assign labels to unannotated proteins. Our method, bi-relational graph based transductive multi-label function annotation (Bi-TMF) uses functional correlation and topological PPI network properties on both the training and testing datasets to predict protein functions through data fusion of the individual kernel result. The main purpose of our proposed method is to enhance the performance of classifier integration for protein function prediction algorithms. Experimental results demonstrate the effectiveness and efficiency of Bi-TMF on multi-sources datasets in yeast, human and mouse benchmarks. Bi-TMF outperforms other recently proposed methods.

  1. Multiple proteins of White spot syndrome virus involved in recognition of beta-integrin.

    PubMed

    Zhang, Jing-Yan; Liu, Qing-Hui; Huang, Jie

    2014-06-01

    The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that beta-integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of beta-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed that envelope proteins VP26, VP31, VP37, VP90 and nucleocapsid protein VP136 interacted with LvInt. RGD-, YGL- and LDV-related peptide functioned as motifs of WSSV proteins binding with beta-integrin. The beta-integrin ligand of RGDT had better blocking effect compared with that of YGL- and LDV-related peptides. In vivo assay indicated that RGD-, LDV- and YGL-related peptides could partially block WSSV infection. These data collectively indicate that multiple proteins were involved in recognition of beta-integrin. Identification of proteins in WSSV that are associated with beta-integrin will assist development of new agents for effective control of the white spot syndrome.

  2. Arabinogalactan proteins are involved in root hair development in barley

    PubMed Central

    Marzec, Marek; Szarejko, Iwona; Melzer, Michael

    2015-01-01

    The arabinogalactan proteins (AGPs) are involved in a range of plant processes, including cell differentiation and expansion. Here, barley root hair mutants and their wild-type parent cultivars were used, as a model system, to reveal the role of AGPs in root hair development. The treatment of roots with different concentrations of βGlcY (a reagent which binds to all classes of AGPs) inhibited or totally suppressed the development of root hairs in all of the cultivars. Three groups of AGP (recognized by the monoclonal antibodies LM2, LM14, and MAC207) were diversely localized in trichoblasts and atrichoblasts of root hair-producing plants. The relevant epitopes were present in wild-type trichoblast cell walls and cytoplasm, whereas in wild-type atrichoblasts and in all epidermal cells of a root hairless mutant, they were only present in the cytoplasm. In all of cultivars the higher expression of LM2, LM14, and MAC207 was observed in trichoblasts at an early stage of development. Additionally, the LM2 epitope was detected on the surface of primordia and root hair tubes in plants able to generate root hairs. The major conclusion was that the AGPs recognized by LM2, LM14, and MAC207 are involved in the differentiation of barley root epidermal cells, thereby implying a requirement for these AGPs for root hair development in barley. PMID:25465033

  3. J domain independent functions of J proteins.

    PubMed

    Ajit Tamadaddi, Chetana; Sahi, Chandan

    2016-07-01

    Heat shock proteins of 40 kDa (Hsp40s), also called J proteins, are obligate partners of Hsp70s. Via their highly conserved and functionally critical J domain, J proteins interact and modulate the activity of their Hsp70 partners. Mutations in the critical residues in the J domain often result in the null phenotype for the J protein in question. However, as more J proteins have been characterized, it is becoming increasingly clear that a significant number of J proteins do not "completely" rely on their J domains to carry out their cellular functions, as previously thought. In some cases, regions outside the highly conserved J domain have become more important making the J domain dispensable for some, if not for all functions of a J protein. This has profound effects on the evolution of such J proteins. Here we present selected examples of J proteins that perform J domain independent functions and discuss this in the context of evolution of J proteins with dispensable J domains and J-like proteins in eukaryotes.

  4. Function and regulation of Rnd proteins.

    PubMed

    Chardin, Pierre

    2006-01-01

    The Rnd proteins, which form a distinct sub-group of the Rho family of small GTP-binding proteins, have been shown to regulate the organization of the actin cytoskeleton in several tissues. In the brain, they participate in neurite extension, whereas in smooth muscle, they modulate contractility. Recent evidence has shown that Rnd3 (RhoE) is also involved in the regulation of cell-cycle progression and transformation, indicating that these proteins might have other, as yet unexplored roles.

  5. Functions of red cell surface proteins.

    PubMed

    Daniels, G

    2007-11-01

    The external membrane of the red cell contains numerous proteins that either cross the lipid bilayer one or more times or are anchored to it through a lipid tail. Many of these proteins express blood group activity. The functions of some of these proteins are known; in others their function can only be surmised from the protein structure or from limited experimental evidence. They are loosely divided into four categories based on their functions: membrane transporters; adhesion molecules and receptors; enzymes; and structural proteins that link the membrane with the membrane skeleton. Some of the proteins carry out more than one of these functions. Some proteins may complete their major functions during erythropoiesis or may only be important under adverse physiological conditions. Furthermore, some might be evolutionary relics and may no longer have significant functions. Polymorphisms or rare changes in red cell surface proteins are often responsible for blood groups. The biological significance of these polymorphisms or the selective pressures responsible for their stability within populations are mostly not known, although exploitation of the proteins by pathogenic micro-organisms has probably played a major role.

  6. STRIPAK complexes: structure, biological function, and involvement in human diseases.

    PubMed

    Hwang, Juyeon; Pallas, David C

    2014-02-01

    The mammalian striatin family consists of three proteins, striatin, S/G2 nuclear autoantigen, and zinedin. Striatin family members have no intrinsic catalytic activity, but rather function as scaffolding proteins. Remarkably, they organize multiple diverse, large signaling complexes that participate in a variety of cellular processes. Moreover, they appear to be regulatory/targeting subunits for the major eukaryotic serine/threonine protein phosphatase 2A. In addition, striatin family members associate with germinal center kinase III kinases as well as other novel components, earning these assemblies the name striatin-interacting phosphatase and kinase (STRIPAK) complexes. Recently, there has been a great increase in functional and mechanistic studies aimed at identifying and understanding the roles of STRIPAK and STRIPAK-like complexes in cellular processes of multiple organisms. These studies have identified novel STRIPAK and STRIPAK-like complexes and have explored their roles in specific signaling pathways. Together, the results of these studies have sparked increased interest in striatin family complexes because they have revealed roles in signaling, cell cycle control, apoptosis, vesicular trafficking, Golgi assembly, cell polarity, cell migration, neural and vascular development, and cardiac function. Moreover, STRIPAK complexes have been connected to clinical conditions, including cardiac disease, diabetes, autism, and cerebral cavernous malformation. In this review, we discuss the expression, localization, and protein domain structure of striatin family members. Then we consider the diverse complexes these proteins and their homologs form in various organisms, emphasizing what is known regarding function and regulation. Finally, we explore possible roles of striatin family complexes in disease, especially cerebral cavernous malformation.

  7. The Arabidopsis PLAT domain protein1 is critically involved in abiotic stress tolerance.

    PubMed

    Hyun, Tae Kyung; van der Graaff, Eric; Albacete, Alfonso; Eom, Seung Hee; Großkinsky, Dominik K; Böhm, Hannah; Janschek, Ursula; Rim, Yeonggil; Ali, Walid Wahid; Kim, Soo Young; Roitsch, Thomas

    2014-01-01

    Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.

  8. The TSG101 protein binds to connexins and is involved in connexin degradation

    SciTech Connect

    Auth, Tanja Schlueter, Sharazad; Urschel, Stephanie; Kussmann, Petra; Sonntag, Stephan; Hoeher, Thorsten; Kreuzberg, Maria M.; Dobrowolski, Radoslaw; Willecke, Klaus

    2009-04-01

    Gap junctions mediate electrical and metabolic communication between cells in almost all tissues and are proposed to play important roles in cellular growth control, differentiation and embryonic development. Gap junctional communication and channel assembly were suggested to be regulated by interaction of connexins with different proteins including kinases and phosphatases. Here, we identified the tumor susceptibility gene 101 (TSG101) protein to bind to the carboxyterminal tail of connexin45 in a yeast two-hybrid protein interaction screen. Glutathione S-transferase pull down experiments and immunoprecipitation revealed that not only connexin45 but also connexin30.2, -36, and -43 carboxyterminal regions were associated with TSG101 protein in pull down analyses and that connexin31, -43 and -45 co-precipitate with endogenous TSG101 protein in lysates from HM1 embryonic stem cells. TSG101 has been shown to be involved in cell cycle control, transcriptional regulation and turnover of endocytosed proteins. Thus, we decided to study the functional role of this interaction. SiRNA mediated knock down of TSG101 in HM1 embryonic stem cells led to increased levels of connexin43 and -45, prolonged half life of these connexins and increased transfer of microinjected Lucifer yellow. Our results suggest that TSG101 is involved in the degradation of connexins via interaction with connexin proteins.

  9. A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae.

    PubMed

    Tomberg, Joshua; Temple, Brenda; Fedarovich, Alena; Davies, Christopher; Nicholas, Robert A

    2012-04-03

    Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the β2a-β2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the β2a-β2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by β-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.

  10. Phylointeractomics reconstructs functional evolution of protein binding

    PubMed Central

    Kappei, Dennis; Scheibe, Marion; Paszkowski-Rogacz, Maciej; Bluhm, Alina; Gossmann, Toni Ingolf; Dietz, Sabrina; Dejung, Mario; Herlyn, Holger; Buchholz, Frank; Mann, Matthias; Butter, Falk

    2017-01-01

    Molecular phylogenomics investigates evolutionary relationships based on genomic data. However, despite genomic sequence conservation, changes in protein interactions can occur relatively rapidly and may cause strong functional diversification. To investigate such functional evolution, we here combine phylogenomics with interaction proteomics. We develop this concept by investigating the molecular evolution of the shelterin complex, which protects telomeres, across 16 vertebrate species from zebrafish to humans covering 450 million years of evolution. Our phylointeractomics screen discovers previously unknown telomere-associated proteins and reveals how homologous proteins undergo functional evolution. For instance, we show that TERF1 evolved as a telomere-binding protein in the common stem lineage of marsupial and placental mammals. Phylointeractomics is a versatile and scalable approach to investigate evolutionary changes in protein function and thus can provide experimental evidence for phylogenomic relationships. PMID:28176777

  11. What induces pocket openings on protein surface patches involved in protein-protein interactions?

    NASA Astrophysics Data System (ADS)

    Eyrisch, Susanne; Helms, Volkhard

    2009-02-01

    We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

  12. Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation

    PubMed Central

    1991-01-01

    A novel protein has been identified which may serve a key function in nucleating spindle microtubule growth in mitosis. This protein, called centrophilin, is sequentially relocated from the centromeres to the centrosomes to the midbody in a manner dependent on the mitotic phase. Centrophilin was initially detected by immunofluorescence with a monoclonal, primate-specific antibody (2D3) raised against kinetochore- enriched chromosome extract from HeLa cells (Valdivia, M. M., and B. R. Brinkley. 1985. J. Cell Biol. 101:1124-1134). Centrophilin forms prominent crescents at the poles of the metaphase spindle, gradually diminishes during anaphase, and bands the equatorial ends of midbody microtubules in telophase. The formation and breakdown of the spindle and midbody correlates in time and space with the aggregation and disaggregation of centrophilin foci. Immunogold EM reveals that centrophilin is a major component of pericentriolar material in metaphase. During recovery from microtubule inhibition, centrophilin foci act as nucleation sites for the assembly of spindle tubules. The 2D3 probe recognizes two high molecular mass polypeptides, 180 and 210 kD, on immunoblots of whole HeLa cell extract. Taken together, these data and the available literature on microtubule dynamics point inevitably to a singular model for control of spindle tubule turnover. PMID:1991791

  13. Predicting protein functions from redundancies in large-scale protein interaction networks

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj Pratim; Liang, Shoudan

    2003-01-01

    Interpreting data from large-scale protein interaction experiments has been a challenging task because of the widespread presence of random false positives. Here, we present a network-based statistical algorithm that overcomes this difficulty and allows us to derive functions of unannotated proteins from large-scale interaction data. Our algorithm uses the insight that if two proteins share significantly larger number of common interaction partners than random, they have close functional associations. Analysis of publicly available data from Saccharomyces cerevisiae reveals >2,800 reliable functional associations, 29% of which involve at least one unannotated protein. By further analyzing these associations, we derive tentative functions for 81 unannotated proteins with high certainty. Our method is not overly sensitive to the false positives present in the data. Even after adding 50% randomly generated interactions to the measured data set, we are able to recover almost all (approximately 89%) of the original associations.

  14. Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair

    PubMed Central

    Alcalay, Myriam; Meani, Natalia; Gelmetti, Vania; Fantozzi, Anna; Fagioli, Marta; Orleth, Annette; Riganelli, Daniela; Sebastiani, Carla; Cappelli, Enrico; Casciari, Cristina; Sciurpi, Maria Teresa; Mariano, Angela Rosa; Minardi, Simone Paolo; Luzi, Lucilla; Muller, Heiko; Di Fiore, Pier Paolo; Frosina, Guido; Pelicci, Pier Giuseppe

    2003-01-01

    Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins. PMID:14660751

  15. Protein function from its emergence to diversity in contemporary proteins

    NASA Astrophysics Data System (ADS)

    Goncearenco, Alexander; Berezovsky, Igor N.

    2015-07-01

    The goal of this work is to learn from nature the rules that govern evolution and the design of protein function. The fundamental laws of physics lie in the foundation of the protein structure and all stages of the protein evolution, determining optimal sizes and shapes at different levels of structural hierarchy. We looked back into the very onset of the protein evolution with a goal to find elementary functions (EFs) that came from the prebiotic world and served as building blocks of the first enzymes. We defined the basic structural and functional units of biochemical reactions—elementary functional loops. The diversity of contemporary enzymes can be described via combinations of a limited number of elementary chemical reactions, many of which are performed by the descendants of primitive prebiotic peptides/proteins. By analyzing protein sequences we were able to identify EFs shared by seemingly unrelated protein superfamilies and folds and to unravel evolutionary relations between them. Binding and metabolic processing of the metal- and nucleotide-containing cofactors and ligands are among the most abundant ancient EFs that became indispensable in many natural enzymes. Highly designable folds provide structural scaffolds for many different biochemical reactions. We show that contemporary proteins are built from a limited number of EFs, making their analysis instrumental for establishing the rules for protein design. Evolutionary studies help us to accumulate the library of essential EFs and to establish intricate relations between different folds and functional superfamilies. Generalized sequence-structure descriptors of the EF will become useful in future design and engineering of desired enzymatic functions.

  16. Protein function from its emergence to diversity in contemporary proteins.

    PubMed

    Goncearenco, Alexander; Berezovsky, Igor N

    2015-06-09

    The goal of this work is to learn from nature the rules that govern evolution and the design of protein function. The fundamental laws of physics lie in the foundation of the protein structure and all stages of the protein evolution, determining optimal sizes and shapes at different levels of structural hierarchy. We looked back into the very onset of the protein evolution with a goal to find elementary functions (EFs) that came from the prebiotic world and served as building blocks of the first enzymes. We defined the basic structural and functional units of biochemical reactions-elementary functional loops. The diversity of contemporary enzymes can be described via combinations of a limited number of elementary chemical reactions, many of which are performed by the descendants of primitive prebiotic peptides/proteins. By analyzing protein sequences we were able to identify EFs shared by seemingly unrelated protein superfamilies and folds and to unravel evolutionary relations between them. Binding and metabolic processing of the metal- and nucleotide-containing cofactors and ligands are among the most abundant ancient EFs that became indispensable in many natural enzymes. Highly designable folds provide structural scaffolds for many different biochemical reactions. We show that contemporary proteins are built from a limited number of EFs, making their analysis instrumental for establishing the rules for protein design. Evolutionary studies help us to accumulate the library of essential EFs and to establish intricate relations between different folds and functional superfamilies. Generalized sequence-structure descriptors of the EF will become useful in future design and engineering of desired enzymatic functions.

  17. 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.

  18. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation

    PubMed Central

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W.; Voisset, Cécile

    2016-01-01

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI+] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI+]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI+] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

  19. Protein carbonylation and muscle function in COPD and other conditions.

    PubMed

    Barreiro, Esther

    2014-01-01

    Skeletal muscle, the most abundant tissue in mammals, is essential for any activity in life. Muscle dysfunction is a common systemic manifestation in highly prevalent conditions such as chronic obstructive pulmonary disease (COPD), cancer cachexia, and sepsis. It has a significant impact on exercise tolerance, thus worsening the patients' quality of life and survival. Among several factors, oxidative stress is a major player in the etiology of skeletal muscle dysfunction associated with those conditions. Whereas low levels of oxidants are absolutely required for normal cell adaptation, high levels of reactive oxygen species (ROS) alter the function and structure of molecules such as proteins, DNA, and lipids. Specifically, protein carbonylation, a common variety of protein oxidation, was shown to alter the function of key enzymes and structural proteins involved in muscle contractile performance. Moreover, increased levels of ROS may also activate proteolytic systems, thus leading to enhanced protein breakdown in several models. In the current review, the specific modifications induced by carbonylation in protein structure and function in muscles have been described. Furthermore, the potential role of ROS in the activation of proteolytic systems in skeletal muscles is also discussed. The review summarizes the effects of protein carbonylation on muscles in several models and conditions such as COPD, disuse muscle atrophy, cancer cachexia, sepsis, and aging. Future research should focus on the elucidation of the specific protein sites modified by ROS in these muscles using redox proteomics analyses and on the assessment of the consequent alterations in protein function and stability.

  20. FunPred-1: protein function prediction from a protein interaction network using neighborhood analysis.

    PubMed

    Saha, Sovan; Chatterjee, Piyali; Basu, Subhadip; Kundu, Mahantapas; Nasipuri, Mita

    2014-12-01

    Proteins are responsible for all biological activities in living organisms. Thanks to genome sequencing projects, large amounts of DNA and protein sequence data are now available, but the biological functions of many proteins are still not annotated in most cases. The unknown function of such non-annotated proteins may be inferred or deduced from their neighbors in a protein interaction network. In this paper, we propose two new methods to predict protein functions based on network neighborhood properties. FunPred 1.1 uses a combination of three simple-yet-effective scoring techniques: the neighborhood ratio, the protein path connectivity and the relative functional similarity. FunPred 1.2 applies a heuristic approach using the edge clustering coefficient to reduce the search space by identifying densely connected neighborhood regions. The overall accuracy achieved in FunPred 1.2 over 8 functional groups involving hetero-interactions in 650 yeast proteins is around 87%, which is higher than the accuracy with FunPred 1.1. It is also higher than the accuracy of many of the state-of-the-art protein function prediction methods described in the literature. The test datasets and the complete source code of the developed software are now freely available at http://code.google.com/p/cmaterbioinfo/ .

  1. Apolipoprotein A-IV: a protein intimately involved in metabolism

    PubMed Central

    Wang, Fei; Kohan, Alison B.; Lo, Chun-Min; Liu, Min; Howles, Philip; Tso, Patrick

    2015-01-01

    The purpose of this review is to summarize our current understanding of the physiological roles of apoA-IV in metabolism, and to underscore the potential for apoA-IV to be a focus for new therapies aimed at the treatment of diabetes and obesity-related disorders. ApoA-IV is primarily synthesized by the small intestine, attached to chylomicrons by enterocytes, and secreted into intestinal lymph during fat absorption. In circulation, apoA-IV is associated with HDL and chylomicron remnants, but a large portion is lipoprotein free. Due to its anti-oxidative and anti-inflammatory properties, and because it can mediate reverse-cholesterol transport, proposed functions of circulating apoA-IV have been related to protection from cardiovascular disease. This review, however, focuses primarily on several properties of apoA-IV that impact other metabolic functions related to food intake, obesity, and diabetes. In addition to participating in triglyceride absorption, apoA-IV can act as an acute satiation factor through both peripheral and central routes of action. It also modulates glucose homeostasis through incretin-like effects on insulin secretion, and by moderating hepatic glucose production. While apoA-IV receptors remain to be conclusively identified, the latter modes of action suggest that this protein holds therapeutic promise for treating metabolic disease. PMID:25640749

  2. Identification and Characterization of Proteins Involved in Rice Urea and Arginine Catabolism1[W

    PubMed Central

    Cao, Feng-Qiu; Werner, Andrea K.; Dahncke, Kathleen; Romeis, Tina; Liu, Lai-Hua; Witte, Claus-Peter

    2010-01-01

    Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (Km = 67 mm, kcat = 490 s−1). The activity depended on the presence of manganese (Kd = 1.3 μm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution. PMID:20631318

  3. Identification and characterization of proteins involved in rice urea and arginine catabolism.

    PubMed

    Cao, Feng-Qiu; Werner, Andrea K; Dahncke, Kathleen; Romeis, Tina; Liu, Lai-Hua; Witte, Claus-Peter

    2010-09-01

    Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K(m) = 67 mm, k(cat) = 490 s(-1)). The activity depended on the presence of manganese (K(d) = 1.3 microm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.

  4. Assigning protein functions by comparative genome analysis protein phylogenetic profiles

    DOEpatents

    Pellegrini, Matteo; Marcotte, Edward M.; Thompson, Michael J.; Eisenberg, David; Grothe, Robert; Yeates, Todd O.

    2003-05-13

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  5. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    PubMed Central

    Gao, Wei-Min; Kuick, Rork; Orchekowski, Randal P; Misek, David E; Qiu, Ji; Greenberg, Alissa K; Rom, William N; Brenner, Dean E; Omenn, Gilbert S; Haab, Brian B; Hanash, Samir M

    2005-01-01

    Background Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Methods Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Results Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Conclusion Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer. PMID:16117833

  6. Using experimental evolution to probe molecular mechanisms of protein function.

    PubMed

    Fischer, Marlies; Kang, Mandeep; Brindle, Nicholas Pj

    2016-02-01

    Directed evolution is a powerful tool for engineering protein function. The process of directed evolution involves iterative rounds of sequence diversification followed by assaying activity of variants and selection. The range of sequence variants and linked activities generated in the course of an evolution are a rich information source for investigating relationships between sequence and function. Key residue positions determining protein function, combinatorial contributors to activity and even potential functional mechanisms have been revealed in directed evolutions. The recent application of high throughput sequencing substantially increases the information that can be retrieved from directed evolution experiments. Combined with computational analysis this additional sequence information has allowed high-resolution analysis of individual residue contributions to activity. These developments promise to significantly enhance the depth of insight that experimental evolution provides into mechanisms of protein function.

  7. Structural determinants of TRIM protein function.

    PubMed

    Esposito, Diego; Koliopoulos, Marios G; Rittinger, Katrin

    2017-02-08

    Tripartite motif (TRIM) proteins constitute one of the largest subfamilies of Really Interesting New Gene (RING) E3 ubiquitin ligases and contribute to the regulation of numerous cellular activities, including innate immune responses. The conserved TRIM harbours a RING domain that imparts E3 ligase activity to TRIM family proteins, whilst a variable C-terminal region can mediate recognition of substrate proteins. The knowledge of the structure of these multidomain proteins and the functional interplay between their constituent domains is paramount to understanding their cellular roles. To date, available structural information on TRIM proteins is still largely restricted to subdomains of many TRIMs in isolation. Nevertheless, applying a combination of structural, biophysical and biochemical approaches has recently allowed important progress to be made towards providing a better understanding of the molecular features that underlie the function of TRIM family proteins and has uncovered an unexpected diversity in the link between self-association and catalytic activity.

  8. A review on protein functionalized carbon nanotubes.

    PubMed

    Nagaraju, Kathyayini; Reddy, Roopa; Reddy, Narendra

    2015-12-18

    Carbon nanotubes (CNTs) have been widely recognized and used for controlled drug delivery and in various other fields due to their unique properties and distinct advantages. Both single-walled carbon nanotubes (SWCNTs) and multiwalled (MWCNTs) carbon nanotubes are used and/or studied for potential applications in medical, energy, textile, composite, and other areas. Since CNTs are chemically inert and are insoluble in water or other organic solvents, they are functionalized or modified to carry payloads or interact with biological molecules. CNTs have been preferably functionalized with proteins because CNTs are predominantly used for medical applications such as delivery of drugs, DNA and genes, and also for biosensing. Extensive studies have been conducted to understand the interactions, cytotoxicity, and potential applications of protein functionalized CNTs but contradicting results have been published on the cytotoxicity of the functionalized CNTs. This paper provides a brief review of CNTs functionalized with proteins, methods used to functionalize the CNTs, and their potential applications.

  9. Hyperhomocysteinemia and bleomycin hydrolase modulate the expression of mouse brain proteins involved in neurodegeneration.

    PubMed

    Suszyńska-Zajczyk, Joanna; Luczak, Magdalena; Marczak, Lukasz; Jakubowski, Hieronim

    2014-01-01

    Homocysteine (Hcy) is a risk factor for Alzheimer's disease (AD). Bleomycin hydrolase (BLMH) participates in Hcy metabolism and is also linked to AD. The inactivation of the Blmh gene in mice causes accumulation of Hcy-thiolactone in the brain and increases susceptibility to Hcy-thiolactone-induced seizures. To gain insight into brain-related Blmh function, we used two-dimensional IEF/SDS-PAGE gel electrophoresis and MALDI-TOF/TOF mass spectrometry to examine brain proteomes of Blmh-/- mice and their Blmh+/+ littermates fed with a hyperhomocysteinemic high-Met or a control diet. We found that: (1) proteins involved in brain-specific function (Ncald, Nrgn, Stmn1, Stmn2), antioxidant defenses (Aop1), cell cycle (RhoGDI1, Ran), and cytoskeleton assembly (Tbcb, CapZa2) were differentially expressed in brains of Blmh-null mice; (2) hyperhomocysteinemia amplified effects of the Blmh-/- genotype on brain protein expression; (3) proteins involved in brain-specific function (Pebp1), antioxidant defenses (Sod1, Prdx2, DJ-1), energy metabolism (Atp5d, Ak1, Pgam-B), and iron metabolism (Fth) showed differential expression in Blmh-null brains only in hyperhomocysteinemic animals; (4) most proteins regulated by the Blmh-/- genotype were also regulated by high-Met diet, albeit in the opposite direction; and (5) the differentially expressed proteins play important roles in neural development, learning, plasticity, and aging and are linked to neurodegenerative diseases, including AD. Taken together, our findings suggest that Blmh interacts with diverse cellular processes from energy metabolism and anti-oxidative defenses to cell cycle, cytoskeleton dynamics, and synaptic plasticity essential for normal brain homeostasis and that modulation of these interactions by hyperhomocysteinemia underlies the involvement of Hcy in AD.

  10. Using the folding landscapes of proteins to understand protein function.

    PubMed

    Giri Rao, V V Hemanth; Gosavi, Shachi

    2016-02-01

    Proteins fold on a biologically-relevant timescale because of a funnel-shaped energy landscape. This landscape is sculpted through evolution by selecting amino-acid sequences that stabilize native interactions while suppressing stable non-native interactions that occur during folding. However, there is strong evolutionary selection for functional residues and these cannot be chosen to optimize folding. Their presence impacts the folding energy landscape in a variety of ways. Here, we survey the effects of functional residues on folding by providing several examples. We then review how such effects can be detected computationally and be used as assays for protein function. Overall, an understanding of how functional residues modulate folding should provide insights into the design of natural proteins and their homeostasis.

  11. Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins.

    PubMed

    Maeda, Yusuke; Kinoshita, Taroh

    2011-10-01

    Glycosylphosphatidylinositol (GPI) is a glycolipid that is covalently attached to proteins as a post-translational modification. Such modification leads to the anchoring of the protein to the outer leaflet of the plasma membrane. Proteins that are decorated with GPIs have unique properties in terms of their physical nature. In particular, these proteins tend to accumulate in lipid rafts, which are critical for the functions and trafficking of GPI-anchored proteins (GPI-APs). Recent studies mainly using mutant cells revealed that various structural remodeling reactions occur to GPIs present in GPI-APs as they are transported from the endoplasmic reticulum to the cell surface. This review examines the recent progress describing the mechanisms of structural remodeling of mammalian GPI-anchors, such as inositol deacylation, glycan remodeling and fatty acid remodeling, with particular focus on their trafficking and functions, as well as the pathogenesis involving GPI-APs and their deficiency.

  12. Protein molecular function prediction by Bayesian phylogenomics.

    PubMed

    Engelhardt, Barbara E; Jordan, Michael I; Muratore, Kathryn E; Brenner, Steven E

    2005-10-01

    We present a statistical graphical model to infer specific molecular function for unannotated protein sequences using homology. Based on phylogenomic principles, SIFTER (Statistical Inference of Function Through Evolutionary Relationships) accurately predicts molecular function for members of a protein family given a reconciled phylogeny and available function annotations, even when the data are sparse or noisy. Our method produced specific and consistent molecular function predictions across 100 Pfam families in comparison to the Gene Ontology annotation database, BLAST, GOtcha, and Orthostrapper. We performed a more detailed exploration of functional predictions on the adenosine-5'-monophosphate/adenosine deaminase family and the lactate/malate dehydrogenase family, in the former case comparing the predictions against a gold standard set of published functional characterizations. Given function annotations for 3% of the proteins in the deaminase family, SIFTER achieves 96% accuracy in predicting molecular function for experimentally characterized proteins as reported in the literature. The accuracy of SIFTER on this dataset is a significant improvement over other currently available methods such as BLAST (75%), GeneQuiz (64%), GOtcha (89%), and Orthostrapper (11%). We also experimentally characterized the adenosine deaminase from Plasmodium falciparum, confirming SIFTER's prediction. The results illustrate the predictive power of exploiting a statistical model of function evolution in phylogenomic problems. A software implementation of SIFTER is available from the authors.

  13. Host membrane proteins involved in the replication of tobamovirus RNA.

    PubMed

    Ishibashi, Kazuhiro; Miyashita, Shuhei; Katoh, Etsuko; Ishikawa, Masayuki

    2012-12-01

    Eukaryotic positive-strand RNA viruses replicate their genomes in membrane-bound replication complexes composed of viral replication proteins and negative-strand RNA templates. These replication proteins are programmed to exhibit RNA polymerase and other replication-related activities only in replication complexes to avoid inducing double-stranded RNA-mediated host defenses. Host membrane components (e.g. proteins and lipids) should play important roles in the activation of replication proteins. Two host membrane proteins are components of the replication complex and activate the replication proteins of tobamoviruses. Interaction analyses using deletion mutants constructed based on structural information suggest a conformational change in replication proteins during the formation of a protein complex with RNA 5'-capping activity.

  14. The HMG-1 box protein family: classification and functional relationships.

    PubMed Central

    Baxevanis, A D; Landsman, D

    1995-01-01

    The abundant and highly-conserved nucleoproteins comprising the high mobility group-1/2 (HMG-1/2) family contains two homologous basic domains of about 75 amino acids. These basic domains, termed HMG-1 boxes, are highly structured and facilitate HMG-DNA interactions. Many proteins that regulate various cellular functions involving DNA binding and whose target DNA sequences share common structural characteristics have been identified as having an HMG-1 box; these proteins include the RNA polymerase I transcription factor UBF, the mammalian testis-determining factor SRY and the mitochondrial transcription factors ABF2 and mtTF1, among others. The sequences of 121 HMG-1 boxes have been compiled and aligned in accordance with thermodynamic results from homology model building (threading) experiments, basing the alignment on structure rather than by using traditional sequence homology methods. The classification of a representative subset of these proteins was then determined using standard least-squares distance methods. The proteins segregate into two groups, the first consisting of HMG-1/2 proteins and the second consisting of proteins containing the HMG-1 box but which are not canonical HMG proteins. The proteins in the second group further segregate based on their function, their ability to bind specific sequences of DNA, or their ability to recognize discrete non-B-DNA structures. The HMG-1 box provides an excellent example of how a specific protein motif, with slight alteration, can be used to recognize DNA in a variety of functional contexts. Images PMID:7784217

  15. Discovering Conformational Sub-States Relevant to Protein Function

    PubMed Central

    Ramanathan, Arvind; Savol, Andrej J.; Langmead, Christopher J.; Agarwal, Pratul K.; Chennubhotla, Chakra S.

    2011-01-01

    Background Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these sub-states present significant challenges for their identification and characterization. Methods and Findings To overcome these challenges we have developed a new computational technique, quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function. Conclusions Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function. PMID:21297978

  16. Structure and function of WD40 domain proteins.

    PubMed

    Xu, Chao; Min, Jinrong

    2011-03-01

    The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates molecular recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymatic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.

  17. NAP-1, Nucleosome assembly protein 1, a histone chaperone involved in Drosophila telomeres.

    PubMed

    López-Panadès, Elisenda; Casacuberta, Elena

    2016-03-01

    Telomere elongation is a function that all eukaryote cells must accomplish in order to guarantee, first, the stability of the end of the chromosomes and second, to protect the genetic information from the inevitable terminal erosion. The targeted transposition of the telomere transposons HeT-A, TART and TAHRE perform this function in Drosophila, while the telomerase mechanism elongates the telomeres in most eukaryotes. In order to integrate telomere maintenance together with cell cycle and metabolism, different components of the cell interact, regulate, and control the proteins involved in telomere elongation. Different partners of the telomerase mechanism have already been described, but in contrast, very few proteins have been related with assisting the telomere transposons of Drosophila. Here, we describe for the first time, the implication of NAP-1 (Nucleosome assembly protein 1), a histone chaperone that has been involved in nuclear transport, transcription regulation, and chromatin remodeling, in telomere biology. We find that Nap-1 and HeT-A Gag, one of the major components of the Drosophila telomeres, are part of the same protein complex. We also demonstrate that their close interaction is necessary to guarantee telomere stability in dividing cells. We further show that NAP-1 regulates the transcription of the HeT-A retrotransposon, pointing to a positive regulatory role of NAP-1 in telomere expression. All these results facilitate the understanding of the transposon telomere maintenance mechanism, as well as the integration of telomere biology with the rest of the cell metabolism.

  18. Functional Importance of Mobile Ribosomal Proteins.

    PubMed

    Chang, Kai-Chun; Wen, Jin-Der; Yang, Lee-Wei

    2015-01-01

    Although the dynamic motions and peptidyl transferase activity seem to be embedded in the rRNAs, the ribosome contains more than 50 ribosomal proteins (r-proteins), whose functions remain largely elusive. Also, the precise forms of some of these r-proteins, as being part of the ribosome, are not structurally solved due to their high flexibility, which hinders the efforts in their functional elucidation. Owing to recent advances in cryo-electron microscopy, single-molecule techniques, and theoretical modeling, much has been learned about the dynamics of these r-proteins. Surprisingly, allosteric regulations have been found in between spatially separated components as distant as those in the opposite sides of the ribosome. Here, we focus on the functional roles and intricate regulations of the mobile L1 and L12 stalks and L9 and S1 proteins. Conformational flexibility also enables versatile functions for r-proteins beyond translation. The arrangement of r-proteins may be under evolutionary pressure that fine-tunes mass distributions for optimal structural dynamics and catalytic activity of the ribosome.

  19. Control of protein function through optochemical translocation.

    PubMed

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

    2014-10-17

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

  20. SEORious business: structural proteins in sieve tubes and their involvement in sieve element occlusion.

    PubMed

    Knoblauch, Michael; Froelich, Daniel R; Pickard, William F; Peters, Winfried S

    2014-04-01

    The phloem provides a network of sieve tubes for long-distance translocation of photosynthates. For over a century, structural proteins in sieve tubes have presented a conundrum since they presumably increase the hydraulic resistance of the tubes while no potential function other than sieve tube or wound sealing in the case of injury has been suggested. Here we summarize and critically evaluate current speculations regarding the roles of these proteins. Our understanding suffers from the suggestive power of images; what looks like a sieve tube plug on micrographs may not actually impede translocation very much. Recent reports of an involvement of SEOR (sieve element occlusion-related) proteins, a class of P-proteins, in the sealing of injured sieve tubes are inconclusive; various lines of evidence suggest that, in neither intact nor injured plants, are SEORs determinative of translocation stoppage. Similarly, the popular notion that P-proteins serve in the defence against phloem sap-feeding insects is unsupported by empirical facts; it is conceivable that in functional sieve tubes, aphids actually could benefit from inducing a plug. The idea that rising cytosolic Ca(2+) generally triggers sieve tube blockage by P-proteins appears widely accepted, despite lacking experimental support. Even in forisomes, P-protein assemblages restricted to one single plant family and the only Ca(2+)-responsive P-proteins known, the available evidence does not unequivocally suggest that plug formation is the cause rather than a consequence of translocation stoppage. We conclude that the physiological roles of structural P-proteins remain elusive, and that in vivo studies of their dynamics in continuous sieve tube networks combined with flow velocity measurements will be required to (hopefully) resolve this scientific roadblock.

  1. Identification of autosomal regions involved in Drosophila Raf function.

    PubMed Central

    Li, W; Noll, E; Perrimon, N

    2000-01-01

    Raf is an essential downstream effector of activated p21(Ras) (Ras) in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified "Raf activator." In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, we conducted a genetic screen to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). We tested a collection of chromosomal deficiencies representing approximately 70% of the autosomal euchromatic genomic regions for their abilities to enhance the lethality associated with a hypomorphic viable allele of Draf, Draf(Su2). Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of Draf(Su2), causing lethality in Draf(Su2) hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3epsilon, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. We demonstrate that Src64B interacts genetically with Draf and that an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, we show that a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, we discovered a genetic interaction between Draf(Su2) and Df(3L)vin5 that revealed a novel role of Draf in limb development. We find that loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs. PMID:11014822

  2. Structure and Function of Lipopolysaccharide Binding Protein

    NASA Astrophysics Data System (ADS)

    Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

    1990-09-01

    The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

  3. Protein conformational populations and functionally relevant substates.

    PubMed

    Ramanathan, Arvind; Savol, Andrej; Burger, Virginia; Chennubhotla, Chakra S; Agarwal, Pratul K

    2014-01-21

    Functioning proteins do not remain fixed in a unique structure, but instead they sample a range of conformations facilitated by motions within the protein. Even in the native state, a protein exists as a collection of interconverting conformations driven by thermodynamic fluctuations. Motions on the fast time scale allow a protein to sample conformations in the nearby area of its conformational landscape, while motions on slower time scales give it access to conformations in distal areas of the landscape. Emerging evidence indicates that protein landscapes contain conformational substates with dynamic and structural features that support the designated function of the protein. Nuclear magnetic resonance (NMR) experiments provide information about conformational ensembles of proteins. X-ray crystallography allows researchers to identify the most populated states along the landscape, and computational simulations give atom-level information about the conformational substates of different proteins. This ability to characterize and obtain quantitative information about the conformational substates and the populations of proteins within them is allowing researchers to better understand the relationship between protein structure and dynamics and the mechanisms of protein function. In this Account, we discuss recent developments and challenges in the characterization of functionally relevant conformational populations and substates of proteins. In some enzymes, the sampling of functionally relevant conformational substates is connected to promoting the overall mechanism of catalysis. For example, the conformational landscape of the enzyme dihydrofolate reductase has multiple substates, which facilitate the binding and the release of the cofactor and substrate and catalyze the hydride transfer. For the enzyme cyclophilin A, computational simulations reveal that the long time scale conformational fluctuations enable the enzyme to access conformational substates that allow

  4. LanCL proteins are not Involved in Lanthionine Synthesis in Mammals.

    PubMed

    He, Chang; Zeng, Min; Dutta, Debapriya; Koh, Tong Hee; Chen, Jie; van der Donk, Wilfred A

    2017-01-20

    LanC-like (LanCL) proteins are mammalian homologs of bacterial LanC enzymes, which catalyze the addition of the thiol of Cys to dehydrated Ser residues during the biosynthesis of lanthipeptides, a class of natural products formed by post-translational modification of precursor peptides. The functions of LanCL proteins are currently unclear. A recent proposal suggested that LanCL1 catalyzes the addition of the Cys of glutathione to protein- or peptide-bound dehydroalanine (Dha) to form lanthionine, analogous to the reaction catalyzed by LanC in bacteria. Lanthionine has been detected in human brain as the downstream metabolite lanthionine ketimine (LK), which has been shown to have neuroprotective effects. In this study, we tested the proposal that LanCL1 is involved in lanthionine biosynthesis by constructing LanCL1 knock-out mice and measuring LK concentrations in their brains using a mass spectrometric detection method developed for this purpose. To investigate whether other LanCL proteins (LanCL2/3) may confer a compensatory effect, triple knock-out (TKO) mice were also generated and tested. Very similar concentrations of LK (0.5-2.5 nmol/g tissue) were found in LanCL1 knock-out, TKO and wild type (WT) mouse brains, suggesting that LanCL proteins are not involved in lanthionine biosynthesis.

  5. LanCL proteins are not Involved in Lanthionine Synthesis in Mammals

    PubMed Central

    He, Chang; Zeng, Min; Dutta, Debapriya; Koh, Tong Hee; Chen, Jie; van der Donk, Wilfred A.

    2017-01-01

    LanC-like (LanCL) proteins are mammalian homologs of bacterial LanC enzymes, which catalyze the addition of the thiol of Cys to dehydrated Ser residues during the biosynthesis of lanthipeptides, a class of natural products formed by post-translational modification of precursor peptides. The functions of LanCL proteins are currently unclear. A recent proposal suggested that LanCL1 catalyzes the addition of the Cys of glutathione to protein- or peptide-bound dehydroalanine (Dha) to form lanthionine, analogous to the reaction catalyzed by LanC in bacteria. Lanthionine has been detected in human brain as the downstream metabolite lanthionine ketimine (LK), which has been shown to have neuroprotective effects. In this study, we tested the proposal that LanCL1 is involved in lanthionine biosynthesis by constructing LanCL1 knock-out mice and measuring LK concentrations in their brains using a mass spectrometric detection method developed for this purpose. To investigate whether other LanCL proteins (LanCL2/3) may confer a compensatory effect, triple knock-out (TKO) mice were also generated and tested. Very similar concentrations of LK (0.5–2.5 nmol/g tissue) were found in LanCL1 knock-out, TKO and wild type (WT) mouse brains, suggesting that LanCL proteins are not involved in lanthionine biosynthesis. PMID:28106097

  6. Endogenous basic fibroblast growth factor isoforms involved in different intracellular protein complexes.

    PubMed Central

    Patry, V; Bugler, B; Maret, A; Potier, M; Prats, H

    1997-01-01

    Four forms of basic fibroblast growth factor (bFGF or FGF-2) result from an alternative initiation of translation involving one AUG (155-amino acid form) and three CUGs (210-, 201- and 196-amino acid forms). These different forms of bFGF show different intracellular biological activities. To identify their intracellular targets, the 210- and 155-amino acid forms of bFGF were independently transfected into CHO cells and their correct subcellular localizations were verified, the 155-amino acid bFGF form being essentially cytoplasmic whereas the 210-amino acid protein was nuclear. The radiation fragmentation method was used to determine the target size of the different bFGF isoforms in the transfected CHO cells and to show that the 210- and 155-amino acids bFGF isoforms were included in protein complexes of 320 and 130 kDa respectively. Similar results were obtained using the SK-Hep1 cell line, which naturally expressed all forms of bFGF. Co-immunoprecipitation assays using different chimaeric bFGF-chloramphenicol acetyltransferase proteins showed that different cellular proteins are associated with different parts of the bFGF molecule. We conclude that bFGF isoforms are involved in different molecular complexes in the cytosol and nucleus, which would reflect different functions for these proteins. PMID:9337877

  7. Identification of ICIS-1, a new protein involved in cilia stability.

    PubMed

    Ponsard, Cecile; Skowron-Zwarg, Marie; Seltzer, Virginie; Perret, Eric; Gallinger, Julia; Fisch, Cathy; Dupuis-Williams, Pascale; Caruso, Nathalie; Middendorp, Sandrine; Tournier, Frederic

    2007-01-01

    Cilia are specialized organelles that exert critical functions in numerous organisms, including that of cell motility, fluid transport and protozoan locomotion. Ciliary architecture and function strictly depend on basal body formation, migration and axoneme elongation. Numerous ultrastructural studies have been undertaken in different species to elucidate the process of ciliogenesis. Recent analyses have led to identification of genes specifically expressed in ciliated organisms, but most proteins involved in ciliogenesis remain uncharacterized. Using human nasal epithelial cells capable of ciliary differentiation in vitro, differential display was carried out to identify new proteins associated with ciliogenesis. We isolated a new gene, ICIS-1 (Involved in CIlia Stability-1), upregulated during mucociliary differentiation. This gene is localized within the TGF-beta1 promoter and is ubiquitously expressed in human tissues. Functional analyses of gene expression inhibition by RNA interference in Paramecium tetraurelia indicated that the ICIS-1 homologue interfered with cilia stability or formation. These findings demonstrate that ICIS-1 is a new protein associated with ciliated cells and potentially related to cilia stability.

  8. The abi proteins and their involvement in bacteriocin self-immunity.

    PubMed

    Kjos, Morten; Snipen, Lars; Salehian, Zhian; Nes, Ingolf F; Diep, Dzung B

    2010-04-01

    The Abi protein family consists of putative membrane-bound metalloproteases. While they are involved in membrane anchoring of proteins in eukaryotes, little is known about their function in prokaryotes. In some known bacteriocin loci, Abi genes have been found downstream of bacteriocin structural genes (e.g., pln locus from Lactobacillus plantarum and sag locus from Streptococcus pyogenes), where they probably are involved in self-immunity. By modifying the profile hidden Markov model used to select Abi proteins in the Pfam protein family database, we show that this family is larger than presently recognized. Using bacteriocin-associated Abi genes as a means to search for novel bacteriocins in sequenced genomes, seven new bacteriocin-like loci were identified in Gram-positive bacteria. One such locus, from Lactobacillus sakei 23K, was selected for further experimental study, and it was confirmed that the bacteriocin-like genes (skkAB) exhibited antimicrobial activity when expressed in a heterologous host and that the associated Abi gene (skkI) conferred immunity against the cognate bacteriocin. Similar investigation of the Abi gene plnI and the Abi-like gene plnL from L. plantarum also confirmed their involvement in immunity to their cognate bacteriocins (PlnEF and PlnJK, respectively). Interestingly, the immunity genes from these three systems conferred a high degree of cross-immunity against each other's bacteriocins, suggesting the recognition of a common receptor. Site-directed mutagenesis demonstrated that the conserved motifs constituting the putative proteolytic active site of the Abi proteins are essential for the immunity function of SkkI, and to our knowledge, this represents a new concept in self-immunity.

  9. Integration of latex protein sequence data provides comprehensive functional overview of latex proteins.

    PubMed

    Cho, Won Kyong; Jo, Yeonhwa; Chu, Hyosub; Park, Sang-Ho; Kim, Kook-Hyung

    2014-03-01

    The laticiferous system is one of the most important conduit systems in higher plants, which produces a milky-like sap known as latex. Latex contains diverse secondary metabolites with various ecological functions. To obtain a comprehensive overview of the latex proteome, we integrated available latex proteins sequences and constructed a comprehensive dataset composed of 1,208 non-redundant latex proteins from 20 various latex-bearing plants. The results of functional analyses revealed that latex proteins are involved in various biological processes, including transcription, translation, protein degradation and the plant response to environmental stimuli. The results of the comparative analysis showed that the functions of the latex proteins are similar to those of phloem, suggesting the functional conservation of plant vascular proteins. The presence of latex proteins in mitochondria and plastids suggests the production of diverse secondary metabolites. Furthermore, using a BLAST search, we identified 854 homologous latex proteins in eight plant species, including three latex-bearing plants, such as papaya, caster bean and cassava, suggesting that latex proteins were newly evolved in vascular plants. Taken together, this study is the largest and most comprehensive in silico analysis of the latex proteome. The results obtained here provide useful resources and information for characterizing the evolution of the latex proteome.

  10. Nano-functionalization of protein microspheres

    NASA Astrophysics Data System (ADS)

    Yoon, Sungkwon; Nichols, William T.

    2014-08-01

    Protein microspheres are promising building blocks for the assembly of complex functional materials. Here we demonstrate a set of three techniques that add functionality to the surface of protein microspheres. In the first technique, a positive surface charge on the protein spheres is deposited by electrostatic adsorption. Negatively charged silica and gold nanoparticle colloids can then electrostatically bind reversibly to the microsphere surface. In the second technique, nanoparticles are covalently anchored to the protein shell using a simple one-pot process. The strong covalent bond between sulfur groups in cysteine in the protein shell irreversibly binds to the gold nanoparticles. In the third technique, surface morphology of the protein microsphere is tuned through hydrodynamic instability at the water-oil interface. This is accomplished through the degree of solubility of the oil phase in water. Taken together these three techniques form a platform to create nano-functionalized protein microspheres, which can then be used as building blocks for the assembly of more complex macroscopic materials.

  11. Physiological functions of MTA family of proteins.

    PubMed

    Sen, Nirmalya; Gui, Bin; Kumar, Rakesh

    2014-12-01

    Although the functional significance of the metastasic tumor antigen (MTA) family of chromatin remodeling proteins in the pathobiology of cancer is fairly well recognized, the physiological role of MTA proteins continues to be an understudied research area and is just beginning to be recognized. Similar to cancer cells, MTA1 also modulates the expression of target genes in normal cells either by acting as a corepressor or coactivator. In addition, physiological functions of MTA proteins are likely to be influenced by its differential expression, subcellular localization, and regulation by upstream modulators and extracellular signals. This review summarizes our current understanding of the physiological functions of the MTA proteins in model systems. In particular, we highlight recent advances of the role MTA proteins play in the brain, eye, circadian rhythm, mammary gland biology, spermatogenesis, liver, immunomodulation and inflammation, cellular radio-sensitivity, and hematopoiesis and differentiation. Based on the growth of knowledge regarding the exciting new facets of the MTA family of proteins in biology and medicine, we speculate that the next burst of findings in this field may reveal further molecular regulatory insights of non-redundant functions of MTA coregulators in the normal physiology as well as in pathological conditions outside cancer.

  12. The endocytic adaptor protein ARH associates with motor and centrosomal proteins and is involved in centrosome assembly and cytokinesis.

    PubMed

    Lehtonen, Sanna; Shah, Mehul; Nielsen, Rikke; Iino, Noriaki; Ryan, Jennifer J; Zhou, Huilin; Farquhar, Marilyn G

    2008-07-01

    Numerous proteins involved in endocytosis at the plasma membrane have been shown to be present at novel intracellular locations and to have previously unrecognized functions. ARH (autosomal recessive hypercholesterolemia) is an endocytic clathrin-associated adaptor protein that sorts members of the LDL receptor superfamily (LDLR, megalin, LRP). We report here that ARH also associates with centrosomes in several cell types. ARH interacts with centrosomal (gamma-tubulin and GPC2 and GPC3) and motor (dynein heavy and intermediate chains) proteins. ARH cofractionates with gamma-tubulin on isolated centrosomes, and gamma-tubulin and ARH interact on isolated membrane vesicles. During mitosis, ARH sequentially localizes to the nuclear membrane, kinetochores, spindle poles and the midbody. Arh(-/-) embryonic fibroblasts (MEFs) show smaller or absent centrosomes suggesting ARH plays a role in centrosome assembly. Rat-1 fibroblasts depleted of ARH by siRNA and Arh(-/-) MEFs exhibit a slower rate of growth and prolonged cytokinesis. Taken together the data suggest that the defects in centrosome assembly in ARH depleted cells may give rise to cell cycle and mitotic/cytokinesis defects. We propose that ARH participates in centrosomal and mitotic dynamics by interacting with centrosomal proteins. Whether the centrosomal and mitotic functions of ARH are related to its endocytic role remains to be established.

  13. The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division.

    PubMed

    Ambrose, J Christian; Shoji, Tsubasa; Kotzer, Amanda M; Pighin, Jamie A; Wasteneys, Geoffrey O

    2007-09-01

    Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.

  14. Analysis of nitrated proteins in Saccharomyces cerevisiae involved in mating signal transduction.

    PubMed

    Kang, Jeong Won; Lee, Na Young; Cho, Kyung-Cho; Lee, Min Young; Choi, Do-Young; Park, Sang-Hyun; Kim, Kwang Pyo

    2015-01-01

    Protein tyrosine nitration (PTN) is a PTM that regulates signal transduction and inflammatory responses, and is related to neurodegenerative and cardiovascular diseases. The cellular function of PTN remains unclear because the low stoichiometry of PTN limits the identification and quantification of nitrated peptides. Effective enrichment is an important aspect of PTN analysis. In this study, we analyzed the in vivo nitroproteome elicited by mating signal transduction in Saccharomyces cerevisiae using a novel chemical enrichment method followed by LC-MS/MS. Nitroproteome profiling successfully identified changes in the nitration states of 14 proteins during mating signal transduction in S. cerevisiae, making this the first reported in vivo nitroproteome in yeast. We investigated the biological functions of these nitroproteins and their relationships to mating signal transduction in S. cerevisiae using a protein-protein interaction network. Our results suggest that PTN and denitration may be involved in nonreactive nitrogen species-mediated signal transduction and can provide clues for understanding the functional roles of PTN in vivo.

  15. TIA-1 Is a Functional Prion-Like Protein.

    PubMed

    Rayman, Joseph B; Kandel, Eric R

    2016-12-21

    Prions are self-propagating protein conformations that are traditionally regarded as agents of neurodegenerative disease in animals. However, it has become evident that prion-like aggregation of endogenous proteins can also occur under normal physiological conditions (e.g., during memory storage or activation of the immune response). In this review, we focus on the functional prion-related protein TIA-1, an RNA-binding protein that is involved in multiple aspects of RNA metabolism but is best understood in terms of its role in stress granule assembly during the cellular stress response. We propose that stress granule formation provides a useful conceptual framework with which to address the positive role of TIA-1 prion-like aggregation. Elucidating the function of TIA-1 prion-like aggregation will advance our understanding of how prion-based molecular switches are used in normal physiological settings.

  16. Evolution and Cellular Function of Monothiol Glutaredoxins: Involvement in Iron-Sulphur Cluster Assembly

    PubMed Central

    Vilella, Felipe; Alves, Rui; Rodríguez-Manzaneque, María Teresa; Bellí, Gemma; Swaminathan, Swarna; Sunnerhagen, Per

    2004-01-01

    A number of bacterial species, mostly proteobacteria, possess monothiol glutaredoxins homologous to the Saccharomyces cerevisiae mitochondrial protein Grx5, which is involved in iron–sulphur cluster synthesis. Phylogenetic profiling is used to predict that bacterial monothiol glutaredoxins also participate in the iron–sulphur cluster (ISC) assembly machinery, because their phylogenetic profiles are similar to the profiles of the bacterial homologues of yeast ISC proteins. High evolutionary co-occurrence is observed between the Grx5 homologues and the homologues of the Yah1 ferredoxin, the scaffold proteins Isa1 and Isa2, the frataxin protein Yfh1 and the Nfu1 protein. This suggests that a specific functional interaction exists between these ISC machinery proteins. Physical interaction analyses using low-definition protein docking predict the formation of strong and specific complexes between Grx5 and several components of the yeast ISC machinery. Two-hybrid analysis has confirmed the in vivo interaction between Grx5 and Isa1. Sequence comparison techniques and cladistics indicate that the other two monothiol glutaredoxins of S. cerevisiae, Grx3 and Grx4, have evolved from the fusion of a thioredoxin gene with a monothiol glutaredoxin gene early in the eukaryotic lineage, leading to differential functional specialization. While bacteria do not contain these chimaeric glutaredoxins, in many eukaryotic species Grx5 and Grx3/4-type monothiol glutaredoxins coexist in the cell. PMID:18629168

  17. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  18. Calreticulin: one protein, one gene, many functions.

    PubMed Central

    Michalak, M; Corbett, E F; Mesaeli, N; Nakamura, K; Opas, M

    1999-01-01

    The endoplasmic reticulum (ER) plays a critical role in the synthesis and chaperoning of membrane-associated and secreted proteins. The membrane is also an important site of Ca(2+) storage and release. Calreticulin is a unique ER luminal resident protein. The protein affects many cellular functions, both in the ER lumen and outside of the ER environment. In the ER lumen, calreticulin performs two major functions: chaperoning and regulation of Ca(2+) homoeostasis. Calreticulin is a highly versatile lectin-like chaperone, and it participates during the synthesis of a variety of molecules, including ion channels, surface receptors, integrins and transporters. The protein also affects intracellular Ca(2+) homoeostasis by modulation of ER Ca(2+) storage and transport. Studies on the cell biology of calreticulin revealed that the ER membrane is a very dynamic intracellular compartment affecting many aspects of cell physiology. PMID:10567207

  19. Structure and Function of a Novel ld-Carboxypeptidase A Involved in Peptidoglycan Recycling

    PubMed Central

    Das, Debanu; Hervé, Mireille; Elsliger, Marc-André; Kadam, Rameshwar U.; Grant, Joanna C.; Chiu, Hsiu-Ju; Knuth, Mark W.; Klock, Heath E.; Miller, Mitchell D.; Godzik, Adam; Lesley, Scott A.; Deacon, Ashley M.

    2013-01-01

    Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling. PMID:24123814

  20. Structure and function of a novel LD-carboxypeptidase a involved in peptidoglycan recycling.

    PubMed

    Das, Debanu; Hervé, Mireille; Elsliger, Marc-André; Kadam, Rameshwar U; Grant, Joanna C; Chiu, Hsiu-Ju; Knuth, Mark W; Klock, Heath E; Miller, Mitchell D; Godzik, Adam; Lesley, Scott A; Deacon, Ashley M; Mengin-Lecreulx, Dominique; Wilson, Ian A

    2013-12-01

    Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling.

  1. Protein receptor for activated C kinase 1 is involved in morphine reward in mice.

    PubMed

    Wan, L; Su, L; Xie, Y; Liu, Y; Wang, Y; Wang, Z

    2009-07-07

    Opiate addiction is associated with upregulation of cAMP signaling in the brain. cAMP-responsive element binding protein (CREB), a nuclear transcription factor, is a downstream component of the extracellular signal-regulated protein kinase (ERK) pathway, which has been shown to regulate different physiological and psychological responses of drug addiction. RACK1, the protein receptor for activated C kinase 1, is a multifunctional scaffolding protein known to be a key regulator of various signaling cascades in the CNS. RACK1 functions specifically in integrin mediated activation of ERK cascade and targets active ERK. We examined if RACK1 is involved in the mechanism of drug addiction by regulating CREB in mouse hippocampus and prefrontal cortex. Several expressions were observed. Chronic administration of morphine made the expression of RACK1 and CREB mRNA increase in hippocampus and prefrontal cortex. The expression of RACK1 and CREB protein was strongly positive in CA1, CA3 and dentate gyrus (DG) of the hippocampus of morphine-treated mice brain, especially the pyramidal neurons in the DG of the hippocampus. Using the small interfering RNA technology, we determined that the expression of CREB mRNA was decreased in hippocampus and prefrontal cortex of morphine-treated mice. The expression of RACK1 and CREB protein was negative in CA1, CA3 and DG of hippocampus. These findings suggest that morphine reward can influence the expression of RACK1 in mouse hippocampus and prefrontal cortex through regulating CREB transcription.

  2. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes

    PubMed Central

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-01-01

    The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains—In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model. PMID:27763556

  3. Structural Interface Forms and Their Involvement in Stabilization of Multidomain Proteins or Protein Complexes.

    PubMed

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-10-18

    The presented analysis concerns the inter-domain and inter-protein interface in protein complexes. We propose extending the traditional understanding of the protein domain as a function of local compactness with an additional criterion which refers to the presence of a well-defined hydrophobic core. Interface areas in selected homodimers vary with respect to their contribution to share as well as individual (domain-specific) hydrophobic cores. The basic definition of a protein domain, i.e., a structural unit characterized by tighter packing than its immediate environment, is extended in order to acknowledge the role of a structured hydrophobic core, which includes the interface area. The hydrophobic properties of interfaces vary depending on the status of interacting domains-In this context we can distinguish: (1) Shared hydrophobic cores (spanning the whole dimer); (2) Individual hydrophobic cores present in each monomer irrespective of whether the dimer contains a shared core. Analysis of interfaces in dystrophin and utrophin indicates the presence of an additional quasi-domain with a prominent hydrophobic core, consisting of fragments contributed by both monomers. In addition, we have also attempted to determine the relationship between the type of interface (as categorized above) and the biological function of each complex. This analysis is entirely based on the fuzzy oil drop model.

  4. Absence of aquaporin-4 in skeletal muscle alters proteins involved in bioenergetic pathways and calcium handling.

    PubMed

    Basco, Davide; Nicchia, Grazia Paola; D'Alessandro, Angelo; Zolla, Lello; Svelto, Maria; Frigeri, Antonio

    2011-04-28

    Aquaporin-4 (AQP4) is a water channel expressed at the sarcolemma of fast-twitch skeletal muscle fibers, whose expression is altered in several forms of muscular dystrophies. However, little is known concerning the physiological role of AQP4 in skeletal muscle and its functional and structural interaction with skeletal muscle proteome. Using AQP4-null mice, we analyzed the effect of the absence of AQP4 on the morphology and protein composition of sarcolemma as well as on the whole skeletal muscle proteome. Immunofluorescence analysis showed that the absence of AQP4 did not perturb the expression and cellular localization of the dystrophin-glycoprotein complex proteins, aside from those belonging to the extracellular matrix, and no alteration was found in sarcolemma integrity by dye extravasation assay. With the use of a 2DE-approach (BN/SDS-PAGE), protein maps revealed that in quadriceps, out of 300 Coomassie-blue detected and matched spots, 19 proteins exhibited changed expression in AQP4(-/-) compared to WT mice. In particular, comparison of the protein profiles revealed 12 up- and 7 down-regulated protein spots in AQP4-/- muscle. Protein identification by MS revealed that the perturbed expression pattern belongs to proteins involved in energy metabolism (i.e. GAPDH, creatine kinase), as well as in Ca(2+) handling (i.e. parvalbumin, SERCA1). Western blot analysis, performed on some significantly changed proteins, validated the 2D results. Together these findings suggest AQP4 as a novel determinant in the regulation of skeletal muscle metabolism and better define the role of this water channel in skeletal muscle physiology.

  5. Absence of Aquaporin-4 in Skeletal Muscle Alters Proteins Involved in Bioenergetic Pathways and Calcium Handling

    PubMed Central

    Basco, Davide; Nicchia, Grazia Paola; D'Alessandro, Angelo; Zolla, Lello; Svelto, Maria; Frigeri, Antonio

    2011-01-01

    Aquaporin-4 (AQP4) is a water channel expressed at the sarcolemma of fast-twitch skeletal muscle fibers, whose expression is altered in several forms of muscular dystrophies. However, little is known concerning the physiological role of AQP4 in skeletal muscle and its functional and structural interaction with skeletal muscle proteome. Using AQP4-null mice, we analyzed the effect of the absence of AQP4 on the morphology and protein composition of sarcolemma as well as on the whole skeletal muscle proteome. Immunofluorescence analysis showed that the absence of AQP4 did not perturb the expression and cellular localization of the dystrophin-glycoprotein complex proteins, aside from those belonging to the extracellular matrix, and no alteration was found in sarcolemma integrity by dye extravasation assay. With the use of a 2DE-approach (BN/SDS-PAGE), protein maps revealed that in quadriceps, out of 300 Coomassie-blue detected and matched spots, 19 proteins exhibited changed expression in AQP4−/− compared to WT mice. In particular, comparison of the protein profiles revealed 12 up- and 7 down-regulated protein spots in AQP4−/− muscle. Protein identification by MS revealed that the perturbed expression pattern belongs to proteins involved in energy metabolism (i.e. GAPDH, creatine kinase), as well as in Ca2+ handling (i.e. parvalbumin, SERCA1). Western blot analysis, performed on some significantly changed proteins, validated the 2D results. Together these findings suggest AQP4 as a novel determinant in the regulation of skeletal muscle metabolism and better define the role of this water channel in skeletal muscle physiology. PMID:21552523

  6. The APSES family proteins in fungi: Characterizations, evolution and functions.

    PubMed

    Zhao, Yong; Su, Hao; Zhou, Jing; Feng, Huihua; Zhang, Ke-Qin; Yang, Jinkui

    2015-08-01

    The APSES protein family belongs to transcriptional factors of the basic helix-loop-helix (bHLH) class, the originally described members (APSES: Asm1p, Phd1p, Sok2p, Efg1p and StuAp) are used to designate this group of proteins, and they have been identified as key regulators of fungal development and other biological processes. APSES proteins share a highly conserved DNA-binding domain (APSES domain) of about 100 amino acids, whose central domain is predicted to form a typical bHLH structure. Besides APSES domain, several APSES proteins also contain additional domains, such as KilA-N and ankyrin repeats. In recent years, an increasing number of APSES proteins have been identified from diverse fungi, and they involve in numerous biological processes, such as sporulation, cellular differentiation, mycelial growth, secondary metabolism and virulence. Most fungi, including Aspergillus fumigatus, Aspergillus nidulans, Candida albicans, Fusarium graminearum, and Neurospora crassa, contain five APSES proteins. However, Cryptococcus neoformans only contains two APSES proteins, and Saccharomyces cerevisiae contains six APSES proteins. The phylogenetic analysis showed the APSES domains from different fungi were grouped into four clades (A, B, C and D), which is consistent with the result of homologous alignment of APSES domains using DNAman. The roles of APSES proteins in clade C have been studied in detail, while little is known about the roles of other APSES proteins in clades A, B and D. In this review, the biochemical properties and functional domains of APSES proteins are predicted and compared, and the phylogenetic relationship among APSES proteins from various fungi are analyzed based on the APSES domains. Moreover, the functions of APSES proteins in different fungi are summarized and discussed.

  7. Protein-protein interaction network-based detection of functionally similar proteins within species.

    PubMed

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

    2012-07-01

    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent.

  8. Involvement of F-BOX proteins in progression and development of human malignancies.

    PubMed

    Uddin, Shahab; Bhat, Ajaz A; Krishnankutty, Roopesh; Mir, Fayaz; Kulinski, Michal; Mohammad, Ramzi M

    2016-02-01

    The Ubiquitin Proteasome System (UPS) is a core regulator with various protein components (ubiquitin-activating E1 enzymes, ubiquitin-conjugating E2 enzymes, ubiquitin-protein E3 ligases, and the 26S proteasome) which work together in a coordinated fashion to ensure the appropriate and efficient proteolysis of target substrates. E3 ubiquitin ligases are essential components of the UPS machinery, working with E1 and E2 enzymes to bind substrates and assist the transport of ubiquitin molecules onto the target protein. As the UPS controls the degradation of several oncogenes and tumor suppressors, dysregulation of this pathway leads to several human malignancies. A major category of E3 Ub ligases, the SCF (Skp-Cullin-F-box) complex, is composed of four principal components: Skp1, Cul1/Cdc53, Roc1/Rbx1/Hrt1, and an F-box protein (FBP). FBPs are the substrate recognition components of SCF complexes and function as adaptors that bring substrates into physical proximity with the rest of the SCF. Besides acting as a component of SCF complexes, FBPs are involved in DNA replication, transcription, cell differentiation and cell death. This review will highlight the recent literature on three well characterized FBPs SKP2, Fbw7, and beta-TRCP. In particular, we will focus on the involvement of these deregulated FBPs in the progression and development of various human cancers. We will also highlight some novel substrates recently identified for these FBPs.

  9. Evolution and diversity of periplasmic proteins involved in copper homeostasis in gamma proteobacteria

    PubMed Central

    2012-01-01

    Background Different systems contributing to copper homeostasis in bacteria have been described in recent years involving periplasmic and transport proteins that provide resistance via metal efflux to the extracellular media (CopA/Cue, Cus, Cut, and Pco). The participation of these proteins in the assembly of membrane, periplasmic and secreted cuproproteins has also been postulated. The integration and interrelation of these systems and their apparent redundancies are less clear since they have been studied in alternative systems. Based on the idea that cellular copper is not free but rather it is transferred via protein-protein interactions, we hypothesized that systems would coevolve and be constituted by set numbers of essential components. Results By the use of a phylogenomic approach we identified the distribution of 14 proteins previously characterized as members of homeostasis systems in the genomes of 268 gamma proteobacteria. Only 3% of the genomes presented the complete systems and 5% of them, all intracellular parasites, lacked the 14 genes. Surprisingly, copper homeostatic pathways did not behave as evolutionary units with particular species assembling different combinations of basic functions. The most frequent functions, and probably because of its distribution the most vital, were copper extrusion from the cytoplasm to the periplasm performed by CopA and copper export from the cytoplasm to the extracellular space performed by CusC, which along with the remaining 12 proteins, assemble in nine different functional repertoires. Conclusions These observations suggest complex evolutionary dynamics and still unexplored interactions to achieve copper homeostasis, challenging some of the molecular transport mechanism proposed for these systems. PMID:23122209

  10. Cellular functions of gamma-secretase-related proteins.

    PubMed

    Haffner, Christof; Haass, Christian

    2006-01-01

    Amyloid-beta peptide (Abeta) is generated by gamma-secretase, a membrane protein complex with an unusual aspartyl protease activity consisting of the four components presenilin, nicastrin, APH-1 and PEN-2. Presenilin is considered the catalytic subunit of this complex since it represents the prototype of the new family of intramembrane-cleaving GxGD-type aspartyl proteases. Recently, five novel members of this family and a nicastrin-like protein were identified. Whereas one of the GxGD-type proteins was shown to be identical with signal peptide peptidase (SPP), the function of the others, now called SPP-like proteins (SPPLs), is not known. We therefore analyzed SPPL2b and SPPL3 and demonstrated that they localize to different subcellular compartments suggesting nonredundant functions. This was supported by different phenotypes obtained in knockdown studies in zebrafish embryos. In addition, these phenotypes could be phenocopied by ectopic expression of putative active site mutants, providing strong evidence for a proteolytic function of SPPL2b and SPPL3. We also identified and characterized the nicastrin-like protein nicalin which, together with the 130-kDa protein NOMO (Nodal modulator), forms a membrane protein complex different from gamma-secretase. We found that during zebrafish embryogenesis this complex is involved in the patterning of the axial mesendoderm, a process controlled by the Nodal signaling pathway.

  11. Computational approaches for inferring the functions of intrinsically disordered proteins

    PubMed Central

    Varadi, Mihaly; Vranken, Wim; Guharoy, Mainak; Tompa, Peter

    2015-01-01

    Intrinsically disordered proteins (IDPs) are ubiquitously involved in cellular processes and often implicated in human pathological conditions. The critical biological roles of these proteins, despite not adopting a well-defined fold, encouraged structural biologists to revisit their views on the protein structure-function paradigm. Unfortunately, investigating the characteristics and describing the structural behavior of IDPs is far from trivial, and inferring the function(s) of a disordered protein region remains a major challenge. Computational methods have proven particularly relevant for studying IDPs: on the sequence level their dependence on distinct characteristics determined by the local amino acid context makes sequence-based prediction algorithms viable and reliable tools for large scale analyses, while on the structure level the in silico integration of fundamentally different experimental data types is essential to describe the behavior of a flexible protein chain. Here, we offer an overview of the latest developments and computational techniques that aim to uncover how protein function is connected to intrinsic disorder. PMID:26301226

  12. Protein function prediction using neighbor relativity in protein-protein interaction network.

    PubMed

    Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

    2013-04-01

    There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network.

  13. Proteins of the corneal stroma: importance in visual function.

    PubMed

    Xuan, Meng; Wang, Shurong; Liu, Xin; He, Yuxi; Li, Ying; Zhang, Yan

    2016-04-01

    The human cornea, consisting of five layers, is the transparent tissue that refracts and transmits light to the lens and retina, providing about two thirds of the refractive power of the eye. The stroma layer comprises nearly 90 % of the thickness of the cornea and thus plays a pivotal role in normal visual function. The bulk of this layer is constituted by proteins in the extracellular martrix secreted by the corneal epithelial, stroma, and endothelial cells. Clinical research has shown that corneal stroma diseases are common and involve conditions such as infections, injuries, and genetic defects, which cause severe visual disturbances or even blindness. To improve our understanding of the basic molecular mechanisms involved in the physiological and pathological activities of the corneal stroma, its proteins have been brought into the limelight to determine their crucial and irreplaceable roles. The data presented in a previous study have demonstrated the presence of 1679 proteins in the stroma, and this data set has subsequently been perfected by utilizing a highly sensitive isobaric peptide-labeling approach. According to their manifestations, these proteins can be classified as a gel-like organic material composed of proteoglycans, enzymes, and hemocyanin-binding proteins and a network of filaments composed of collagen, elastin, keratin, vimentin, and interconnected filaments comprising fibronectin and laminin. The aim of this review is to describe some corneal stroma proteins by highlighting their major functions and valuable applications in ophthalmologic research toward the better characterization and treatment of eye diseases.

  14. Protein Nitration in Placenta – Functional Significance

    PubMed Central

    Webster, RP; Roberts, VHJ; Myatt, L

    2009-01-01

    Crucial roles of the placenta are disrupted in early and mid-trimester pregnancy loss, preeclampsia, eclampsia and intrauterine growth restriction. The pathophysiology of these disorders includes a relative hypoxia of the placenta, ischemia/reperfusion injury, an inflammatory response and oxidative stress. Reactive oxygen species including nitric oxide (NO), carbon monoxide and superoxide have been shown to participate in trophoblast invasion, regulation of placental vascular reactivity and other events. Superoxide, which regulates expression of redox sensitive genes, has been implicated in up-regulation of transcription factors, antioxidant production, angiogenesis, proliferation and matrix remodeling. When superoxide and nitric oxide are present in abundance, their interaction yields peroxynitrite a potent pro-oxidant, but also alters levels of nitric oxide, which in turn affect physiological functions. The peroxynitrite anion is extremely unstable thus evidence of its formation in vivo has been indirect via the occurrence of nitrated moieties including nitrated lipids and nitrotyrosine residues in proteins. Formation of 3-nitrotyrosine (protein nitration) is a “molecular fingerprint” of peroxynitrite formation. Protein nitration has been widely reported in a number of pathological states associated with inflammation but is reported to occur in normal physiology and is thought of as a prevalent, functionally relevant post-translational modification of proteins. Nitration of proteins can give either no effect, a gain or a loss of function. Nitration of a range of placental proteins is found in normal pregnancy but increased in pathologic pregnancies. Evidence is presented for nitration of placental signal transduction enzymes and transporters. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic settings. PMID:18851882

  15. Differentiation of HL60 cells: involvement of protein phosphorylation

    SciTech Connect

    Spearman, T.N.; Fontana, J.A.; Butcher, F.R.; Durham, J.P.

    1986-05-01

    The addition of retinoic acid (RA) to the human promyelocytic leukemic cell line HL60 in culture results in the cessation of growth and the acquisition of a more mature phenotype. Previous work in these laboratories has demonstrated a concomitant increase in the activity of calcium-dependent, phospholipid-sensitive protein kinase (PK-C). HL60 cells were incubated with /sup 32/P-P/sub i/ in the absence and presence of RA, homogenized, and aliquots subjected to two-dimensional electrophoresis. A comparison of autoradiograms made from these gels revealed several phosphoproteins whose radiolabeling was affected by RA. The radiolabeling of one particular phosphoprotein (49kd, pI 4.8) was found to be increased prior to phenotypic evidence of differentiation. It was demonstrated via incubating HL60 cytosol with /sup 32/P -ATP and Ca/sup 2 +/ in the absence and presence of phosphatidylserine and resolving the labeled proteins as above that this protein is phosphorylated by PK-C. The labeling of this protein was also increased by RA in other leukemic cell lines which showed phenotypic evidence of differentiation while no effect was seen in HL60 sublines resistant to RA or in mature neutrophils (the end product of myeloid differentiation). These results suggest that this protein may be an important intermediate in myeloid differentiation.

  16. β1-Integrin Cytoplasmic Subdomains Involved in Dominant Negative Function

    PubMed Central

    Retta, S. Francesco; Balzac, Fiorella; Ferraris, Piercarlo; Belkin, Alexey M.; Fässler, Reinhard; Humphries, Martin J.; De Leo, Giacomo; Silengo, Lorenzo; Tarone, Guido

    1998-01-01

    The β1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms (“common” region) and a distal subdomain specific for each isoform (“variable” region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used β1A and β1B isoforms as well as four mutants lacking the entire cytoplasmic domain (β1TR), the variable region (β1COM), or the common region (β1ΔCOM-B and β1ΔCOM-A). By expressing these constructs in Chinese hamster ovary and β1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227–238, 1996), we show that β1B, β1COM, β1ΔCOM-B, and β1ΔCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, β1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that β1B interferes in a dominant negative manner with β1A and β3/β5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the β1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the β1B isoform. PMID:9529373

  17. beta1-integrin cytoplasmic subdomains involved in dominant negative function.

    PubMed

    Retta, S F; Balzac, F; Ferraris, P; Belkin, A M; Fässler, R; Humphries, M J; De Leo, G; Silengo, L; Tarone, G

    1998-04-01

    The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

  18. Modular protein domains: an engineering approach toward functional biomaterials.

    PubMed

    Lin, Charng-Yu; Liu, Julie C

    2016-08-01

    Protein domains and peptide sequences are a powerful tool for conferring specific functions to engineered biomaterials. Protein sequences with a wide variety of functionalities, including structure, bioactivity, protein-protein interactions, and stimuli responsiveness, have been identified, and advances in molecular biology continue to pinpoint new sequences. Protein domains can be combined to make recombinant proteins with multiple functionalities. The high fidelity of the protein translation machinery results in exquisite control over the sequence of recombinant proteins and the resulting properties of protein-based materials. In this review, we discuss protein domains and peptide sequences in the context of functional protein-based materials, composite materials, and their biological applications.

  19. Internalization of isolated functional mitochondria: involvement of macropinocytosis

    PubMed Central

    Kitani, Tomoya; Kami, Daisuke; Matoba, Satoaki; Gojo, Satoshi

    2014-01-01

    In eukaryotic cells, mitochondrial dysfunction is associated with a variety of human diseases. Delivery of exogenous functional mitochondria into damaged cells has been proposed as a mechanism of cell transplant and physiological repair for damaged tissue. We here demonstrated that isolated mitochondria can be transferred into homogeneic and xenogeneic cells by simple co-incubation using genetically labelled mitochondria, and elucidated the mechanism and the effect of direct mitochondrial transfer. Intracellular localization of exogenous mitochondria was confirmed by PCR, real-time PCR, live fluorescence imaging, three-dimensional reconstruction imaging, continuous time-lapse microscopic observation, flow cytometric analysis and immunoelectron microscopy. Isolated homogeneic mitochondria were transferred into human uterine endometrial gland-derived mesenchymal cells in a dose-dependent manner. Moreover, mitochondrial transfer rescued the mitochondrial respiratory function and improved the cellular viability in mitochondrial DNA-depleted cells and these effects lasted several days. Finally, we discovered that mitochondrial internalization involves macropinocytosis. In conclusion, these data support direct transfer of exogenous mitochondria as a promising approach for the treatment of various diseases. PMID:24912369

  20. Internalization of isolated functional mitochondria: involvement of macropinocytosis.

    PubMed

    Kitani, Tomoya; Kami, Daisuke; Matoba, Satoaki; Gojo, Satoshi

    2014-08-01

    In eukaryotic cells, mitochondrial dysfunction is associated with a variety of human diseases. Delivery of exogenous functional mitochondria into damaged cells has been proposed as a mechanism of cell transplant and physiological repair for damaged tissue. We here demonstrated that isolated mitochondria can be transferred into homogeneic and xenogeneic cells by simple co-incubation using genetically labelled mitochondria, and elucidated the mechanism and the effect of direct mitochondrial transfer. Intracellular localization of exogenous mitochondria was confirmed by PCR, real-time PCR, live fluorescence imaging, three-dimensional reconstruction imaging, continuous time-lapse microscopic observation, flow cytometric analysis and immunoelectron microscopy. Isolated homogeneic mitochondria were transferred into human uterine endometrial gland-derived mesenchymal cells in a dose-dependent manner. Moreover, mitochondrial transfer rescued the mitochondrial respiratory function and improved the cellular viability in mitochondrial DNA-depleted cells and these effects lasted several days. Finally, we discovered that mitochondrial internalization involves macropinocytosis. In conclusion, these data support direct transfer of exogenous mitochondria as a promising approach for the treatment of various diseases.

  1. Protein function prediction using domain families

    PubMed Central

    2013-01-01

    Here we assessed the use of domain families for predicting the functions of whole proteins. These 'functional families' (FunFams) were derived using a protocol that combines sequence clustering with supervised cluster evaluation, relying on available high-quality Gene Ontology (GO) annotation data in the latter step. In essence, the protocol groups domain sequences belonging to the same superfamily into families based on the GO annotations of their parent proteins. An initial test based on enzyme sequences confirmed that the FunFams resemble enzyme (domain) families much better than do families produced by sequence clustering alone. For the CAFA 2011 experiment, we further associated the FunFams with GO terms probabilistically. All target proteins were first submitted to domain superfamily assignment, followed by FunFam assignment and, eventually, function assignment. The latter included an integration step for multi-domain target proteins. The CAFA results put our domain-based approach among the top ten of 31 competing groups and 56 prediction methods, confirming that it outperforms simple pairwise whole-protein sequence comparisons. PMID:23514456

  2. The lipocalin protein family: structure and function.

    PubMed Central

    Flower, D R

    1996-01-01

    The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at the sequence level; however, most lipocalins share three characteristic conserved sequence motifs, the kernel lipocalins, while a group of more divergent family members, the outlier lipocalins, share only one. Belying this sequence dissimilarity, lipocalin crystal structures are highly conserved and comprise a single eight-stranded continuously hydrogen-bonded antiparallel beta-barrel, which encloses an internal ligand-binding site. Together with two other families of ligand-binding proteins, the fatty-acid-binding proteins (FABPs) and the avidins, the lipocalins form part of an overall structural superfamily: the calycins. Members of the lipocalin family are characterized by several common molecular-recognition properties: the ability to bind a range of small hydrophobic molecules, binding to specific cell-surface receptors and the formation of complexes with soluble macromolecules. The varied biological functions of the lipocalins are mediated by one or more of these properties. In the past, the lipocalins have been classified as transport proteins; however, it is now clear that the lipocalins exhibit great functional diversity, with roles in retinol transport, invertebrate cryptic coloration, olfaction and pheromone transport, and prostaglandin synthesis. The lipocalins have also been implicated in the regulation of cell homoeostasis and the modulation of the immune response, and, as carrier proteins, to act in the general clearance of endogenous and exogenous compounds. PMID:8761444

  3. Senescence Marker Protein 30: Functional and Structural Insights to its Unknown Physiological Function

    PubMed Central

    Scott, Stephanie H.; Bahnson, Brian J.

    2011-01-01

    Senescence marker protein 30 (SMP30) is a multifunctional protein involved in cellular Ca2+ homeostasis and the biosynthesis of ascorbate in non-primate mammals. The primary structure of the protein is highly conserved among vertebrates, suggesting the existence of a significant physiological function common to all mammals, including primates. Enzymatic activities of SMP30 include aldonolactone and organophosphate hydrolysis. Protective effects against apoptosis and oxidative stress have been reported. X-ray crystallography revealed that SMP30 is a six-bladed β-propeller with structural similarity to paraoxonase 1, another protein with lactonase and organophosphate hydrolase activities. SMP30 has recently been tied to several physiological conditions including osteoporosis, liver fibrosis, diabetes, and cancer. This review aims to describe the recent advances made toward understanding the connection between molecular structure, enzymatic activity and physiological function of this highly conserved, multifaceted protein. PMID:22844387

  4. Hierarchical Ensemble Methods for Protein Function Prediction

    PubMed Central

    2014-01-01

    Protein function prediction is a complex multiclass multilabel classification problem, characterized by multiple issues such as the incompleteness of the available annotations, the integration of multiple sources of high dimensional biomolecular data, the unbalance of several functional classes, and the difficulty of univocally determining negative examples. Moreover, the hierarchical relationships between functional classes that characterize both the Gene Ontology and FunCat taxonomies motivate the development of hierarchy-aware prediction methods that showed significantly better performances than hierarchical-unaware “flat” prediction methods. In this paper, we provide a comprehensive review of hierarchical methods for protein function prediction based on ensembles of learning machines. According to this general approach, a separate learning machine is trained to learn a specific functional term and then the resulting predictions are assembled in a “consensus” ensemble decision, taking into account the hierarchical relationships between classes. The main hierarchical ensemble methods proposed in the literature are discussed in the context of existing computational methods for protein function prediction, highlighting their characteristics, advantages, and limitations. Open problems of this exciting research area of computational biology are finally considered, outlining novel perspectives for future research. PMID:25937954

  5. Modification of sorghum proteins for enhanced functionality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum is the third most widely produced crop in the United States (U.S.) and fifth in the world during fiscal year 2006/07(USDA-FAS, 2007). The use of sorghum in foods faces functional and nutritional constraints due, mainly, to the rigidity of the protein bodies. The disruption and modificatio...

  6. A Novel RNA-Binding Protein Involves ABA Signaling by Post-transcriptionally Repressing ABI2

    PubMed Central

    Xu, Jianwen; Chen, Yihan; Qian, Luofeng; Mu, Rong; Yuan, Xi; Fang, Huimin; Huang, Xi; Xu, Enshun; Zhang, Hongsheng; Huang, Ji

    2017-01-01

    The Stress Associated RNA-binding protein 1 (SRP1) repressed by ABA, salt and cold encodes a C2C2-type zinc finger protein in Arabidopsis. The knock-out mutation in srp1 reduced the sensitivity of seed to ABA and salt stress during germination and post-germinative growth stages. In contrast, SRP1-overexpressing seedlings were more sensitive to ABA and salt compared to wild type plants. In the presence of ABA, the transcript levels of ABA signaling and germination-related genes including ABI3. ABI5. EM1 and EM6 were less induced in srp1 compared to WT. Interestingly, expression of ABI2 encoding a protein phosphatase 2C protein were significantly up-regulated in srp1 mutants. By in vitro analysis, SRP1 was identified as a novel RNA-binding protein directly binding to 3′UTR of ABI2 mRNA. Moreover, transient expression assay proved the function of SRP1 in reducing the activity of luciferase whose coding sequence was fused with the ABI2 3’UTR. Together, it is suggested that SRP1 is involved in the ABA signaling by post-transcriptionally repressing ABI2 expression in Arabidopsis. PMID:28174577

  7. Posttranslational Modification Assays on Functional Protein Microarrays.

    PubMed

    Neiswinger, Johnathan; Uzoma, Ijeoma; Cox, Eric; Rho, HeeSool; Jeong, Jun Seop; Zhu, Heng

    2016-10-03

    Protein microarray technology provides a straightforward yet powerful strategy for identifying substrates of posttranslational modifications (PTMs) and studying the specificity of the enzymes that catalyze these reactions. Protein microarray assays can be designed for individual enzymes or a mixture to establish connections between enzymes and substrates. Assays for four well-known PTMs-phosphorylation, acetylation, ubiquitylation, and SUMOylation-have been developed and are described here for use on functional protein microarrays. Phosphorylation and acetylation require a single enzyme and are easily adapted for use on an array. The ubiquitylation and SUMOylation cascades are very similar, and the combination of the E1, E2, and E3 enzymes plus ubiquitin or SUMO protein and ATP is sufficient for in vitro modification of many substrates.

  8. Purification of IFT particle proteins and preparation of recombinant proteins for structural and functional analysis.

    PubMed

    Behal, Robert H; Betleja, Ewelina; Cole, Douglas G

    2009-01-01

    Intraflagellar transport (IFT) is characterized by a robust bidirectional movement of large proteinaceous particles along the length of eukaryotic cilia and flagella. Essential for the assembly and function of the organelle, IFT is believed to transport a large array of ciliary components in and out of the organelle. Biochemical analysis of the proteins involved with this transport has been largely dependent on the ability to isolate suitable quantities of intact cilia or flagella. One model organism, Chlamydomonas reinhardtii, has proven to be especially well-suited for such endeavors. Indeed, many of the IFT particle proteins were initially identified through biochemical analysis of green algae. This chapter describes some of the most effective methods for the purification of IFT particle proteins from Chlamydomonas flagella. This chapter also describes complementary approaches where recombinant IFT proteins are generated with affinity tags that allow rapid and specific purification. The recombinant proteins can be used to analyze protein-protein interactions and can be directly delivered to mutant cells to analyze functional domains. Although the techniques described here are focused entirely on Chlamydomonas IFT proteins, the approaches, especially regarding recombinant proteins, should be applicable to the study of IFT machinery in other model organisms.

  9. Involvement of Protein Kinase C-δ in Vascular Permeability in Acute Lung Injury.

    PubMed

    Ahn, Jong J; Jung, Jong P; Park, Soon E; Lee, Minhyun; Kwon, Byungsuk; Cho, Hong R

    2015-08-01

    Pulmonary edema is a major cause of mortality due to acute lung injury (ALI). The involvement of protein kinase C-δ (PKC-δ) in ALI has been a controversial topic. Here we investigated PKC-δ function in ALI using PKC-δ knockout (KO) mice and PKC inhibitors. Our results indicated that although the ability to produce proinflammatory mediators in response to LPS injury in PKC-δ KO mice was similar to that of control mice, they showed enhanced recruitment of neutrophils to the lung and more severe pulmonary edema. PKC-δ inhibition promoted barrier dysfunction in an endothelial cell layer in vitro, and administration of a PKC-δ-specific inhibitor significantly increased steady state vascular permeability. A neutrophil transmigration assay indicated that the PKC-δ inhibition increased neutrophil transmigration through an endothelial monolayer. This suggests that PKC-δ inhibition induces structural changes in endothelial cells, allowing extravasation of proteins and neutrophils.

  10. The H-NS protein is involved in the biogenesis of flagella in Escherichia coli.

    PubMed Central

    Bertin, P; Terao, E; Lee, E H; Lejeune, P; Colson, C; Danchin, A; Collatz, E

    1994-01-01

    The function of the flagellum-chemotaxis regulon requires the expression of many genes and is positively regulated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex. In this paper, we show that motile behavior was affected in Escherichia coli hns mutants. The loss of motility resulted from a complete lack of flagella. A decrease in the level of transcription of the flhD and fliA genes, which are both required for the synthesis of flagella, was observed in the presence of an hns mutation. Furthermore, the Fla- phenotype was not reversed to the wild type in the presence of a cfs mutation which renders the flagellum synthesis independent of the cAMP-CAP complex. These results suggest that the H-NS protein acts as a positive regulator of genes involved in the biogenesis of flagella by a mechanism independent of the cAMP-CAP pathway. Images PMID:8071234

  11. Diversity and functions of protein glycosylation in insects.

    PubMed

    Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

    2017-04-01

    The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes.

  12. Characterization and Modulation of Proteins Involved in Sulfur Mustard Vesication

    DTIC Science & Technology

    2000-06-01

    SM may induce apoptosis as well. Recent evidence has revealed that Bcl-2 can complex with both the Caenorhabditis elegans death proteins 3 and 4 (Ced-3...the product of a gene required for programmed Tris-HCl (pH 6.8), and 0.02% bromophenol blue. Samples were re- cell death in Caenorhabditis elegans [17...biochemical or mor- which is required for apoptosis in Caenorhabditis elegans (8). phological changes characteristic of apoptosis when In human

  13. cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-Telomeric Roles of Arabidopsis Telomerase.

    PubMed

    Dokládal, Ladislav; Honys, David; Rana, Rajiv; Lee, Lan-Ying; Gelvin, Stanton B; Sýkorová, Eva

    2015-01-01

    Telomerase-reverse transcriptase (TERT) plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE) TERT domain and identified a nuclear-localized protein that contains an RNA recognition motif (RRM). This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

  14. Development of neurodevelopmental disorders: a regulatory mechanism involving bromodomain-containing proteins.

    PubMed

    Li, Junlin; Zhao, Guifang; Gao, Xiaocai

    2013-02-20

    Neurodevelopmental disorders are classified as diseases that cause abnormal functions of the brain or central nervous system. Children with neurodevelopmental disorders show impaired language and speech abilities, learning and memory damage, and poor motor skills. However, we still know very little about the molecular etiology of these disorders. Recent evidence implicates the bromodomain-containing proteins (BCPs) in the initiation and development of neurodevelopmental disorders. BCPs have a particular domain, the bromodomain (Brd), which was originally identified as specifically binding acetyl-lysine residues at the N-terminus of histone proteins in vitro and in vivo. Other domains of BCPs are responsible for binding partner proteins to form regulatory complexes. Once these complexes are assembled, BCPs alter chromosomal states and regulate gene expression. Some BCP complexes bind nucleosomes, are involved in basal transcription regulation, and influence the transcription of many genes. However, most BCPs are involved in targeting. For example, some BCPs function as a recruitment platform or scaffold through their Brds-binding targeting sites. Others are recruited to form a complex to bind the targeting sites of their partners. The regulation mediated by these proteins is especially critical during normal and abnormal development. Mutant BCPs or dysfunctional BCP-containing complexes are implicated in the initiation and development of neurodevelopmental disorders. However, the pathogenic molecular mechanisms are not fully understood. In this review, we focus on the roles of regulatory BCPs associated with neurodevelopmental disorders, including mental retardation, Fragile X syndrome (FRX), Williams syndrome (WS), Rett syndrome and Rubinstein-Taybi syndrome (RTS). A better understanding of the molecular pathogenesis, based upon the roles of BCPs, will lead to screening of targets for the treatment of neurodevelopmental disorders.

  15. An Ehrlichia chaffeensis tandem repeat protein interacts with multiple host targets involved in cell signaling, transcriptional regulation, and vesicle trafficking.

    PubMed

    Wakeel, Abdul; Kuriakose, Jeeba A; McBride, Jere W

    2009-05-01

    Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes forming cytoplasmic membrane-bound microcolonies called morulae. To survive and replicate within phagocytes, E. chaffeensis exploits the host cell by modulating a number of host cell processes, but the ehrlichial effector proteins involved are unknown. In this study, we determined that p47, a secreted, differentially expressed, tandem repeat (TR) protein, interacts with multiple host proteins associated with cell signaling, transcriptional regulation, and vesicle trafficking. Yeast two-hybrid analysis revealed that p47 interacts with polycomb group ring finger 5 (PCGF5) protein, Src protein tyrosine kinase FYN (FYN), protein tyrosine phosphatase non-receptor type 2 (PTPN2), and adenylate cyclase-associated protein 1 (CAP1). p47 interaction with these proteins was further confirmed by coimmunoprecipitation assays and colocalization in HeLa cells transfected with p47-green fluorescent fusion protein (AcGFP1-p47). Moreover, confocal microscopy demonstrated p47-expressing dense-cored (DC) ehrlichiae colocalized with PCGF5, FYN, PTPN2, and CAP1. An amino-terminally truncated form of p47 containing TRs interacted only with PCGF5 and not with FYN, PTPN2, and CAP1, indicating differences in p47 domains that are involved in these interactions. These results demonstrate that p47 is involved in a complex network of interactions involving numerous host cell proteins. Furthermore, this study provides a new insight into the molecular and functional distinction of DC ehrlichiae, as well as the effector proteins involved in facilitating ehrlichial survival in mononuclear phagocytes.

  16. Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

    PubMed

    Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

    2014-01-01

    G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.

  17. Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase.

    PubMed Central

    Xu, Kui; Belunis, Charles; Chu, Wei; Weber, David; Podlaski, Frank; Huang, Kuo-Sen; Reed, Steven I; Vassilev, Lyubomir T

    2003-01-01

    The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2. PMID:12529174

  18. SitesIdentify: a protein functional site prediction tool

    PubMed Central

    2009-01-01

    Background The rate of protein structures being deposited in the Protein Data Bank surpasses the capacity to experimentally characterise them and therefore computational methods to analyse these structures have become increasingly important. Identifying the region of the protein most likely to be involved in function is useful in order to gain information about its potential role. There are many available approaches to predict functional site, but many are not made available via a publicly-accessible application. Results Here we present a functional site prediction tool (SitesIdentify), based on combining sequence conservation information with geometry-based cleft identification, that is freely available via a web-server. We have shown that SitesIdentify compares favourably to other functional site prediction tools in a comparison of seven methods on a non-redundant set of 237 enzymes with annotated active sites. Conclusion SitesIdentify is able to produce comparable accuracy in predicting functional sites to its closest available counterpart, but in addition achieves improved accuracy for proteins with few characterised homologues. SitesIdentify is available via a webserver at http://www.manchester.ac.uk/bioinformatics/sitesidentify/ PMID:19922660

  19. DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions

    PubMed Central

    Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

    2016-01-01

    Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. PMID:27604871

  20. Experimental and bioinformatic approaches for interrogating protein-protein interactions to determine protein function.

    PubMed

    Droit, Arnaud; Poirier, Guy G; Hunter, Joanna M

    2005-04-01

    An ambitious goal of proteomics is to elucidate the structure, interactions and functions of all proteins within cells and organisms. One strategy to determine protein function is to identify the protein-protein interactions. The increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. A challenge for bioinformatics is to explore this disparate data and to uncover biologically relevant interactions and pathways. In parallel, there is clearly a need for the development of approaches that can predict novel protein-protein interaction networks in silico. Here, we present an overview of different experimental and bioinformatic methods to elucidate protein-protein interactions.

  1. Not all mitochondrial carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1.

    PubMed

    Crichton, Paul G; Parker, Nadeene; Vidal-Puig, Antonio J; Brand, Martin D

    2009-12-15

    The mPTP (mitochondrial permeability transition pore) is a non-specific channel that is formed in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltage-dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the mitochondrial carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in de-energized mitochondria from wild-type and Ucp1KO (Ucp1-knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80% inhibited, compared with approximately 60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT.

  2. Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

    PubMed Central

    Sheu, Yi-Jun; Santos, Beatriz; Fortin, Nathalie; Costigan, Christine; Snyder, Michael

    1998-01-01

    The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Δ, pea2Δ, and bud6Δ mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain

  3. A VAMP-associated protein, PVA31 is involved in leaf senescence in Arabidopsis

    PubMed Central

    Ichikawa, Mie; Nakai, Yusuke; Arima, Keita; Nishiyama, Sayo; Hirano, Tomoko; Sato, Masa H

    2015-01-01

    VAMP-associated proteins (VAPs) are highly conserved among eukaryotes. Here, we report a functional analysis of one of the VAPs, PVA31, and demonstrate its novel function on leaf senescence in Arabidopsis. The expression of PVA31 is highly induced in senescence leaves, and localizes to the plasma membrane as well as the ARA7-positive endosomes. Yeast two-hybrid analysis demonstrates that PVA31 is interacted with the plasma membrane localized-VAMP proteins, VAMP721/722/724 but not with the endosome-localized VAMPs, VAMP711 and VAMP727, indicating that PVA31 is associated with VAMP721/722/724 on the plasma membrane. Strong constitutive expression of PVA31 under the control of the Cauliflower mosaic virus 35S promoter induces the typical symptom of leaf senescence earlier than WT in normal growth and an artificially induced senescence conditions. In addition, the marker genes for the SA-mediated signaling pathways, PR-1, is promptly expressed with elicitor application. These data indicate that PVA31-overexpressing plants exhibit the early senescence phenotype in their leaves, and suggest that PVA31 is involved in the SA-mediated programmed cell death process during leaf senescence and PR-protein secretion during pathogen infection in Arabidopsis. PMID:25897470

  4. Arabidopsis Membrane Steroid Binding Protein 1 Is Involved in Inhibition of Cell ElongationW⃞

    PubMed Central

    Yang, Xiao-Hua; Xu, Zhi-Hong; Xue, Hong-Wei

    2005-01-01

    A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220–amino acid protein that can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants. PMID:15608331

  5. Shewanella oneidensis MR-1 sensory box protein involved in aerobic and anoxic growth.

    PubMed

    Sundararajan, A; Kurowski, J; Yan, T; Klingeman, D M; Joachimiak, M P; Zhou, J; Naranjo, B; Gralnick, J A; Fields, M W

    2011-07-01

    Although little is known of potential function for conserved signaling proteins, it is hypothesized that such proteins play important roles to coordinate cellular responses to environmental stimuli. In order to elucidate the function of a putative sensory box protein (PAS domains) in Shewanella oneidensis MR-1, the physiological role of SO3389 was characterized. The predicted open reading frame (ORF) encodes a putative sensory box protein that has PAS, GGDEF, and EAL domains, and an in-frame deletion mutant was constructed (ΔSO3389) with approximately 95% of the ORF deleted. Under aerated conditions, wild-type and mutant cultures had similar growth rates, but the mutant culture had a lower growth rate under static, aerobic conditions. Oxygen consumption rates were lower for mutant cultures (1.5-fold), and wild-type cultures also maintained lower dissolved oxygen concentrations under aerated growth conditions. When transferred to anoxic conditions, the mutant did not grow with fumarate, iron(III), or dimethyl sulfoxide (DMSO) as electron acceptors. Biochemical assays demonstrated the expression of different c-type cytochromes as well as decreased fumarate reductase activity in the mutant transferred to anoxic growth conditions. Transcriptomic studies showed the inability of the mutant to up-express and down-express genes, including c-type cytochromes (e.g., SO4047/SO4048, SO3285/SO3286), reductases (e.g., SO0768, SO1427), and potential regulators (e.g., SO1329). The complemented strain was able to grow when transferred from aerobic to anoxic growth conditions with the tested electron acceptors. The modeled structure for the SO3389 PAS domains was highly similar to the crystal structures of FAD-binding PAS domains that are known O2/redox sensors. Based on physiological, genomic, and bioinformatic results, we suggest that the sensory box protein, SO3389, is an O2/redox sensor that is involved in optimization of aerobic growth and transitions to anoxia in S

  6. Folding funnels, binding funnels, and protein function.

    PubMed Central

    Tsai, C. J.; Kumar, S.; Ma, B.; Nussinov, R.

    1999-01-01

    Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly

  7. Functional protein microarrays by electrohydrodynamic jet printing.

    PubMed

    Shigeta, Kazuyo; He, Ying; Sutanto, Erick; Kang, Somi; Le, An-Phong; Nuzzo, Ralph G; Alleyne, Andrew G; Ferreira, Placid M; Lu, Yi; Rogers, John A

    2012-11-20

    This paper reports the use of advanced forms of electrohydrodynamic jet (e-jet) printing for creating micro- and nanoscale patterns of proteins on various surfaces ranging from flat silica substrates to structured plasmonic crystals, suitable for micro/nanoarray analysis and other applications in both fluorescent and plasmonic detection modes. The approaches function well with diverse classes of proteins, including streptavidin, IgG, fibrinogen, and γ-globulin. Detailed study reveals that the printing process does not adversely alter the protein structure or function, as demonstrated in the specific case of streptavidin through measurements of its binding specificity to biotin-modified DNA. Multinozzle printing systems enable several types of proteins (up to four currently) to be patterned on a single substrate, in rapid fashion and with excellent control over spatial dimensions and registration. High-speed, pulsed operational modes allow large-area printing, with narrow statistical distributions of drop size and spacing in patterns that include millions of droplets. The process is also compatible with the structured surfaces of plasmonic crystal substrates to enable detection without fluorescence. These collective characteristics suggest potential utility of e-jet techniques in wide-ranging areas of biotechnology, where its compatibility with various biomaterials and substrates with different topographies and surface chemistries, and ability to form deposits that range from thick films to submonolayer coatings, derive from the remote, noncontacting physical material transfer mode of operation.

  8. Lipid transfer proteins: classification, nomenclature, structure, and function.

    PubMed

    Salminen, Tiina A; Blomqvist, Kristina; Edqvist, Johan

    2016-11-01

    The non-specific lipid transfer proteins (LTPs) constitute a large protein family found in all land plants. They are small proteins characterized by a tunnel-like hydrophobic cavity, which makes them suitable for binding and transporting various lipids. The LTPs are abundantly expressed in most tissues. In general, they are synthesized with an N-terminal signal peptide that localizes the protein to spaces exterior to the plasma membrane. The in vivo functions of LTPs are still disputed, although evidence has accumulated for a role in the synthesis of lipid barrier polymers, such as cuticular waxes, suberin, and sporopollenin. There are also reports suggesting that LTPs are involved in signaling during pathogen attacks. LTPs are considered as key proteins for the plant's survival and colonization of land. In this review, we aim to present an overview of the current status of LTP research and also to discuss potential future applications of these proteins. We update the knowledge on 3D structures and lipid binding and review the most recent data from functional investigations, such as from knockout or overexpressing experiments. We also propose and argument for a novel system for the classification and naming of the LTPs.

  9. Using co-expression analysis and stress-based screens to uncover Arabidopsis peroxisomal proteins involved in drought response

    DOE PAGES

    Li, Jiying; Hu, Jianping; Bassham, Diane

    2015-09-14

    Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Usingmore » microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their coexpression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Lastly, our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to

  10. Using co-expression analysis and stress-based screens to uncover Arabidopsis peroxisomal proteins involved in drought response

    SciTech Connect

    Li, Jiying; Hu, Jianping; Bassham, Diane

    2015-09-14

    Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Using microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their coexpression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Lastly, our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to

  11. Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major

    PubMed Central

    Aoki, Juliana Ide; Coelho, Adriano Cappellazzo; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Sanchez, Eduardo Milton Ramos; Nerland, Audun Helge; Floeter-Winter, Lucile Maria; Cotrim, Paulo Cesar

    2016-01-01

    Background Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. Methodology/Principal findings After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. Conclusions/Significance This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how

  12. A Wiskott-Aldrich syndrome protein is involved in endocytosis in Aspergillus nidulans.

    PubMed

    Hoshi, Hiro-Omi; Zheng, Lu; Ohta, Akinori; Horiuchi, Hiroyuki

    2016-09-01

    Endocytosis is vital for hyphal tip growth in filamentous fungi and is involved in the tip localization of various membrane proteins. To investigate the function of a Wiskott-Aldrich syndrome protein (WASP) in endocytosis of filamentous fungi, we identified a WASP ortholog-encoding gene, wspA, in Aspergillus nidulans and characterized it. The wspA product, WspA, localized to the tips of germ tubes during germination and actin rings in the subapical regions of mature hyphae. wspA is essential for the growth and functioned in the polarity establishment and maintenance during germination of conidia. We also investigated its function in endocytosis and revealed that endocytosis of SynA, a synaptobrevin ortholog that is known to be endocytosed at the subapical regions of hyphal tips in A. nidulans, did not occur when wspA expression was repressed. These results suggest that WspA plays roles in endocytosis at hyphal tips and polarity establishment during germination.

  13. PARP12, an interferon-stimulated gene involved in the control of protein translation and inflammation.

    PubMed

    Welsby, Iain; Hutin, David; Gueydan, Cyril; Kruys, Veronique; Rongvaux, Anthony; Leo, Oberdan

    2014-09-19

    Transcriptome analyses have recently identified PARP12, a member of a large family of ADP-ribosyl transferases, as an interferon-induced gene (ISG), whose function remains incompletely characterized. We demonstrate herein that PARP12 is a genuine ISG, whose expressed protein displays at least two distinct subcellular locations and related functions. Upon ectopic expression or exposure to oxidative stress, PARP12 is recruited to stress-granules (SGs), known sites of mRNA translational arrest. Accordingly, PARP12 was found to block mRNA translation, possibly upon association to the translational machinery. Both the N-terminal domain (containing an RNA-binding domain characterized by the presence of five CCCH-type Zn-fingers) and integrity of the catalytic domain are required for this suppressive function. In contrast, stimulation with LPS leads to the localization of PARP12 to p62/SQSTM1 (an adaptor protein involved in innate signaling and autophagy) containing structures, unrelated to SGs. Deletion of the N-terminal domain promotes the association of the protein to p62/SQSTM1, suggesting that the RNA-binding domain is responsible for the subcellular localization of PARP12. Association to p62/SQSTM1 was found to correlate with increased NF-κB signaling, suggesting a role for PARP12 in inflammation. Collectively, these observations suggest that PARP12 can alternate between two distinct subcellular compartments associated to two distinct cellular functions. The present work therefore identifies PARP12 as an ISG with a potential role in cellular defenses against viral infections.

  14. Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid

    SciTech Connect

    Guo, Jianjun; Wang, Shucai; Valerius, Oliver; Hall, Hardy; Zeng, Qingning; Li, Jian-Feng; Weston, David; Ellis, Brian; Chen, Jay

    2011-01-01

    Earlier studies have shown that RACK1 functions as a negative regulator of ABA responses in Arabidopsis, but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, co-expression analysis revealed that >80% of the genes co-expressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1 s function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and yeast RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B and RACK1C, complemented the growth defects of the S. cerevisiae cpc2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor 6 (eIF6), whose mammalian homologue is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.

  15. The Amyloid Precursor Protein Controls PIKfyve Function.

    PubMed

    Balklava, Zita; Niehage, Christian; Currinn, Heather; Mellor, Laura; Guscott, Benjamin; Poulin, Gino; Hoflack, Bernard; Wassmer, Thomas

    2015-01-01

    While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease.

  16. Domain mobility in proteins: functional and evolutionary implications.

    PubMed

    Basu, Malay Kumar; Poliakov, Eugenia; Rogozin, Igor B

    2009-05-01

    A substantial fraction of eukaryotic proteins contains multiple domains, some of which show a tendency to occur in diverse domain architectures and can be considered mobile (or 'promiscuous'). These promiscuous domains are typically involved in protein-protein interactions and play crucial roles in interaction networks, particularly those contributing to signal transduction. They also play a major role in creating diversity of protein domain architecture in the proteome. It is now apparent that promiscuity is a volatile and relatively fast-changing feature in evolution, and that only a few domains retain their promiscuity status throughout evolution. Many such domains attained their promiscuity status independently in different lineages. Only recently, we have begun to understand the diversity of protein domain architectures and the role the promiscuous domains play in evolution of this diversity. However, many of the biological mechanisms of protein domain mobility remain shrouded in mystery. In this review, we discuss our present understanding of protein domain promiscuity, its evolution and its role in cellular function.

  17. Functionalized nanoparticle probes for protein detection

    NASA Astrophysics Data System (ADS)

    Park, Do Hyun; Lee, Jae-Seung

    2015-05-01

    In this Review, we discuss representative studies of recent advances in the development of nanoparticle-based protein detection methods, with a focus on the properties and functionalization of nanoparticle probes, as well as their use in detection schemes. We have focused on functionalized nanoparticle probes because they offer a number of advantages over conventional assays and because their use for detecting protein targets for diagnostic purposed has been demonstrated. In this report, we discuss nanoparticle probes classified by material type (gold, silver, silica, semiconductor, carbon, and virus) and surface functionality (antibody, aptamer, and DNA), which play a critical role in enhancing the sensitivity, selectivity, and efficiency of the detection systems. In particular, the synergistic function of each component of the nanoparticle probe is emphasized in terms of specific chemical and physical properties. This research area is in its early stages with many milestones to reach before nanoparticle probes are successfully applied in the field; however, the substantial ongoing efforts of researchers underline the great promise offered by nanoparticlebased probes for future applications. [Figure not available: see fulltext.

  18. Photoreactive synthetic regulator of protein function and methods of use thereof

    SciTech Connect

    Trauner, Dirk; Isacoff, Ehud Y; Kramer, Richard H; Banghart, Matthew R; Fortin, Doris L; Mourot, Alexandre

    2015-03-31

    The present disclosure provides a photoreactive synthetic regulator of protein function. The present disclosure further provides a light-regulated polypeptide that includes a subject synthetic regulator. Also provided are cells and membranes comprising a subject light-regulated polypeptide. The present disclosure further provides methods of modulating protein function, involving use of light.

  19. Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors*

    PubMed Central

    Cameron, Kate; Najmudin, Shabir; Alves, Victor D.; Bayer, Edward A.; Smith, Steven P.; Bule, Pedro; Waller, Helen; Ferreira, Luís M. A.; Gilbert, Harry J.; Fontes, Carlos M. G. A.

    2015-01-01

    Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼1012 m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes. PMID:25855788

  20. Functional characterization of the alphavirus TF protein.

    PubMed

    Snyder, Jonathan E; Kulcsar, Kirsten A; Schultz, Kimberly L W; Riley, Catherine P; Neary, Jacob T; Marr, Scott; Jose, Joyce; Griffin, Diane E; Kuhn, Richard J

    2013-08-01

    Alphavirus dogma has long dictated the production of a discrete set of structural proteins during infection of a cell: capsid, pE2, 6K, and E1. However, bioinformatic analyses of alphavirus genomes (A. E. Firth, B. Y. Chung, M. N. Fleeton, and J. F. Atkins, Virol. J. 5:108, 2008) suggested that a ribosomal frameshifting event occurs during translation of the alphavirus structural polyprotein. Specifically, a frameshift event is suggested to occur during translation of the 6K gene, yielding production of a novel protein, termed transframe (TF), comprised of a C-terminal extension of the 6K protein in the -1 open reading frame (ORF). Here, we validate the findings of Firth and colleagues with respect to the production of the TF protein and begin to characterize the function of TF. Using a mass spectrometry-based approach, we identified TF in purified preparations of both Sindbis and Chikungunya virus particles. We next constructed a panel of Sindbis virus mutants with mutations which alter the production, size, or sequence of TF. We demonstrate that TF is not absolutely required in culture, although disrupting TF production leads to a decrease in virus particle release in both mammalian and insect cells. In a mouse neuropathogenesis model, mortality was <15% in animals infected with the TF mutants, whereas mortality was 95% in animals infected with the wild-type virus. Using a variety of additional assays, we demonstrate that TF retains ion-channel activity analogous to that of 6K and that lack of production of TF does not affect genome replication, particle infectivity, or envelope protein transit to the cell surface. The TF protein therefore represents a previously uncharacterized factor important for alphavirus assembly.

  1. Effects of Radiation and Dietary Iron on Expression of Genes and Proteins Involved in Drug Metabolism

    NASA Technical Reports Server (NTRS)

    Faust, K. M.; Wotring, V. E.

    2014-01-01

    Liver function, especially the rate of metabolic enzyme activities, determines the concentration of circulating drugs and the duration of their efficacy. Most pharmaceuticals are metabolized by the liver, and clinically-used medication doses are given with normal liver function in mind. A drug overdose can result in the case of a liver that is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism, we want to understand any effects of spaceflight on the enzymes of the liver. Dietary factors and exposure to radiation are aspects of spaceflight that are potential oxidative stressors and both can be modeled in ground experiments. In this experiment, we examined the effects of high dietary iron and low dose gamma radiation (individually and combined) on the gene expression of enzymes involved in drug metabolism, redox homeostasis, and DNA repair. METHODS All procedures were approved by the JSC Animal Care and Use Committee. Male Sprague-Dawley rats were divided into 4 groups (n=8); control, high Fe diet (650 mg iron/kg), radiation (fractionated 3 Gy exposure from a Cs- 137 source) and combined high Fe diet + radiation exposure. Animals were euthanized 24h after the last treatment of radiation; livers were removed immediately and flash -frozen in liquid nitrogen. Expression of genes thought to be involved in redox homeostasis, drug metabolism and DNA damage repair was measured by RT-qPCR. Where possible, protein expression of the same genes was measured by western blotting. All data are expressed as % change in expression normalized to reference gene expression; comparisons were then made of each treatment group to the sham exposed/ normal diet control group. Data was considered significant at p< 0

  2. Defining the boundaries: structure and function of LOB domain proteins.

    PubMed

    Majer, Christine; Hochholdinger, Frank

    2011-01-01

    The plant-specific LBD (Lateral Organ Boundaries Domain) gene family is essential in the regulation of plant lateral organ development and is involved in the regulation of anthocyanin and nitrogen metabolism. LBD proteins contain a characteristic LOB domain composed of a C-motif required for DNA-binding, a conserved glycine residue, and a leucine-zipper-like sequence required for protein-protein interactions. Recently, several LBD genes associated with mutant phenotypes related to almost all aspects of plant development, including embryo, root, leaf, and inflorescence development have been functionally characterized. These novel insights contribute to a better understanding of the molecular definition of boundaries between organs or boundaries between organs and meristems and the regulation of these processes by environmental cues and phytohormones.

  3. Targeting functional motifs of a protein family

    NASA Astrophysics Data System (ADS)

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β -lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β -lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β -lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  4. Targeting functional motifs of a protein family.

    PubMed

    Bhadola, Pradeep; Deo, Nivedita

    2016-10-01

    The structural organization of a protein family is investigated by devising a method based on the random matrix theory (RMT), which uses the physiochemical properties of the amino acid with multiple sequence alignment. A graphical method to represent protein sequences using physiochemical properties is devised that gives a fast, easy, and informative way of comparing the evolutionary distances between protein sequences. A correlation matrix associated with each property is calculated, where the noise reduction and information filtering is done using RMT involving an ensemble of Wishart matrices. The analysis of the eigenvalue statistics of the correlation matrix for the β-lactamase family shows the universal features as observed in the Gaussian orthogonal ensemble (GOE). The property-based approach captures the short- as well as the long-range correlation (approximately following GOE) between the eigenvalues, whereas the previous approach (treating amino acids as characters) gives the usual short-range correlations, while the long-range correlations are the same as that of an uncorrelated series. The distribution of the eigenvector components for the eigenvalues outside the bulk (RMT bound) deviates significantly from RMT observations and contains important information about the system. The information content of each eigenvector of the correlation matrix is quantified by introducing an entropic estimate, which shows that for the β-lactamase family the smallest eigenvectors (low eigenmodes) are highly localized as well as informative. These small eigenvectors when processed gives clusters involving positions that have well-defined biological and structural importance matching with experiments. The approach is crucial for the recognition of structural motifs as shown in β-lactamase (and other families) and selectively identifies the important positions for targets to deactivate (activate) the enzymatic actions.

  5. Mechanosensitive Molecular Networks Involved in Transducing Resistance Exercise-Signals into Muscle Protein Accretion

    PubMed Central

    Rindom, Emil; Vissing, Kristian

    2016-01-01

    Loss of skeletal muscle myofibrillar protein with disease and/or inactivity can severely deteriorate muscle strength and function. Strategies to counteract wasting of muscle myofibrillar protein are therefore desirable and invite for considerations on the potential superiority of specific modes of resistance exercise and/or the adequacy of low load resistance exercise regimens as well as underlying mechanisms. In this regard, delineation of the potentially mechanosensitive molecular mechanisms underlying muscle protein synthesis (MPS), may contribute to an understanding on how differentiated resistance exercise can transduce a mechanical signal into stimulation of muscle accretion. Recent findings suggest specific upstream exercise-induced mechano-sensitive myocellular signaling pathways to converge on mammalian target of rapamycin complex 1 (mTORC1), to influence MPS. This may e.g. implicate mechanical activation of signaling through a diacylglycerol kinase (DGKζ)-phosphatidic acid (PA) axis or implicate integrin deformation to signal through a Focal adhesion kinase (FAK)-Tuberous Sclerosis Complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) axis. Moreover, since initiation of translation is reliant on mRNA, it is also relevant to consider potentially mechanosensitive signaling pathways involved in muscle myofibrillar gene transcription and whether some of these pathways converge with those affecting mTORC1 activation for MPS. In this regard, recent findings suggest how mechanical stress may implicate integrin deformation and/or actin dynamics to signal through a Ras homolog gene family member A protein (RhoA)-striated muscle activator of Rho signaling (STARS) axis or implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP) signaling through a small mother of decapentaplegic (Smad) axis. PMID:27909410

  6. Mechanosensitive Molecular Networks Involved in Transducing Resistance Exercise-Signals into Muscle Protein Accretion.

    PubMed

    Rindom, Emil; Vissing, Kristian

    2016-01-01

    Loss of skeletal muscle myofibrillar protein with disease and/or inactivity can severely deteriorate muscle strength and function. Strategies to counteract wasting of muscle myofibrillar protein are therefore desirable and invite for considerations on the potential superiority of specific modes of resistance exercise and/or the adequacy of low load resistance exercise regimens as well as underlying mechanisms. In this regard, delineation of the potentially mechanosensitive molecular mechanisms underlying muscle protein synthesis (MPS), may contribute to an understanding on how differentiated resistance exercise can transduce a mechanical signal into stimulation of muscle accretion. Recent findings suggest specific upstream exercise-induced mechano-sensitive myocellular signaling pathways to converge on mammalian target of rapamycin complex 1 (mTORC1), to influence MPS. This may e.g. implicate mechanical activation of signaling through a diacylglycerol kinase (DGKζ)-phosphatidic acid (PA) axis or implicate integrin deformation to signal through a Focal adhesion kinase (FAK)-Tuberous Sclerosis Complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) axis. Moreover, since initiation of translation is reliant on mRNA, it is also relevant to consider potentially mechanosensitive signaling pathways involved in muscle myofibrillar gene transcription and whether some of these pathways converge with those affecting mTORC1 activation for MPS. In this regard, recent findings suggest how mechanical stress may implicate integrin deformation and/or actin dynamics to signal through a Ras homolog gene family member A protein (RhoA)-striated muscle activator of Rho signaling (STARS) axis or implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP) signaling through a small mother of decapentaplegic (Smad) axis.

  7. Critical importance of RAB proteins for synaptic function.

    PubMed

    Mignogna, Maria Lidia; D'Adamo, Patrizia

    2017-02-01

    Neurons are highly polarized cells that exhibit one of the more complex morphology and function. Neuronal intracellular trafficking plays a key role in dictating the directionality and specificity of vesicle formation, transport and fusion, allowing the transmission of information in sophisticate cellular network. Thus, the integrity of protein trafficking and spatial organization is especially important in neuronal cells. RAB proteins, small monomeric GTPases belonging to the RAS superfamily, spatially and temporally orchestrate specific vesicular trafficking steps. In this review we summarise the known roles of RAB GTPases involved in the maintenance of neuronal vesicular trafficking in the central nervous system. In particular, we discriminate the axonal pre-synaptic trafficking and dendritic post-synaptic trafficking, to better underlie how a correct orchestration of vesicle movement is necessary to maintain neuronal polarity and then, to permit an accurate architecture and functionality of synaptic activity.

  8. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

    PubMed Central

    Neira, José L.; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L.; Abián, Olga

    2017-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs. PMID:28054562

  9. A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum.

    PubMed

    Jia, P; Zhang, C; Huang, X P; Poda, M; Akbas, F; Lemanski, S L; Erginel-Unaltuna, N; Lemanski, L F

    2008-11-01

    The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

  10. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma.

    PubMed

    Neira, José L; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L; Abián, Olga

    2017-01-05

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the 'fuzzy' interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

  11. HNE-modified proteins in Down syndrome: Involvement in development of Alzheimer disease neuropathology.

    PubMed

    Barone, Eugenio; Head, Elizabeth; Butterfield, D Allan; Perluigi, Marzia

    2016-11-10

    Down syndrome (DS), trisomy of chromosome 21, is the most common genetic form of intellectual disability. The neuropathology of DS involves multiple molecular mechanisms, similar to AD, including the deposition of beta-amyloid (Aβ) into senile plaques and tau hyperphosphorylationg in neurofibrillary tangles. Interestingly, many genes encoded by chromosome 21, in addition to being primarily linked to amyloid-beta peptide (Aβ) pathology, are responsible for increased oxidative stress (OS) conditions that also result as a consequence of reduced antioxidant system efficiency. However, redox homeostasis is disturbed by overproduction of Aβ, which accumulates into plaques across the lifespan in DS as well as in AD, thus generating a vicious cycle that amplifies OS-induced intracellular changes. The present review describes the current literature that demonstrates the accumulation of oxidative damage in DS with a focus on the lipid peroxidation by-product, 4-hydroxy-2-nonenal (HNE). HNE reacts with proteins and can irreversibly impair their functions. We suggest that among different post-translational modifications, HNE-adducts on proteins accumulate in DS brain and play a crucial role in causing the impairment of glucose metabolism, neuronal trafficking, protein quality control and antioxidant response. We hypothesize that dysfunction of these specific pathways contribute to accelerated neurodegeneration associated with AD neuropathology.

  12. Adaptive expression pattern of different proteins involved in cellular calcium homeostasis in denervated rat vas deferens.

    PubMed

    Quintas, Luis Eduardo M; Cunha, Valéria M N; Scaramello, Christianne B V; da Silva, Cláudia L M; Caricati-Neto, Afonso; Lafayette, Simone S L; Jurkiewicz, Aron; Noël, François

    2005-11-21

    The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+-Mg2+)ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+-Mg2+)ATPase expression. Finally, [3H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens.

  13. Looking for prosocial genes: ITRAQ analysis of proteins involved in MDMA-induced sociability in mice.

    PubMed

    Kuteykin-Teplyakov, Konstantin; Maldonado, Rafael

    2014-11-01

    Social behavior plays a fundamental role in life of many animal species, allowing the interaction between individuals and sharing of experiences, needs, and goals across them. In humans, some neuropsychiatric diseases, including anxiety, posttraumatic stress disorder and autism spectrum disorders, are often characterized by impaired sociability. Here we report that N-Methyl-3,4-methylenedioxyamphetamine (MDMA, "Ecstasy") at low dose (3mg/kg) has differential effects on mouse social behavior. In some animals, MDMA promotes sociability without hyperlocomotion, whereas in other mice it elevates locomotor activity without affecting sociability. Both WAY-100635, a selective antagonist of 5-HT1A receptor, and L-368899, a selective oxytocin receptor antagonist, abolish prosocial effects of MDMA. Differential quantitative analysis of brain proteome by isobaric tag for relative and absolute quantification technology (iTRAQ) revealed 21 specific proteins that were highly correlated with sociability, and allowed to distinguish between entactogenic prosocial and hyperlocomotor effects of MDMA on proteome level. Our data suggest particular relevance of neurotransmission mediated by GABA B receptor, as well as proteins involved in energy maintenance for MDMA-induced sociability. Functional association network for differentially expressed proteins in cerebral cortex, hippocampus and amygdala were identified. These results provide new information for understanding the neurobiological substrate of sociability and may help to discover new therapeutic approaches to modulate social behavior in patients suffering from social fear and low sociability.

  14. Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

    NASA Astrophysics Data System (ADS)

    Neira, José L.; Bintz, Jennifer; Arruebo, María; Rizzuti, Bruno; Bonacci, Thomas; Vega, Sonia; Lanas, Angel; Velázquez-Campoy, Adrián; Iovanna, Juan L.; Abián, Olga

    2017-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ‘fuzzy’ interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs.

  15. Amyloid precursor protein in Drosophila glia regulates sleep and genes involved in glutamate recycling.

    PubMed

    Farca Luna, Abud Jose; Perier, Magali; Seugnet, Laurent

    2017-03-17

    The Amyloid Precursor Protein (App) plays a crucial role in Alzheimer disease (AD) via the production and deposition of toxic β-amyloid peptides. App is heavily expressed in neurons where the vast majority of studies investigating its function have been carried out, while almost nothing is known about its function in glia, where it is also expressed, and can potentially participate in the regulation of neuronal physiology. In this report, we investigated whether Appl, the Drosophila homolog of App, could influence sleep-wake regulation when its function is manipulated in glial cells. Appl inhibition in astrocyte-like and cortex glia resulted in higher sleep amounts and longer sleep bout duration during the night, while overexpression had the opposite effect. These sleep phenotypes were not the result of developmental defects, and were correlated with changes in expression in Glutamine Synthetase (GS) in astrocyte-like glia, and in changes in the gap-junction component innexin2 in cortex glia. Downregulating both GS and innexin2, but not either one individually, resulted in higher sleep amounts, similarly to Appl inhibition. Consistent with these results the expression of GS and innexin2 are increased following sleep deprivation indicating that these two genes are dynamically linked to vigilance states. Interestingly, the reduction of GS expression and the sleep phenotype observed upon Appl inhibition could be rescued by increasing the expression of the glutamate transporter dEaat1. In contrast, reducing dEaat1 expression severely disrupted sleep. These results associate glutamate recycling, sleep and a glial function for the App family proteins.StatementThe Amyloid Precursor Protein (App) has been intensively studied for its implication in Alzheimer Disease (AD). The attributed functions of App are linked to the physiology and cellular biology of neurons where the protein is predominantly expressed. Consequences on glia in AD are generally thought to be secondary

  16. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    PubMed

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  17. A generating function for certain coefficients involving several complex variables.

    PubMed

    Srivastava, H M

    1970-10-01

    In an attempt to unify a number of generating functions for certain classes of generalized hypergeometric polynomials, Lagrange's expansion formula is applied to prove a generating relation for an n-dimensional polynomial with arbitrary coefficients. It is also shown how these coefficients can be specialized to obtain the generalized Lauricella function as a generating function for a class of generalized hypergeometric polynomials of several complex variables.

  18. Fragment complementation and co-immunoprecipitation assays for understanding R protein structure and function.

    PubMed

    Moffett, Peter

    2011-01-01

    Plant disease resistance (R) proteins confer protection against specific pathogens or pathogen isolates. R proteins function by recognizing pathogen-encoded avirulence (Avr) proteins and translating this recognition event into an initiation of downstream signaling pathways. Key to understanding this process is the study of the protein-protein interactions involving R proteins. Recognition and signaling mechanisms are mediated by both intramolecular interactions that take place between different domains of R proteins as well as intermolecular interactions between R proteins and additional plant proteins. These processes have been studied in part by using Agrobacterium-mediated transient expression of R protein fragments in Nicotiana benthamiana which allows for the rapid assessment of functionality. Furthermore, pairs of proteins or protein fragments can be transiently expressed as fusions with different epitope tags. One putative protein partner is subjected to immunoprecipitation. Subsequent immunoblotting is performed to determine whether the second protein has remained associated (or co-immunoprecipitated) with the first, indicating a protein-protein interaction. This technique has contributed substantially to structure-function analyses of R proteins and to the characterization of interactions between R proteins and other plant proteins.

  19. The MagA Protein of Magnetospirilla Is Not Involved in Bacterial Magnetite Biomineralization

    PubMed Central

    Uebe, René; Henn, Verena

    2012-01-01

    Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria. PMID:22194451

  20. AN ODORANT-BINDING PROTEIN INVOLVED IN PERCEPTION OF HOST PLANT ODORANTS IN LOCUST Locusta migratoria.

    PubMed

    Li, Jia; Zhang, Long; Wang, Xiaoqi

    2016-04-01

    Locusts, Locusta migratoria (Orthoptera: Acrididae), are extremely destructive agricultural pests, but very little is known of their molecular aspects of perception to host plant odorants including related odorant-binding proteins (OBPs), though several OBPs have been identified in locust. To elucidate the function of LmigOBP1, the first OBP identified from locust, RNA interference was employed in this study to silence LmigOBP1, which was achieved by injection of dsRNA targeting LmigOBP1 into the hemolymph of male nymphs. Compared with LmigOBP1 normal nymphs, LmigOBP1 knockdown nymphs significantly decreased food (maize leaf, Zea mays) consumption and electro-antennography responses to five maize leaf volatiles, ((Z)-3-hexenol, linalool, nonanal, decanal, and (Z)-3-hexenyl acetate). These suggest that LmigOBP1 is involved in perception of host plant odorants.

  1. A novel pax-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

    PubMed

    Wang, Yi-Ting; Pan, Yu-Jiao; Cho, Chao-Cheng; Lin, Bo-Chi; Su, Li-Hsin; Huang, Yu-Chang; Sun, Chin-Hung

    2010-10-15

    Giardia lamblia differentiates into infectious cysts to survive outside of the host. It is of interest to identify factors involved in up-regulation of cyst wall proteins (CWPs) during this differentiation. Pax proteins are important regulators of development and cell differentiation in Drosophila and vertebrates. No member of this gene family has been reported to date in yeast, plants, or protozoan parasites. We have identified a pax-like gene (pax1) encoding a putative paired domain in the G. lamblia genome. Epitope-tagged Pax1 localized to nuclei during both vegetative growth and encystation. Recombinant Pax1 specifically bound to the AT-rich initiator elements of the encystation-induced cwp1 to -3 and myb2 genes. Interestingly, overexpression of Pax1 increased cwp1 to -3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of the transactivation function of Pax1. Our results indicate that the Pax family has been conserved during evolution, and Pax1 could up-regulate the key encystation-induced genes to regulate differentiation of the protozoan eukaryote, G. lamblia.

  2. Multivesicular Bodies in Neurons: Distribution, Protein Content, and Trafficking Functions

    PubMed Central

    VON BARTHELD, CHRISTOPHER S.; ALTICK, AMY L.

    2011-01-01

    Summary Multivesicular bodies (MVBs) are intracellular endosomal organelles characterized by multiple internal vesicles that are enclosed within a single outer membrane. MVBs were initially regarded as purely prelysosomal structures along the degradative endosomal pathway of internalized proteins. MVBs are now known to be involved in numerous endocytic and trafficking functions, including protein sorting, recycling, transport, storage, and release. This review of neuronal MVBs summarizes their research history, morphology, distribution, accumulation of cargo and constitutive proteins, transport, and theories of functions of MVBs in neurons and glia. Due to their complex morphologies, neurons have expanded trafficking and signaling needs, beyond those of “geometrically simpler” cells, but it is not known whether neuronal MVBs perform additional transport and signaling functions. This review examines the concept of compartment-specific MVB functions in endosomal protein trafficking and signaling within synapses, axons, dendrites and cell bodies. We critically evaluate reports of the accumulation of neuronal MVBs based on evidence of stress-induced MVB formation. Furthermore, we discuss potential functions of neuronal and glial MVBs in development, in dystrophic neuritic syndromes, injury, disease, and aging. MVBs may play a role in Alzheimer’s, Huntington’s, and Niemann-Pick diseases, some types of frontotemporal dementia, prion and virus trafficking, as well as in adaptive responses of neurons to trauma and toxin or drug exposure. Functions of MVBs in neurons have been much neglected, and major gaps in knowledge currently exist. Developing truly MVB-specific markers would help to elucidate the roles of neuronal MVBs in intra- and intercellular signaling of normal and diseased neurons. PMID:21216273

  3. Microarray Technology Reveals Potentially Novel Genes and Pathways Involved in Non-Functioning Pituitary Adenomas

    PubMed Central

    Qiao, X; Wang, H; Wang, X; Zhao, B; Liu, J

    2016-01-01

    Abstract Microarray data of non-functioning pituitary adenomas (NFPAs) were analyzed to disclose novel genes and pathways involved in NFPA tumorigenesis. Raw microarray data were downloaded from Gene Expression Omnibus. Data pre-treatment and differential analysis were conducted using packages in R. Functional and pathway enrichment analyses were performed using package GOs-tats. A protein-protein interaction (PPI) network was constructed using server STRING and Cytoscape. Known genes involved in pituitary adenomas (PAs), were obtained from the Comparative Toxicogenomics Database. A total of 604 differentially expressed genes (DEGs) were identifed between NFPAs and controls, including 177 up- and 427 down-regulated genes. Jak-STAT and p53 signaling pathways were significantly enriched by DEGs. The PPI network of DEGs was constructed, containing 99 up- and 288 down-regulated known disease genes (e.g. EGFR and ESR1) as well as 16 up- and 17 down-regulated potential novel NFPAs-related genes (e.g. COL4A5, LHX3, MSN, and GHSR). Genes like COL4A5, LHX3, MSN, and GHSR and pathways such as p53 signaling and Jak-STAT signaling, might participate in NFPA development. Although further validations are required, these findings might provide guidance for future basic and therapy researches. PMID:28289583

  4. Transcription Factor Functional Protein-Protein Interactions in Plant Defense Responses

    PubMed Central

    Alves, Murilo S.; Dadalto, Silvana P.; Gonçalves, Amanda B.; de Souza, Gilza B.; Barros, Vanessa A.; Fietto, Luciano G.

    2014-01-01

    Responses to biotic stress in plants lead to dramatic reprogramming of gene expression, favoring stress responses at the expense of normal cellular functions. Transcription factors are master regulators of gene expression at the transcriptional level, and controlling the activity of these factors alters the transcriptome of the plant, leading to metabolic and phenotypic changes in response to stress. The functional analysis of interactions between transcription factors and other proteins is very important for elucidating the role of these transcriptional regulators in different signaling cascades. In this review, we present an overview of protein-protein interactions for the six major families of transcription factors involved in plant defense: basic leucine zipper containing domain proteins (bZIP), amino-acid sequence WRKYGQK (WRKY), myelocytomatosis related proteins (MYC), myeloblastosis related proteins (MYB), APETALA2/ ETHYLENE-RESPONSIVE ELEMENT BINDING FACTORS (AP2/EREBP) and no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC) (NAC). We describe the interaction partners of these transcription factors as molecular responses during pathogen attack and the key components of signal transduction pathways that take place during plant defense responses. These interactions determine the activation or repression of response pathways and are crucial to understanding the regulatory networks that modulate plant defense responses. PMID:28250372

  5. Involvement of TREK-1 activity in astrocyte function and neuroprotection under simulated ischemia conditions.

    PubMed

    Wu, Xiao; Liu, Yang; Chen, Xiaojing; Sun, Qian; Tang, Ronghua; Wang, Wei; Yu, Zhiyuan; Xie, Minjie

    2013-03-01

    Astrocytes play a fundamental role in the pathogenesis of ischemic neuronal death. The optimal operation of electrogenic astrocytic transporters and exchangers for some well-defined astrocyte brain homeostatic functions depends on the presence of K(+) channels in the cell membranes and the hyperpolarized membrane potential. Our previous study showed that astrocytes functionally express two-pore domain K(+) channel TREK-1, which helps to set the negative resting membrane potential. However, the roles of TREK-1 on astrocytic function under normal and ischemic conditions remain unclear. In this study, we investigated the expression of TREK-1 protein on cultured astrocytes and the effect of TREK-1 activity on astrocytic glutamate clearance capacity and release of s100β after simulated ischemic insult. TREK-1 immunoreactivity was up-regulated after hypoxia. Suppression of TREK-1 activity inhibited the glutamate clearance capability, enhanced the inflammatory secretion of astrocytes derived s100β and led to increased neuronal apoptosis after ischemic insult. Our results suggest that TREK-1 activity is involved in astrocytic function and neuronal survival. This would provide evidence showing astrocytic TREK-1 involvement in ischemia pathology which may serve as a potential therapeutic target in stroke.

  6. STAT5 proteins are involved in down-regulation of iron regulatory protein 1 gene expression by nitric oxide.

    PubMed

    Starzynski, Rafal Radoslaw; Gonçalves, Ana Sofia; Muzeau, Françoise; Tyrolczyk, Zofia; Smuda, Ewa; Drapier, Jean-Claude; Beaumont, Carole; Lipinski, Pawel

    2006-12-01

    RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-gamma-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1-DNA and STAT-DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression.

  7. Interfacial interactions involved in the biological assembly of Chandipura virus nucleocapsid protein.

    PubMed

    Sreejith, R; Gulati, Sahil; Gupta, Sanjay

    2013-06-01

    The biological assembly of Chandipura virus nucleocapsid (N) protein has been modeled and the amino acid residues involved in specific intermolecular interactions among N monomers during oligomerisation have been predicted.

  8. Multiple functions of microsomal triglyceride transfer protein

    PubMed Central

    2012-01-01

    Microsomal triglyceride transfer protein (MTP) was first identified as a major cellular protein capable of transferring neutral lipids between membrane vesicles. Its role as an essential chaperone for the biosynthesis of apolipoprotein B (apoB)-containing triglyceride-rich lipoproteins was established after the realization that abetalipoproteinemia patients carry mutations in the MTTP gene resulting in the loss of its lipid transfer activity. Now it is known that it also plays a role in the biosynthesis of CD1, glycolipid presenting molecules, as well as in the regulation of cholesterol ester biosynthesis. In this review, we will provide a historical perspective about the identification, purification and characterization of MTP, describe methods used to measure its lipid transfer activity, and discuss tissue expression and function. Finally, we will review the role MTP plays in the assembly of apoB-lipoprotein, the regulation of cholesterol ester synthesis, biosynthesis of CD1 proteins and propagation of hepatitis C virus. We will also provide a brief overview about the clinical potentials of MTP inhibition. PMID:22353470

  9. CombFunc: predicting protein function using heterogeneous data sources.

    PubMed

    Wass, Mark N; Barton, Geraint; Sternberg, Michael J E

    2012-07-01

    Only a small fraction of known proteins have been functionally characterized, making protein function prediction essential to propose annotations for uncharacterized proteins. In recent years many function prediction methods have been developed using various sources of biological data from protein sequence and structure to gene expression data. Here we present the CombFunc web server, which makes Gene Ontology (GO)-based protein function predictions. CombFunc incorporates ConFunc, our existing function prediction method, with other approaches for function prediction that use protein sequence, gene expression and protein-protein interaction data. In benchmarking on a set of 1686 proteins CombFunc obtains precision and recall of 0.71 and 0.64 respectively for gene ontology molecular function terms. For biological process GO terms precision of 0.74 and recall of 0.41 is obtained. CombFunc is available at http://www.sbg.bio.ic.ac.uk/combfunc.

  10. Involvement of fission yeast Pdc2 in RNA degradation and P-body function.

    PubMed

    Wang, Chun-Yu; Wang, Yi-Ting; Hsiao, Wan-Yi; Wang, Shao-Win

    2017-04-01

    In this study we identified Pdc2, the fission yeast ortholog of human Pat1b protein, which forms a complex with Lsm1-7 and plays a role in coupling deadenylation and decapping. The involvement of Pdc2 in RNA degradation and P-body function was also determined. We found that Pdc2 interacts with Dcp2 and is required for decapping in vivo. Although not absolutely essential for P-body assembly, overexpression of Pdc2 enhanced P-body formation even in the absence of Pdc1, the fission yeast functional homolog of human Edc4 protein, indicating that Pdc2 also plays a role in P-body formation. Intriguingly, in the absence of Pdc2, Lsm1 was found to accumulate in the nucleus, suggesting that Pdc2 shuttling between nucleus and cytoplasm plays a role in decreasing the nuclear concentration of Lsm1 to increase Lsm1 in the cytoplasm. Furthermore, unlike other components of P-bodies, the deadenylase Ccr4 did not accumulate in P-bodies in cells growing under favorable conditions and was only recruited to P-bodies after deprivation of glucose in a Pdc2-Lsm1-dependent manner, indicating a function of Pdc2 in cellular response to environmental stress. In supporting this idea, pdc2 mutants are defective in recovery from glucose starvation with a much longer time to re-enter the cell cycle. In keeping with the notion that Pat1 is a nucleocytoplasmic protein, functioning also in the nucleus, we found that Pdc2 physically and genetically interacts with the nuclear 5'-3' exonuclease Dhp1. A function of Pdc2-Lsm1, in concert with Dhp1, regulating RNA by promoting its decapping/destruction in the nucleus was suggested.

  11. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

    PubMed Central

    Talay, S R; Valentin-Weigand, P; Jerlström, P G; Timmis, K N; Chhatwal, G S

    1992-01-01

    The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to

  12. Dietary proteins and functional gastrointestinal disorders.

    PubMed

    Boettcher, Erica; Crowe, Sheila E

    2013-05-01

    Food intolerance is a common complaint amongst patients with functional gastrointestinal (GI) disorders (FGIDs), including those with irritable bowel syndrome (IBS), functional dyspepsia, as well as gastroesophageal reflux disease. Although there has been a longstanding interest in the possible role of food allergy in IBS, there are limited data supporting the association. However, the prevalence of food allergy is sufficiently high that patients with FGID may also have food allergies or hypersensitivities. Food intolerances or sensitivities are reactions to foods, which are not due to immunological mechanisms. Lactose intolerance is common in the general population and can mimic symptoms of FGID or coexist with FGID. As discussed in other articles in this series, other carbohydrate intolerances may be responsible for symptom generation in patients with IBS and perhaps other FGIDs. There is a great interest in the role of a major dietary protein, gluten, in the production of symptoms that are very similar to those of patients with celiac disease without the enteropathy that characterizes celiac disease. Emerging research into a syndrome known as nonceliac gluten sensitivity suggests a heterogeneous condition with some features of celiac disease but often categorized as FGIDs, including IBS. This article summarizes the role of dietary proteins in the symptoms and pathophysiology of FGIDs.

  13. Protein tyrosine phosphatases: structure-function relationships.

    PubMed

    Tabernero, Lydia; Aricescu, A Radu; Jones, E Yvonne; Szedlacsek, Stefan E

    2008-03-01

    Structural analysis of protein tyrosine phosphatases (PTPs) has expanded considerably in the last several years, producing more than 200 structures in this class of enzymes (from 35 different proteins and their complexes with ligands). The small-medium size of the catalytic domain of approximately 280 residues plus a very compact fold makes it amenable to cloning and overexpression in bacterial systems thus facilitating crystallographic analysis. The low molecular weight PTPs being even smaller, approximately 150 residues, are also perfect targets for NMR analysis. The availability of different structures and complexes of PTPs with substrates and inhibitors has provided a wealth of information with profound effects in the way we understand their biological functions. Developments in mammalian expression technology recently led to the first crystal structure of a receptor-like PTP extracellular region. Altogether, the PTP structural work significantly advanced our knowledge regarding the architecture, regulation and substrate specificity of these enzymes. In this review, we compile the most prominent structural traits that characterize PTPs and their complexes with ligands. We discuss how the data can be used to design further functional experiments and as a basis for drug design given that many PTPs are now considered strategic therapeutic targets for human diseases such as diabetes and cancer.

  14. Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector, Ixodes scapularis.

    PubMed

    Villar, Margarita; Ayllón, Nieves; Kocan, Katherine M; Bonzón-Kulichenko, Elena; Alberdi, Pilar; Blouin, Edmour F; Weisheit, Sabine; Mateos-Hernández, Lourdes; Cabezas-Cruz, Alejandro; Bell-Sakyi, Lesley; Vancová, Marie; Bílý, Tomáš; Meyer, Damien F; Sterba, Jan; Contreras, Marinela; Rudenko, Nataliia; Grubhoffer, Libor; Vázquez, Jesús; de la Fuente, José

    2015-01-01

    Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10-15% and 65-71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface

  15. Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector, Ixodes scapularis

    PubMed Central

    Kocan, Katherine M.; Bonzón-Kulichenko, Elena; Alberdi, Pilar; Blouin, Edmour F.; Weisheit, Sabine; Mateos-Hernández, Lourdes; Cabezas-Cruz, Alejandro; Bell-Sakyi, Lesley; Vancová, Marie; Bílý, Tomáš; Meyer, Damien F.; Sterba, Jan; Contreras, Marinela; Rudenko, Nataliia; Grubhoffer, Libor; Vázquez, Jesús; de la Fuente, José

    2015-01-01

    Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10–15% and 65–71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface

  16. Identification of Resting State Networks Involved in Executive Function.

    PubMed

    Connolly, Joanna; McNulty, Jonathan P; Boran, Lorraine; Roche, Richard A P; Delany, David; Bokde, Arun L W

    2016-06-01

    The structural networks in the human brain are consistent across subjects, and this is reflected also in that functional networks across subjects are relatively consistent. These findings are not only present during performance of a goal oriented task but there are also consistent functional networks during resting state. It suggests that goal oriented activation patterns may be a function of component networks identified using resting state. The current study examines the relationship between resting state networks measured and patterns of neural activation elicited during a Stroop task. The association between the Stroop-activated networks and the resting state networks was quantified using spatial linear regression. In addition, we investigated if the degree of spatial association of resting state networks with the Stroop task may predict performance on the Stroop task. The results of this investigation demonstrated that the Stroop activated network can be decomposed into a number of resting state networks, which were primarily associated with attention, executive function, visual perception, and the default mode network. The close spatial correspondence between the functional organization of the resting brain and task-evoked patterns supports the relevance of resting state networks in cognitive function.

  17. Proteomic profile of carbonylated proteins in rat liver: exercise attenuated oxidative stress may be involved in fatty liver improvement.

    PubMed

    Hu, Xiaofei; Duan, Zhigui; Hu, Hui; Li, Guolin; Yan, Siyu; Wu, Jinfeng; Wang, Jun; Yin, Dazhong; Xie, Qingji

    2013-05-01

    To screen target proteins of oxidative stress which mediate the effects of exercise on preventing nonalcoholic fatty liver disease (NAFLD), the methods for selecting carbonylated proteins were modified, and carbonylated proteins were profiled. The results showed that treadmill training reduced oxidative stress and the levels of intrahepatic triglyceride (IHTG). The changes in IHTG showed a significant positive correlation with oxidative stress as indicated by malondialdehyde level. Further results from proteomics illustrated that 17 functional proteins were susceptible to oxidative modification, and exercise protected three proteins from carbonylation. The latter three proteins may serve as both direct target proteins of oxidative stress and mediators contributing to the beneficial effects of exercise. In particular, a long-chain specific acyl-CoA dehydrogenase (ACADL) which was a key enzyme in lipid metabolism was not carbonylated and with higher activities in exercise group. These findings indicate that this modified technique is practical and powerful in selecting carbonylated proteins. Long-term treadmill training is effective in ameliorating oxidative stress and preventing the accumulation of IHTG. Among the 17 target proteins of oxidative modification, three proteins contribute to the beneficial effects of exercise. Preventing ACADL from carbonylation may be involved in the physiological mechanism of exercise-induced NAFLD improvement.

  18. Human DCXR - another 'moonlighting protein' involved in sugar metabolism, carbonyl detoxification, cell adhesion and male fertility?

    PubMed

    Ebert, Bettina; Kisiela, Michael; Maser, Edmund

    2015-02-01

    Dicarbonyl/L-xylulose reductase (DCXR; SDR20C1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily catalyzes the reduction of α-dicarbonyl compounds and monosaccharides. Its role in the metabolism of L-xylulose has been known since 1970, when essential pentosuria was found to be associated with DCXR deficiency. Despite its early discovery, our knowledge about the role of human DCXR in normal physiology and pathophysiology is still incomplete. Sporadic studies have demonstrated aberrant expression in several cancers, but their physiological significance is unknown. In reproductive medicine, where DCXR is commonly referred to as 'sperm surface protein P34H', it serves as marker for epididymal sperm maturation and is essential for gamete interaction and successful fertilization. DCXR exhibits a multifunctional nature, both acting as a carbonyl reductase and also performing non-catalytic functions, possibly resulting from interactions with other proteins. Recent observations associate DCXR with a role in cell adhesion, pointing to a novel function involving tumour progression and possibly metastasis. This review summarizes the current knowledge about human DCXR and its orthologs from mouse and Caenorhabditis elegans (DHS-21) with an emphasis on its multifunctional characteristics. Due to its close structural relationship with DCXR, carbonyl reductase 2 (Cbr2), a tetrameric enzyme found in several non-primate species is also discussed. Similar to human DCXR, Cbr2 from golden hamster (P26h) and cow (P25b) is essential for sperm-zona pellucida interaction and fertilization. Because of the apparent similarity of these two proteins and the inconsistent use of alternative names previously, we provide an overview of the systematic classification of DCXR and Cbr2 and a phylogenetic analysis to illustrate their ancestry.

  19. Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

    NASA Astrophysics Data System (ADS)

    Kim, Young Eun; Kim, Yu-Na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

    2015-05-01

    Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein-protein interactions and tools to manipulate receptor clustering on live cell surfaces.

  20. Rosetta stone method for detecting protein function and protein-protein interactions from genome sequences

    DOEpatents

    Eisenberg, David; Marcotte, Edward M.; Pellegrini, Matteo; Thompson, Michael J.; Yeates, Todd O.

    2002-10-15

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  1. Mapping the H+ (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation

    PubMed Central

    Merkulova, Maria; Păunescu, Teodor G.; Azroyan, Anie; Marshansky, Vladimir; Breton, Sylvie; Brown, Dennis

    2015-01-01

    V-ATPases (H+ ATPases) are multisubunit, ATP-dependent proton pumps that regulate pH homeostasis in virtually all eukaryotes. They are involved in key cell biological processes including vesicle trafficking, endosomal pH sensing, membrane fusion and intracellular signaling. They also have critical systemic roles in renal acid excretion and blood pH balance, male fertility, bone remodeling, synaptic transmission, olfaction and hearing. Furthermore, V-ATPase dysfunction either results in or aggravates various other diseases, but little is known about the complex protein interactions that regulate these varied V-ATPase functions. Therefore, we performed a proteomic analysis to identify V-ATPase associated proteins and construct a V-ATPase interactome. Our analysis using kidney tissue revealed V-ATPase-associated protein clusters involved in protein quality control, complex assembly and intracellular trafficking. ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27 and 9 subunits of the chaperonin containing TCP1 complex (CCT) were found to interact with V-ATPase for the first time in this study. Knockdown of two interacting proteins, DMXL1 and WDR7, inhibited V-ATPase-mediated intracellular vesicle acidification in a kidney cell line, providing validation for the utility of our interactome as a screen for functionally important novel V-ATPase-regulating proteins. Our data, therefore, provide new insights and directions for the analysis of V-ATPase cell biology and (patho)physiology. PMID:26442671

  2. Involvement of hypothalamic AMP-activated protein kinase in leptin-induced sympathetic nerve activation.

    PubMed

    Tanida, Mamoru; Yamamoto, Naoki; Shibamoto, Toshishige; Rahmouni, Kamal

    2013-01-01

    In mammals, leptin released from the white adipose tissue acts on the central nervous system to control feeding behavior, cardiovascular function, and energy metabolism. Central leptin activates sympathetic nerves that innervate the kidney, adipose tissue, and some abdominal organs in rats. AMP-activated protein kinase (AMPK) is essential in the intracellular signaling pathway involving the activation of leptin receptors (ObRb). We investigated the potential of AMPKα2 in the sympathetic effects of leptin using in vivo siRNA injection to knockdown AMPKα2 in rats, to produce reduced hypothalamic AMPKα2 expression. Leptin effects on body weight, food intake, and blood FFA levels were eliminated in AMPKα2 siRNA-treated rats. Leptin-evoked enhancements of the sympathetic nerve outflows to the kidney, brown and white adipose tissues were attenuated in AMPKα2 siRNA-treated rats. To check whether AMPKα2 was specific to sympathetic changes induced by leptin, we examined the effects of injecting MT-II, a melanocortin-3 and -4 receptor agonist, on the sympathetic nerve outflows to the kidney and adipose tissue. MT-II-induced sympatho-excitation in the kidney was unchanged in AMPKα2 siRNA-treated rats. However, responses of neural activities involving adipose tissue to MT-II were attenuated in AMPKα2 siRNA-treated rats. These results suggest that hypothalamic AMPKα2 is involved not only in appetite and body weight regulation but also in the regulation of sympathetic nerve discharges to the kidney and adipose tissue. Thus, AMPK might function not only as an energy sensor, but as a key molecule in the cardiovascular, thermogenic, and lipolytic effects of leptin through the sympathetic nervous system.

  3. TMEM16 proteins: unknown structure and confusing functions

    PubMed Central

    Picollo, Alessandra; Malvezzi, Mattia; Accardi, Alessio

    2014-01-01

    The TMEM16 family of membrane proteins, also known as anoctamins, play key roles in a variety of physiological functions that range from ion transport, to phospholipid scrambling and to regulating other ion channels. The first two family members to be functionally characterized, TMEM16A (ANO1) and TMEM16B (ANO2), form Ca2+-activated Cl− channels (CaCCs) and are important for transepithelial ion transport, olfaction, phototransduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. The role(s) of other family members, such as TMEM16C (ANO3), TMEM16D (ANO4), TMEM16F (ANO6), TMEM16G (ANO7), and TMEM16J (ANO9), remain poorly understood and controversial. These homologues were reported to be phospholipid scramblases, ion channels, to have both functions or to be regulatory subunits of other channels. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused by impaired Ca2+-dependent externalization of phosphatidylserine in activated platelets, suggesting that this homologue might be a scramblase. However, overexpression of TMEM16F has also been associated with a remarkable number of different ion channel types, raising the possibility that this protein might be involved in both ion and lipid transport. The recent identification of an ancestral TMEM16 homologue with intrinsic channel and scramblase activities supports this hypothesis. Thus, the TMEM16 family might have diverged in two or three different subclasses, channels, scramblases and dual function channel/scramblases. The structural bases and functional implication of such a functional diversity within a single protein family remain to be elucidated and the links between TMEM16 functions and human physiology and pathologies need to be investigated. PMID:25451786

  4. Modulation of lipoprotein receptor functions by intracellular adaptor proteins.

    PubMed

    Stolt, Peggy C; Bock, Hans H

    2006-10-01

    Members of the low density lipoprotein (LDL) receptor gene family are critically involved in a wide range of physiological processes including lipid and vitamin homeostasis, cellular migration, neurodevelopment, and synaptic plasticity, to name a few. Lipoprotein receptors exert these diverse biological functions by acting as cellular uptake receptors or by inducing intracellular signaling cascades. It was discovered that a short sequence in the intracellular region of all lipoprotein receptors, Asn-Pro-X-Tyr (NPXY) is important for mediating either endocytosis or signal transduction events, and that this motif serves as a binding site for phosphotyrosine-binding (PTB) domain containing scaffold proteins. These molecular adaptors connect the transmembrane receptors with the endocytosis machinery and regulate cellular trafficking, or function as assembly sites for dynamic multi-protein signaling complexes. Whereas the LDL receptor represents the archetype of an endocytic lipoprotein receptor, the structurally closely related apolipoprotein E receptor 2 (apoER2) and very low density lipoprotein (VLDL) receptor activate a kinase-dependent intracellular signaling cascade after binding to the neuronal signaling molecule Reelin. This review focuses on two related PTB domain containing adaptor proteins that mediate these divergent lipoprotein receptor responses, ARH (autosomal recessive hypercholesterolemia protein) and Dab1 (disabled-1), and discusses the structural and molecular basis of this different behaviour.

  5. Beneficial effects of soy protein consumption for renal function.

    PubMed

    Anderson, James W

    2008-01-01

    Alterations in dietary protein intake have an important role in prevention and management of several forms of kidney disease. Using soy protein instead of animal protein reduces development of kidney disease in animals. Reducing protein intake preserves kidney function in persons with early diabetic kidney disease. Our clinical observations led us to the soy-protein hypothesis that "substitution of soy protein for animal protein results in less hyperfiltration and glomerular hypertension with resulting protection from diabetic nephropathy." These components of soy protein may lead to the benefits: specific peptides, amino acids, and isoflavones. Substituting soy protein for animal protein usually decreases hyperfiltration in diabetic subjects and may reduce urine albumin excretion. Limited data are available on effects of soy peptides, isoflavones, and other soy components on renal function on renal function in diabetes. Further studies are required to discern the specific benefits of soy protein and its components on renal function in diabetic subjects.

  6. Characterization of the functional properties of carob germ proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins from the carob germ were identified as having gluten-like proteins in 1935. While some biochemical characterization of carob germ proteins and their functionality has been carried out, relatively little has been done when compared to proteins such as gluten. Carob germ proteins were separ...

  7. The LIM protein LIMD1 influences osteoblast differentiation and function

    SciTech Connect

    Luderer, Hilary F.; Bai Shuting; Longmore, Gregory D.

    2008-09-10

    The balance between bone resorption and bone formation involves the coordinated activities of osteoblasts and osteoclasts. Communication between these two cell types is essential for maintenance of normal bone homeostasis; however, the mechanisms regulating this cross talk are not completely understood. Many factors that mediate differentiation and function of both osteoblasts and osteoclasts have been identified. The LIM protein Limd1 has been implicated in the regulation of stress osteoclastogenesis through an interaction with the p62/sequestosome protein. Here we show that Limd1 also influences osteoblast progenitor numbers, differentiation, and function. Limd1{sup -/-} calvarial osteoblasts display increased mineralization and accelerated differentiation. While no significant differences in osteoblast number or function were detected in vivo, bone marrow stromal cells isolated from Limd1{sup -/-} mice contain significantly more osteoblast progenitors compared to wild type controls when cultured ex vivo. Furthermore, we observed a significant increase in nuclear {beta}-catenin staining in differentiating Limd1{sup -/-} calvarial osteoblasts suggesting that Limd1 is a negative regulator of canonical Wnt signaling in osteoblasts. These results demonstrate that Limd1 influences not only stress osteoclastogenesis but also osteoblast function and osteoblast progenitor commitment. Together, these data identify Limd1 as a novel regulator of both bone osetoclast and bone osteoblast development and function.

  8. Chemistry-based functional proteomics to identify novel deubiquitylating enzymes involved in viral infection.

    PubMed

    Lei, Yunlong; Xie, Ke; Huang, Kai; Wu, Hong; Huang, Canhua

    2012-05-01

    Ubiquitylation is a reversible post-translational modification pathway that regulates a variety of cellular processes including protein degradation and trafficking, intracellular localization, DNA repair, immune response and cellcycle progression. Deubiquitylating enzymes (DUBs) can remove the ubiquitin from the modified proteins and reverse the ubiquitylation-induced biological processes; hence it isn't hard to understand that viral pathogens take advantage of the host cell ubiquitin system through disturbing DUBs, for infection and replication. Although accumulated virus-related DUBs have been defined, but how viruses regulate their expression and activities is poor understand because of limitation of technologies. Recently, chemistry-based functional proteomics, which can not only monitor the alteration of abundance but also changes in activity of enzymes, was used to study the function of DUBs involved in virus infection and held much promise. Theses works suggest that chemistry-based functional proteomics is a potent strategy for high throughput screening of virus-related DUBs and exploring their roles in virus infection.

  9. CombFunc: predicting protein function using heterogeneous data sources

    PubMed Central

    Wass, Mark N.; Barton, Geraint; Sternberg, Michael J. E.

    2012-01-01

    Only a small fraction of known proteins have been functionally characterized, making protein function prediction essential to propose annotations for uncharacterized proteins. In recent years many function prediction methods have been developed using various sources of biological data from protein sequence and structure to gene expression data. Here we present the CombFunc web server, which makes Gene Ontology (GO)-based protein function predictions. CombFunc incorporates ConFunc, our existing function prediction method, with other approaches for function prediction that use protein sequence, gene expression and protein–protein interaction data. In benchmarking on a set of 1686 proteins CombFunc obtains precision and recall of 0.71 and 0.64 respectively for gene ontology molecular function terms. For biological process GO terms precision of 0.74 and recall of 0.41 is obtained. CombFunc is available at http://www.sbg.bio.ic.ac.uk/combfunc. PMID:22641853

  10. A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

    NASA Astrophysics Data System (ADS)

    Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

    2000-05-01

    RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

  11. Urothelial function reconsidered: A role in urinary protein secretion

    PubMed Central

    Deng, Fang-Ming; Ding, Mingxiao; Lavker, Robert M.; Sun, Tung-Tien

    2001-01-01

    Mammalian bladder epithelium functions as an effective permeability barrier. We demonstrate here that this epithelium can also function as a secretory tissue directly involved in modifying urinary protein composition. Our data indicate that normal bovine urothelium synthesizes, as its major differentiation products, two well-known proteases: tissue-type plasminogen activator and urokinase, as well as a serine protease inhibitor, PP5. Moreover, we demonstrate that the urothelium secretes these proteins in a polarized fashion into the urine via a cAMP- and calcium-regulated pathway. Urinary plasminogen activators of ruminants are therefore urothelium derived rather then kidney derived as in some other species; this heterogeneity may have evolved in response to different physiological or dietary factors. In conjunction with our recent finding that transgenic mouse urothelium can secrete ectopically expressed human growth hormone into the urine, our data establish that normal mammalian urothelium can function not only as a permeability barrier but also as a secretor of urinary proteins that can play physiological or pathological roles in the urinary tract. PMID:11136252

  12. Membrane Proteins in Four Acts: Function Precedes Structure Determination

    PubMed Central

    Cramer, W. A.; Zakharov, S. D.; Hasan, S. Saif; Zhang, H.; Baniulis, D.; Zhalnina, M. V.; Soriano, G. M.; Sharma, O.; Rochet, J. C.; Ryan, C.; Whitelegge., J.; Kurisu, G.; Yamashita, E.

    2011-01-01

    Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/ proton translocation). (1) Crystal structures of the eight subunit heterooligomeric trans-membrane dimeric cytochrome b6f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of seventeen monotopic and polytopic hetero-subunits. (II) β-barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B12 binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins (1). A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a “fishing pole” model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83 Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained (2). A crystal structure of the N-terminal translocation domain of colicin E3

  13. Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex

    PubMed Central

    Rust, René C.; Landmann, Lukas; Gosert, Rainer; Tang, Bor Luen; Hong, Wanjin; Hauri, Hans-Peter; Egger, Denise; Bienz, Kurt

    2001-01-01

    Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway. PMID:11559814

  14. Involvement of the cellular prion protein in the migration of brain microvascular endothelial cells.

    PubMed

    Watanabe, Takuya; Yasutaka, Yuki; Nishioku, Tsuyoshi; Kusakabe, Sae; Futagami, Koujiro; Yamauchi, Atsushi; Kataoka, Yasufumi

    2011-06-01

    The conversion of cellular prion protein (PrP(C)) to its protease-resistant isoform is involved in the pathogenesis of prion disease. Although PrP(C) is a ubiquitous glycoprotein that is present in various cell types, the physiological role of PrP(C) remains obscure. The present study aimed to determine whether PrP(C) mediates migration of brain microvascular endothelial cells. Small interfering RNAs (siRNAs) targeting PrP(C) were transfected into a mouse brain microvascular endothelial cell line (bEND.3 cells). siPrP1, selected among three siRNAs, reduced mRNA and protein levels of PrP(C) in bEND.3 cells. Cellular migration was evaluated with a scratch-wound assay. siPrP1 suppressed migration without significantly affecting cellular proliferation. This study provides the first evidence that PrP(C) may be necessary for brain microvascular endothelial cells to migrate into damaged regions in the brain. This function of PrP(C) in the brain endothelium may be a mechanism by which the neurovascular unit recovers from an injury such as an ischemic insult.

  15. Sendai virus trailer RNA binds TIAR, a cellular protein involved in virus-induced apoptosis.

    PubMed

    Iseni, Frédéric; Garcin, Dominique; Nishio, Machiko; Kedersha, Nancy; Anderson, Paul; Kolakofsky, Daniel

    2002-10-01

    Sendai virus (SeV) leader (le) and trailer (tr) RNAs are short transcripts generated during abortive antigenome and genome synthesis, respectively. Recom binant SeV (rSeV) that express tr-like RNAs from the leader region are non-cytopathic and, moreover, prevent wild-type SeV from inducing apoptosis in mixed infections. These rSeV thus appear to have gained a function. Here we report that tr RNA binds to a cellular protein with many links to apoptosis (TIAR) via the AU-rich sequence 5' UUUUAAAUUUU. Duplication of this AU-rich sequence alone within the le RNA confers TIAR binding on this le* RNA and a non-cytopathic phenotype to these rSeV in cell culture. Transgenic overexpression of TIAR during SeV infection promotes apoptosis and reverses the anti-apoptotic effects of le* RNA expression. More over, TIAR overexpression and SeV infection act synergistically to induce apoptosis. These short viral RNAs may act by sequestering TIAR, a multivalent RNA recognition motif (RRM) family RNA-binding protein involved in SeV-induced apoptosis. In this view, tr RNA is not simply a by-product of abortive genome synthesis, but is also an antigenome transcript that modulates the cellular antiviral response.

  16. Analysis of Amyloid Precursor Protein Function in Drosophila melanogaster

    PubMed Central

    Cassar, Marlène; Kretzschmar, Doris

    2016-01-01

    The Amyloid precursor protein (APP) has mainly been investigated in connection with its role in Alzheimer’s Disease (AD) due to its cleavage resulting in the production of the Aβ peptides that accumulate in the plaques characteristic for this disease. However, APP is an evolutionary conserved protein that is not only found in humans but also in many other species, including Drosophila, suggesting an important physiological function. Besides Aβ, several other fragments are produced by the cleavage of APP; large secreted fragments derived from the N-terminus and a small intracellular C-terminal fragment. Although these fragments have received much less attention than Aβ, a picture about their function is finally emerging. In contrast to mammals, which express three APP family members, Drosophila expresses only one APP protein called APP-like or APPL. Therefore APPL functions can be studied in flies without the complication that other APP family members may have redundant functions. Flies lacking APPL are viable but show defects in neuronal outgrowth in the central and peripheral nervous system (PNS) in addition to synaptic changes. Furthermore, APPL has been connected with axonal transport functions. In the adult nervous system, APPL, and more specifically its secreted fragments, can protect neurons from degeneration. APPL cleavage also prevents glial death. Lastly, APPL was found to be involved in behavioral deficits and in regulating sleep/activity patterns. This review, will describe the role of APPL in neuronal development and maintenance and briefly touch on its emerging function in circadian rhythms while an accompanying review will focus on its role in learning and memory formation. PMID:27507933

  17. Enhanced functional connectivity involving the ventromedial hypothalamus following methamphetamine exposure.

    PubMed

    Zuloaga, Damian G; Iancu, Ovidiu D; Weber, Sydney; Etzel, Desiree; Marzulla, Tessa; Stewart, Blair; Allen, Charles N; Raber, Jacob

    2015-01-01

    Methamphetamine (MA) consumption causes disruption of many biological rhythms including the sleep-wake cycle. This circadian effect is seen shortly following MA exposure and later in life following developmental MA exposure. MA phase shifts, entrains the circadian clock and can also alter the entraining effect of light by currently unknown mechanisms. We analyzed and compared immunoreactivity of the immediate early gene c-Fos, a marker of neuronal activity, to assess neuronal activation 2 h following MA exposure in the light and dark phases. We used network analyses of correlation patterns derived from global brain immunoreactivity patterns of c-Fos, to infer functional connectivity between brain regions. There were five distinct patterns of neuronal activation. In several brain areas, neuronal activation following exposure to MA was stronger in the light than the dark phase, highlighting the importance of considering circadian periods of increased effects of MA in defining experimental conditions and understanding the mechanisms underlying detrimental effects of MA exposure to brain function. Functional connectivity between the ventromedial hypothalamus (VMH) and other brain areas, including the paraventricular nucleus of the hypothalamus and basolateral and medial amygdala, was enhanced following MA exposure, suggesting a role for the VMH in the effects of MA on the brain.

  18. Pathway logic modeling of protein functional domains in signal transduction.

    PubMed

    Talcott, C; Eker, S; Knapp, M; Lincoln, P; Laderoute, K

    2004-01-01

    Protein functional domains (PFDs) are consensus sequences within signaling molecules that recognize and assemble other signaling components into complexes. Here we describe the application of an approach called Pathway Logic to the symbolic modeling signal transduction networks at the level of PFDs. These models are developed using Maude, a symbolic language founded on rewriting logic. Models can be queried (analyzed) using the execution, search and model-checking tools of Maude. We show how signal transduction processes can be modeled using Maude at very different levels of abstraction involving either an overall state of a protein or its PFDs and their interactions. The key insight for the latter is our algebraic representation of binding interactions as a graph.

  19. Adsorption of DNA binding proteins to functionalized carbon nanotube surfaces with and without DNA wrapping.

    PubMed

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-02-15

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH2 groups (CONH2-SWNT) exhibited very strong interactions between the CONH2-SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  20. Export of virulence proteins by malaria-infected erythrocytes involves remodeling of host actin cytoskeleton.

    PubMed

    Rug, Melanie; Cyrklaff, Marek; Mikkonen, Antti; Lemgruber, Leandro; Kuelzer, Simone; Sanchez, Cecilia P; Thompson, Jennifer; Hanssen, Eric; O'Neill, Matthew; Langer, Christine; Lanzer, Michael; Frischknecht, Friedrich; Maier, Alexander G; Cowman, Alan F

    2014-11-27

    Following invasion of human red blood cells (RBCs) by the malaria parasite, Plasmodium falciparum, a remarkable process of remodeling occurs in the host cell mediated by trafficking of several hundred effector proteins to the RBC compartment. The exported virulence protein, P falciparum erythrocyte membrane protein 1 (PfEMP1), is responsible for cytoadherence of infected cells to host endothelial receptors. Maurer clefts are organelles essential for protein trafficking, sorting, and assembly of protein complexes. Here we demonstrate that disruption of PfEMP1 trafficking protein 1 (PfPTP1) function leads to severe alterations in the architecture of Maurer's clefts. Furthermore, 2 major surface antigen families, PfEMP1 and STEVOR, are no longer displayed on the host cell surface leading to ablation of cytoadherence to host receptors. PfPTP1 functions in a large complex of proteins and is required for linking of Maurer's clefts to the host actin cytoskeleton.

  1. Evaluation of function predictions by PFP, ESG, and PSI-BLAST for moonlighting proteins

    PubMed Central

    2012-01-01

    Background Advancements in function prediction algorithms are enabling large scale computational annotation for newly sequenced genomes. With the increase in the number of functionally well characterized proteins it has been observed that there are many proteins involved in more than one function. These proteins characterized as moonlighting proteins show varied functional behavior depending on the cell type, localization in the cell, oligomerization, multiple binding sites, etc. The functional diversity shown by moonlighting proteins may have significant impact on the traditional sequence based function prediction methods. Here we investigate how well diverse functions of moonlighting proteins can be predicted by some existing function prediction methods. Results We have analyzed the performances of three major sequence based function prediction methods, PSI-BLAST, the Protein Function Prediction (PFP), and the Extended Similarity Group (ESG) on predicting diverse functions of moonlighting proteins. In predicting discrete functions of a set of 19 experimentally identified moonlighting proteins, PFP showed overall highest recall among the three methods. Although ESG showed the highest precision, its recall was lower than PSI-BLAST. Recall by PSI-BLAST greatly improved when BLOSUM45 was used instead of BLOSUM62. Conclusion We have analyzed the performances of PFP, ESG, and PSI-BLAST in predicting the functional diversity of moonlighting proteins. PFP shows overall better performance in predicting diverse moonlighting functions as compared with PSI-BLAST and ESG. Recall by PSI-BLAST greatly improved when BLOSUM45 was used. This analysis indicates that considering weakly similar sequences in prediction enhances the performance of sequence based AFP methods in predicting functional diversity of moonlighting proteins. The current study will also motivate development of novel computational frameworks for automatic identification of such proteins. PMID:23173871

  2. The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

    PubMed Central

    Wenz, Lena-Sophie; Opaliński, Łukasz; Schuler, Max-Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; van der Laan, Martin; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

    2014-01-01

    The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane. PMID:24781695

  3. Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling

    PubMed Central

    DeBonis, Salvatore; Neumann, Emmanuelle; Skoufias, Dimitrios A.

    2015-01-01

    TPPP/p25 is a microtubule-associated protein, detected in protein inclusions associated with various neurodegenerative diseases. Deletion analysis data show that TPPP/p25 has two microtubule binding sites, both located in intrinsically disordered domains, one at the N-terminal and the other in the C-terminal domain. In copolymerization assays the full-length protein exhibits microtubule stimulation and bundling activity. In contrast, at the same ratio relative to tubulin, truncated forms of TPPP/p25 exhibit either lower or no microtubule stimulation and no bundling activity, suggesting a cooperative phenomenon which is enhanced by the presence of the two binding sites. The binding characteristics of the N- and C-terminally truncated proteins to taxol-stabilized microtubules are similar to the full-length protein. However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules. Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors. The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules. PMID:26289831

  4. Spindle function in Xenopus oocytes involves possible nanodomain calcium signaling

    PubMed Central

    Li, Ruizhen; Leblanc, Julie; He, Kevin; Liu, X. Johné

    2016-01-01

    Intracellular calcium transients are a universal phenomenon at fertilization and are required for egg activation, but the exact role of Ca2+ in second-polar-body emission remains unknown. On the other hand, similar calcium transients have not been demonstrated during oocyte maturation, and yet, manipulating intracellular calcium levels interferes with first-polar-body emission in mice and frogs. To determine the precise role of calcium signaling in polar body formation, we used live-cell imaging coupled with temporally precise intracellular calcium buffering. We found that BAPTA-based calcium chelators cause immediate depolymerization of spindle microtubules in meiosis I and meiosis II. Surprisingly, EGTA at similar or higher intracellular concentrations had no effect on spindle function or polar body emission. Using two calcium probes containing permutated GFP and the calcium sensor calmodulin (Lck-GCaMP3 and GCaMP3), we demonstrated enrichment of the probes at the spindle but failed to detect calcium increase during oocyte maturation at the spindle or elsewhere. Finally, endogenous calmodulin was found to colocalize with spindle microtubules throughout all stages of meiosis. Our results—most important, the different sensitivities of the spindle to BAPTA and EGTA—suggest that meiotic spindle function in frog oocytes requires highly localized, or nanodomain, calcium signaling. PMID:27582389

  5. Involvement of autophagy in NK cell development and function.

    PubMed

    López-Soto, Alejandro; Bravo-San Pedro, José Manuel; Kroemer, Guido; Galluzzi, Lorenzo; Gonzalez, Segundo

    2017-03-04

    Natural killer (NK) cells are the prototypical members of the recently identified family of innate lymphoid cells (ILCs). Thanks to their cytotoxic and secretory functions, NK cells play a key role in the immune response to cells experiencing various forms of stress, including viral infection and malignant transformation. Autophagy is a highly conserved network of degradative pathways that participate in the maintenance of cellular and organismal homeostasis as they promote adaptation to adverse microenvironmental conditions. The relevance of autophagy in the development and functionality of cellular components of the adaptive immune system is well established. Conversely, whether autophagy also plays an important role in the biology of ILC populations such as NK cells has long remained elusive. Recent experimental evidence shows that ablating Atg5 (autophagy-related 5, an essential component of the autophagic machinery) in NK cells and other specific ILC populations results in progressive mitochondrial damage, reactive oxygen species (ROS) overgeneration, and regulated cell death, hence interrupting ILC development. Moreover, disrupting the interaction of ATG7 with phosphorylated FOXO1 (forkhead box O1) in the cytosol of immature NK cells prevents autophagic responses that are essential for NK cell maturation. These findings suggest that activating autophagy may support the maturation of NK cells and other ILCs that manifest antiviral and anticancer activity.

  6. Structure of lpg0406, a carboxymuconolactone decarboxylase family protein possibly involved in antioxidative response from Legionella pneumophila.

    PubMed

    Chen, Xiaofang; Hu, Yanjin; Yang, Bo; Gong, Xiaojian; Zhang, Nannan; Niu, Liwen; Wu, Yun; Ge, Honghua

    2015-12-01

    Lpg0406, a hypothetical protein from Legionella pneumophila, belongs to carboxymuconolactone decarboxylase (CMD) family. We determined the crystal structure of lpg0406 both in its apo and reduced form. The structures reveal that lpg0406 forms a hexamer and have disulfide exchange properties. The protein has an all-helical fold with a conserved thioredoxin-like active site CXXC motif and a proton relay system similar to that of alkylhydroperoxidase from Mycobacterium tuberculosis (MtAhpD), suggesting that lpg0406 might function as an enzyme with peroxidase activity and involved in antioxidant defense. A comparison of the size and the surface topology of the putative substrate-binding region between lpg0406 and MtAhpD indicates that the two enzymes accommodate the different substrate preferences. The structural findings will enhance understanding of the CMD family protein structure and its various functions.

  7. 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/.

  8. Structure and function of gap junction proteins: role of gap junction proteins in embryonic heart development.

    PubMed

    Ahir, Bhavesh K; Pratten, Margaret K

    2014-01-01

    Intercellular (cell-to-cell) communication is a crucial and complex mechanism during embryonic heart development. In the cardiovascular system, the beating of the heart is a dynamic and key regulatory process, which is functionally regulated by the coordinated spread of electrical activity through heart muscle cells. Heart tissues are composed of individual cells, each bearing specialized cell surface membrane structures called gap junctions that permit the intercellular exchange of ions and low molecular weight molecules. Gap junction channels are essential in normal heart function and they assist in the mediated spread of electrical impulses that stimulate synchronized contraction (via an electrical syncytium) of cardiac tissues. This present review describes the current knowledge of gap junction biology. In the first part, we summarise some relevant biochemical and physiological properties of gap junction proteins, including their structure and function. In the second part, we review the current evidence demonstrating the role of gap junction proteins in embryonic development with particular reference to those involved in embryonic heart development. Genetics and transgenic animal studies of gap junction protein function in embryonic heart development are considered and the alteration/disruption of gap junction intercellular communication which may lead to abnormal heart development is also discussed.

  9. Defense-related proteins involved in sugarcane responses to biotic stress.

    PubMed

    Souza, Thais P; Dias, Renata O; Silva-Filho, Marcio C

    2017-02-20

    Sugarcane is one of the most important agricultural crops in the world. However, pathogen infection and herbivore attack cause constant losses in yield. Plants respond to pathogen infection by inducing the expression of several protein types, such as glucanases, chitinases, thaumatins, peptidase inhibitors, defensins, catalases and glycoproteins. Proteins induced by pathogenesis are directly or indirectly involved in plant defense, leading to pathogen death or inducing other plant defense responses. Several of these proteins are induced in sugarcane by different pathogens or insects and have antifungal or insecticidal activity. In this review, defense-related proteins in sugarcane are described, with their putative mechanisms of action, pathogen targets and biotechnological perspectives.

  10. Deciphering primordial cyanobacterial genome functions from protein network analysis.

    PubMed

    Harel, Arye; Karkar, Slim; Cheng, Shu; Falkowski, Paul G; Bhattacharya, Debashish

    2015-03-02

    The Great Oxidation Event (GOE) ∼2.4 billion years ago resulted from the accumulation of oxygen by the ancestors of cyanobacteria [1-3]. Cyanobacteria continue to play a significant role in primary production [4] and in regulating the global marine and limnic nitrogen cycles [5, 6]. Relatively little is known, however, about the evolutionary history and gene content of primordial cyanobacteria [7, 8]. To address these issues, we used protein similarity networks [9], containing proteomes from 48 cyanobacteria as the test group, and reference proteomes from 84 microbes representing four distinct metabolic groups from most reducing to most oxidizing: methanogens, obligate anaerobes (nonmethanogenic), facultative aerobes, and obligate aerobes. These four metabolic groups represent extant bioinformatic proxies for ancient redox chemistries, extending from an anoxic origin through the GOE and ultimately to obligate aerobes [10-13]. Analysis of the network metric degree showed a strong relationship between cyanobacteria and obligate anaerobes, from which cyanobacteria presumably arose, for core functions that include translation, photosynthesis, energy conservation, and environmental interactions. These data were used to reconstruct primordial functions in cyanobacteria that included nine gene families involved in photosynthesis, hydrogenases, and proteins involved in defense from environmental stress. The presence of 60% of these genes in both reaction center I (RC-I) and RC-II-type bacteria may be explained by selective loss of either RC in the evolutionary history of some photosynthetic lineages. Finally, the network reveals that cyanobacteria occupy a unique position among prokaryotes as a hub between anaerobes and obligate aerobes.

  11. Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly.

    PubMed

    Georgiadou, Michaella; Castagnini, Marta; Karimova, Gouzel; Ladant, Daniel; Pelicic, Vladimir

    2012-06-01

    The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system. In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery. Here we have used a bacterial two-hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis. We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact. We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N-terminal motif in PilN is essential for both PilM-PilN interactions and pilus assembly. Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies.

  12. Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis1[OPEN

    PubMed Central

    Bunsupa, Somnuk; Hanada, Kousuke; Maruyama, Akira; Aoyagi, Kaori; Komatsu, Kana; Ueno, Hideki; Yamashita, Madoka; Sasaki, Ryosuke; Oikawa, Akira; Yamazaki, Mami

    2016-01-01

    Lycopodium alkaloids (LAs) are derived from lysine (Lys) and are found mainly in Huperziaceae and Lycopodiaceae. LAs are potentially useful against Alzheimer’s disease, schizophrenia, and myasthenia gravis. Here, we cloned the bifunctional lysine/ornithine decarboxylase (L/ODC), the first gene involved in LA biosynthesis, from the LA-producing plants Lycopodium clavatum and Huperzia serrata. We describe the in vitro and in vivo functional characterization of the L. clavatum L/ODC (LcL/ODC). The recombinant LcL/ODC preferentially catalyzed the decarboxylation of l-Lys over l-ornithine (l-Orn) by about 5 times. Transient expression of LcL/ODC fused with the amino or carboxyl terminus of green fluorescent protein, in onion (Allium cepa) epidermal cells and Nicotiana benthamiana leaves, showed LcL/ODC localization in the cytosol. Transgenic tobacco (Nicotiana tabacum) hairy roots and Arabidopsis (Arabidopsis thaliana) plants expressing LcL/ODC enhanced the production of a Lys-derived alkaloid, anabasine, and cadaverine, respectively, thus, confirming the function of LcL/ODC in plants. In addition, we present an example of the convergent evolution of plant Lys decarboxylase that resulted in the production of Lys-derived alkaloids in Leguminosae (legumes) and Lycopodiaceae (clubmosses). This convergent evolution event probably occurred via the promiscuous functions of the ancestral Orn decarboxylase, which is an enzyme involved in the primary metabolism of polyamine. The positive selection sites were detected by statistical analyses using phylogenetic trees and were confirmed by site-directed mutagenesis, suggesting the importance of those sites in granting the promiscuous function to Lys decarboxylase while retaining the ancestral Orn decarboxylase function. This study contributes to a better understanding of LA biosynthesis and the molecular evolution of plant Lys decarboxylase. PMID:27303024

  13. Coupled cluster Green function: Model involving single and double excitations

    SciTech Connect

    Bhaskaran-Nair, Kiran; Kowalski, Karol; Shelton, William A.

    2016-04-14

    In this paper we report on the parallel implementation of the coupled-cluster (CC) Green function formulation (GF-CC) employing single and double excitations in the cluster operator (GF-CCSD). The detailed description of the underlying algorithm is provided, including the structure of ionization-potential- and electron-affinity-type intermediate tensors which enable to formulate GF-CC approach in a computationally feasible form. Several examples including calculations of ionization-potentials and electron a*ffinities for benchmark systems, which are juxtaposed against the experimental values, provide an illustration of the accuracies attainable in the GFCCSD simulations. We also discuss the structure of the CCSD self energies and discuss approximation that are geared to reduce the computational cost while maintaining the pole structure of the full GF-CCSD approach.

  14. Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction

    PubMed Central

    Watterson, D. Martin; Grum-Tokars, Valerie L.; Roy, Saktimayee M.; Schavocky, James P.; Bradaric, Brinda Desai; Bachstetter, Adam D.; Xing, Bin; Dimayuga, Edgardo; Saeed, Faisal; Zhang, Hong; Staniszewski, Agnieszka; Pelletier, Jeffrey C.; Minasov, George; Anderson, Wayne F.; Arancio, Ottavio; Van Eldik, Linda J.

    2013-01-01

    Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will

  15. Signatures of nitrogen limitation in the elemental composition of the proteins involved in the metabolic apparatus.

    PubMed

    Acquisti, Claudia; Kumar, Sudhir; Elser, James J

    2009-07-22

    Nitrogen (N) is a fundamental component of nucleotides and amino acids and is often a limiting nutrient in natural ecosystems. Thus, study of the N content of biomolecules may establish important connections between ecology and genomics. However, while significant differences in the elemental composition of whole organisms are well documented, how the flux of nutrients in the cell has shaped the evolution of different cellular processes remains poorly understood. By examining the elemental composition of major functional classes of proteins in four multicellular eukaryotic model organisms, we find that the catabolic machinery shows substantially lower N content than the anabolic machinery and the rest of the proteome. This pattern suggests that ecological selection for N conservation specifically targets cellular components that are highly expressed in response to nutrient limitation. We propose that the RNA component of the anabolic machineries is the mechanistic force driving the elemental imbalance we found, and that RNA functions as an intracellular nutrient reservoir that is degraded and recycled during starvation periods. A comparison of the elemental composition of the anabolic and catabolic machineries in species that have experienced different levels of N limitation in their evolutionary history (animals versus plants) suggests that selection for N conservation has preferentially targeted the catabolic machineries of plants, resulting in a lower N content of the proteins involved in their catabolic processes. These findings link the composition of major cellular components to the environmental factors that trigger the activation of those components, suggesting that resource availability has constrained the atomic composition and the molecular architecture of the biotic processes that enable cells to respond to reduced nutrient availability.

  16. How special is the biochemical function of native proteins?

    PubMed

    Skolnick, Jeffrey; Gao, Mu; Zhou, Hongyi

    2016-01-01

    Native proteins perform an amazing variety of biochemical functions, including enzymatic catalysis, and can engage in protein-protein and protein-DNA interactions that are essential for life. A key question is how special are these functional properties of proteins. Are they extremely rare, or are they an intrinsic feature? Comparison to the properties of compact conformations of artificially generated compact protein structures selected for thermodynamic stability but not any type of function, the artificial (ART) protein library, demonstrates that a remarkable number of the properties of native-like proteins are recapitulated. These include the complete set of small molecule ligand-binding pockets and most protein-protein interfaces. ART structures are predicted to be capable of weakly binding metabolites and cover a significant fraction of metabolic pathways, with the most enriched pathways including ancient ones such as glycolysis. Native-like active sites are also found in ART proteins. A small fraction of ART proteins are predicted to have strong protein-protein and protein-DNA interactions. Overall, it appears that biochemical function is an intrinsic feature of proteins which nature has significantly optimized during evolution. These studies raise questions as to the relative roles of specificity and promiscuity in the biochemical function and control of cells that need investigation.

  17. Autophagy-linked FYVE protein (Alfy) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS).

    PubMed

    Han, Huihui; Wei, Wanyi; Duan, Weisong; Guo, Yansu; Li, Yi; Wang, Jie; Bi, Yue; Li, Chunyan

    2015-03-01

    Autophagy-linked FYVE (Alfy) is a protein implicated in the selective degradation of aggregated proteins. In our present study, we found that Alfy was recruited into the aggregated G93A-SOD1 in transgenic mice with amyotrophic lateral sclerosis (ALS). We demonstrated that Alfy overexpression could decrease the expression of mutant proteins via the autophagosome-lysosome pathway, and thereby, the toxicity of mutant proteins was reduced. The clearance of the mutant proteins in NSC34 cells was significantly inhibited in an Alfy knockdown cellular model. We therefore deduced that Alfy translocalization likely is involved in the pathogenesis of ALS. Alfy may be developed into a useful target for ALS therapy.

  18. The involvement of FANCM, FANCI, and checkpoint proteins in the interstrand DNA crosslink repair pathway is conserved in C. elegans.

    PubMed

    Lee, Kyong Yun; Chung, Kee Yang; Koo, Hyeon-Sook

    2010-04-04

    Fanconi anemia (FA) patients are specifically defective in the repair of interstrand DNA crosslinks (ICLs), a complex process involving at least 13 FA proteins and other repair/checkpoint proteins. Of the 13 FA proteins, FANCD1/BRCA2, FANCD2, and FANCJ were previously found to be functionally conserved in C. elegans. We have also identified C. elegans homologs of FANCM and FANCI, and determined their epistatic relationships with homologs of FANCD2, checkpoint proteins, and RAD51 upon DNA crosslinking. The counterparts of FANCM, FANCI, and three checkpoint proteins (RPA, ATR and CHK1) are required for focus formation and ubiquitination associated with FANCD2 in C. elegans. However, C. elegans FANCM affects neither RPA focus formation nor CHK1 phosphorylation induced by ICLs, unlike the reported role of human FANCM, which influences ATR-CHK1 signaling at stalled replication forks. Although focus formation by both FANCD2 and RAD51 requires ATR-CHK1 signaling, FANCD2 and RAD51 acted independently in the formation of their respective foci. Thus, the FANCD2 activation pathway involving FANCM, FANCI, and the checkpoint proteins is conserved in C. elegans but with distinct differences.

  19. EXTRACELLULAR HEAT SHOCK PROTEINS: A NEW LOCATION, A NEW FUNCTION

    PubMed Central

    De Maio, Antonio; Vazquez, Daniel

    2015-01-01

    The expression of heat shock proteins (hsp) is a basic and well conserved cellular response to an array of stresses. These proteins are involved in the repair of cellular damage induced by the stress, which is necessary for the salutary resolution from the insult. Moreover, they confer protection from subsequent insults, which has been coined stress tolerance. Since these proteins are expressed in subcellular compartments, it was thought that their function during stress conditions was circumscribed to the intracellular environment. However, it is now well established that hsp can also be present outside cells where they appear to display a function different than the well understood chaperone role. Extracellular hsp act as alert stress signals priming other cells, particularly of the immune system, to avoid the propagation of the insult and favor resolution. Since the majority of hsp do not possess a secretory peptide signal, they are likely be exported by a non-classical secretory pathway. Different mechanisms have been proposed to explain the export of hsp, including translocation across the plasma membrane and release associated with lipid vesicles, as well as the passive release after cell death by necrosis. Extracellular hsp appear in various flavors, including membrane-bound and membrane-free forms. All of these variants of extracellular hsp suggest that their interactions with cells may be quite diverse, both in target cell types and the activation signaling pathways. This review addresses some of our current knowledge about the release and relevance of extracellular hsp. PMID:23807250

  20. Fragile X syndrome: From protein function to therapy.

    PubMed

    Bagni, Claudia; Oostra, Ben A

    2013-11-01

    Fragile X syndrome (FXS) is the leading monogenic cause of intellectual disability and autism. The FMR1 gene contains a CGG repeat present in the 5'-untranslated region which can be unstable upon transmission to the next generation. The repeat is up to 55 CGGs long in the normal population. In patients with fragile X syndrome (FXS), a repeat length exceeding 200 CGGs generally leads to methylation of the repeat and the promoter region, which is accompanied by silencing of the FMR1 gene. The disease is a result of lack of expression of the fragile X mental retardation protein leading to severe symptoms, including intellectual disability, hyperactivity, and autistic-like behavior. The FMR1 protein (FMRP) has a number of functions. The translational dysregulation of a subset of mRNAs targeted by FMRP is probably the major contribution to FXS. FMRP is also involved in mRNA transport to synapses where protein synthesis occurs. For some FMRP-bound mRNAs, FMRP is a direct modulator of mRNA stability either by sustaining or preventing mRNA decay. Increased knowledge about the role of FMRP has led to the identification of potential treatments for fragile X syndrome that were often tested first in the different animal models. This review gives an overview about the present knowledge of the function of FMRP and the therapeutic strategies in mouse and man.

  1. Protein-protein interactions of the LIM-only protein FHL2 and functional implication of the interactions relevant in cardiovascular disease.

    PubMed

    Tran, M Khang; Kurakula, Kondababu; Koenis, Duco S; de Vries, Carlie J M

    2016-02-01

    FHL2 belongs to the LIM-domain only proteins and contains four and a half LIM domains, each of which are composed of two zinc finger structures. FHL2 exhibits specific interaction with proteins exhibiting diverse functions, including transmembrane receptors, transcription factors and transcription co-regulators, enzymes, and structural proteins. The function of these proteins is regulated by FHL2, which modulates intracellular signal transduction pathways involved in a plethora of cellular tasks. The present review summarizes the current knowledge on the protein interactome of FHL2 and provides an overview of the functional implication of these interactions in apoptosis, migration, and regulation of nuclear receptor function. FHL2 was originally identified in the heart and there is extensive literature available on the role of FHL2 in the cardiovascular system, which is also summarized in this review.

  2. Functional involvement of cerebral cortex in adult sleepwalking.

    PubMed

    Oliviero, A; Della Marca, G; Tonali, P A; Pilato, F; Saturno, E; Dileone, M; Rubino, M; Di Lazzaro, V

    2007-08-01

    The pathophysiology of adult sleepwalking is still poorly understood. However, it is widely accepted that sleepwalking is a disorder of arousal. Arousal circuits widely project to the cortex, including motor cortex. We hypothesized that functional abnormality of these circuits could lead to changes in cortical excitability in sleepwalkers, even during wakefulness. We used transcranial magnetic stimulation (TMS) to examine the excitability of the human motor cortex during wakefulness in a group of adult sleepwalkers. When compared with the healthy control group, short interval intracortical inhibition (SICI), cortical silent period (CSP) duration, and short latency afferent inhibition (SAI) were reduced in adult sleepwalkers during wakefulness. Mean CSP duration was shorter in patients than in controls (80.9 +/- 41 ms vs. 139.4 +/- 37 ms; p = 0.0040). Mean SICI was significantly reduced in patients than in controls (73.5 +/- 38.4% vs. 36.7 +/- 13.1%; p = 0.0061). Mean SAI was also significantly reduced in patients than in controls (65.8 +/- 14.2% vs. 42.8 +/- 16.9%; p = 0.0053). This neurophysiological study suggests that there are alterations in sleepwalkers consistent with an impaired efficiency of inhibitory circuits during wakefulness. This inhibitory impairment could represent the neurophysiological correlate of brain "abnormalities" of sleepwalkers like "immaturity" of some neural circuits, synapses, or receptors.

  3. Pressure-temperature folding landscape in proteins involved in neurodegenerative diseases and cancer.

    PubMed

    Cordeiro, Yraima; Foguel, Debora; Silva, Jerson L

    2013-12-15

    High hydrostatic pressure (HHP) is a valuable tool to study processes such as protein folding, protein hydration and protein-protein interactions. HHP is a nondestructive technique because it reversibly affects internal cavities excluded from the solvent present in the hydrophobic core of proteins. HHP allows the solvation of buried amino acid side chains, thus shifting the equilibrium towards states of the studied molecule or molecular ensemble that occupy smaller volumes. HHP has long been used to dissociate multimeric proteins and protein aggregates and allows investigation of intermediate folding states, some of which are formed by proteins involved in human degenerative diseases, such as spongiform encephalopathies and Parkinson's disease, as well as cancer. When coupled with nuclear magnetic resonance and spectroscopic methods such as infrared and fluorescence spectroscopy, HHP treatment facilitates the understanding of protein folding and misfolding processes; the latter is related to protein aggregation into amyloid or amorphous species. In this review, we will address how HHP provides information about intermediate folding states and the aggregation processes of p53, which is related to cancer, and prion proteins, transthyretin and α-synuclein, which are related to human degenerative diseases.

  4. Amphioxus SARM involved in neural development may function as a suppressor of TLR signaling.

    PubMed

    Yuan, Shaochun; Wu, Kui; Yang, Manyi; Xu, Liqun; Huang, Ling; Liu, Huiling; Tao, Xin; Huang, Shengfeng; Xu, Anlong

    2010-06-15

    Among five Toll/IL-1R resistance adaptors, sterile alpha and Toll/IL-1R resistance motif containing protein (SARM) is the only one conserved from Caenorhabditis elegans to human. However, its physiologic roles are hardly understood, and its involvement in TLR signaling remains debatable. In this study, we first demonstrated a predominant expression of amphioxus SARM (Branchiostoma belcheri tsingtauense SARM) in neural cells during embryogenesis and its predominant expression in the digestive system from larva to adult, suggesting its primitive role in neural development and a potential physiologic role in immunity. We further found that B. belcheri tsingtauense SARM was localized in mitochondria and could attenuate the TLR signaling via interacting with amphioxus MyD88 and tumor necrosis receptor associated factor 6. Thus, amphioxus SARM appears unique in that it may play dual functions in neural development and innate immunity by targeting amphioxus TLR signaling.

  5. Modelling protein functional domains in signal transduction using Maude

    NASA Technical Reports Server (NTRS)

    Sriram, M. G.

    2003-01-01

    Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.

  6. Photo Control of Protein Function Using Photoactive Yellow Protein.

    PubMed

    Reis, Jakeb M; Woolley, G Andrew

    2016-01-01

    Photoswitchable proteins are becoming increasingly common tools for manipulating cellular processes with high spatial and temporal precision. Photoactive yellow protein (PYP) is a small, water-soluble protein that undergoes a blue light induced change in conformation. It can serve as a scaffold for designing new tools to manipulate biological processes, but with respect to other protein scaffolds it presents some technical challenges. Here, we present practical information on how to overcome these, including how to synthesize the PYP chromophore, how to express and purify PYP, and how to screen for desired activity.

  7. Toxicant-induced acceleration of epididymal sperm transit: androgen-dependent proteins may be involved.

    PubMed

    Klinefelter, G R; Suarez, J D

    1997-01-01

    protein profile in homogenates of the caput/corpus epididymidis revealed treatment-related diminutions in two proteins CC9 (M(r) = 42 kDa, pI = 4.2) and CC34 (M(r) = 35 kDa, pI = 5.5), and the level of each of these proteins in the caput/corpus was significantly correlated with the decrease in caput/corpus sperm number. Thus, both CEMS and HFLUT accelerate sperm transit through the proximal segment of the epididymis; and, while this effect is not dependent on the testis, it may involve a lesion in androgen-dependent epididymal function.

  8. Integrated protein function prediction by mining function associations, sequences, and protein–protein and gene–gene interaction networks

    PubMed Central

    Cao, Renzhi; Cheng, Jianlin

    2016-01-01

    Motivations 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. Results 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. PMID:26370280

  9. Dynamics of Arabidopsis SUN proteins during mitosis and their involvement in nuclear shaping.

    PubMed

    Oda, Yoshihisa; Fukuda, Hiroo

    2011-05-01

    The nuclear envelope (NE) is a highly active structure with a specific set of nuclear envelope proteins acting in diverse cellular events. SUN proteins are conserved NE proteins among eukaryotes. Although they form nucleocytoplasmic linkage complexes in metazoan cells, their functions in the plant kingdom are unknown. To understand the function of plant SUN proteins, in this study we first investigated the dynamics of Arabidopsis SUN proteins during mitosis in Arabidopsis roots and cultured cells. For this purpose, we performed dual and triple visualization of these proteins, microtubules, chromosomes, and endoplasmic reticulum (ER) in cultured cells, and observed their dynamics during mitosis using a high-speed spinning disk confocal microscope. The localizations of SUN proteins changed dynamically during mitosis, tightly coupled with NE dynamics. Moreover, NE re-formation marked with SUN proteins is temporally and spatially coordinated with plant-specific microtubule structures such as phragmoplasts. Finally, the analysis with gene knockdowns of AtSUN1 and AtSUN2 indicated that they are necessary for the maintenance and/or formation of polarized nuclear shape in root hairs. These results suggest that Arabidopsis SUN proteins function in the maintenance or formation of nuclear shape as components of the nucleocytoskeletal complex.

  10. Functional interactions between a glutamine synthetase promoter and MYB proteins.

    PubMed

    Gómez-Maldonado, Josefa; Avila, Concepción; Torre, Fernando; Cañas, Rafael; Cánovas, Francisco M; Campbell, Malcolm M

    2004-08-01

    In Scots pine (Pinus sylvestris), ammonium assimilation is catalysed by glutamine synthetase (GS) [EC 6.3.1.2], which is encoded by two genes, PsGS1a and PsGS1b. PsGS1b is expressed in the vascular tissue throughout the plant body, where it is believed to play a role in recycling ammonium released by various facets of metabolism. The mechanisms that may underpin the transcriptional regulation of PsGS1b were explored. The PsGS1b promoter contains a region that is enriched in previously characterized cis-acting elements, known as AC elements. Pine nuclear proteins bound these AC element-rich regions in a tissue-specific manner. As previous experiments had shown that R2R3-MYB transcription factors could interact with AC elements, the capacity of the AC elements in the PsGS1b promoter to interact with MYB proteins was examined. Two MYB proteins from loblolly pine (Pinus taeda), PtMYB1 and PtMYB4, bound to the PsGS1b promoter were able to activate transcription from this promoter in yeast, arabidopsis and pine cells. Immunolocalization experiments revealed that the two MYB proteins were most abundant in cells previously shown to accumulate PsGS1b transcripts. Immunoprecipitation analysis and supershift electrophoretic mobility shift assays implicated these same two proteins in the formation of complexes between pine nuclear extracts and the PsGS1b promoter. Given that these MYB proteins were previously shown to have the capacity to activate gene expression related to lignin biosynthesis, we hypothesize that they may function to co-regulate lignification, a process that places significant demands on nitrogen recycling, and GS, the major enzyme involved in the nitrogen recycling pathway.

  11. Aquaporin-Mediated Water and Hydrogen Peroxide Transport Is Involved in Normal Human Spermatozoa Functioning

    PubMed Central

    Laforenza, Umberto; Pellavio, Giorgia; Marchetti, Anna Lisa; Omes, Claudia; Todaro, Federica; Gastaldi, Giulia

    2016-01-01

    Different aquaporins (AQPs) are expressed in human sperm cells and with a different localization. Their function has been related to cell volume control in response to the osmotic changes encountered passing from the epididymal fluid to the cervical mucus or involved in the end stage of cytoplasm removal during sperm maturation. Recently, AQPs have also shown hydrogen peroxide (H2O2) permeability properties. Here, we investigate the expression, localization and functioning of AQPs in human sperm cells with particular attention to their role as peroxiporins in reactive oxygen species (ROS) scavenging in both normospermic and sub-fertile human subjects. Western blotting and immunocytochemistry were used to confirm and clarify the AQPs expression and localization. Water and H2O2 permeability was tested by stopped flow light scattering method and by the CM-H2DCFDA (5-(and-6)-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate, acetyl ester) H2O2 fluorescence probe, respectively. AQP3, -7, -8, and -11 proteins were found in human sperm cells and localized in the head (AQP7), in the middle piece (AQP8) and in the tail (AQP3 and -11) in both the plasma membrane and in intracellular structures. Sperm cells showed water and H2O2 permeability which was reversibly inhibited by H2O2, heat stress and the AQP inhibitor HgCl2. Reduced functionality was observed in patients with compromised basal semen parameters. Present findings suggest that AQPs are involved in both volume regulation and ROS elimination. The relationship between sperm number and motility and AQP functioning was also demonstrated. PMID:28042826

  12. Karyopherins: potential biological elements involved in the delayed graft function in renal transplant recipients

    PubMed Central

    2014-01-01

    Background Immediately after renal transplantation, patients experience rapid and significant improvement of their clinical conditions and undergo considerable systemic and cellular modifications. However, some patients present a slow recovery of the renal function commonly defined as delayed graft function (DGF). Although clinically well characterized, the molecular mechanisms underlying this condition are not totally defined, thus, we are currently missing specific clinical markers to predict and to make early diagnosis of this event. Methods We investigated, using a pathway analysis approach, the transcriptomic profile of peripheral blood mononuclear cells (PBMC) from renal transplant recipients with DGF and with early graft function (EGF), before (T0) and 24 hours (T24) after transplantation. Results Bioinformatics/statistical analysis showed that 15 pathways (8 up-regulated and 7 down-regulated) and 11 pathways (5 up-regulated and 6 down-regulated) were able to identify DGF patients at T0 and T24, respectively. Interestingly, the most up-regulated pathway at both time points was NLS-bearing substrate import into nucleus, which includes genes encoding for several subtypes of karyopherins, a group of proteins involved in nucleocytoplasmic transport. Signal transducers and activators of transcription (STAT) utilize karyopherins-alpha (KPNA) for their passage from cytoplasm into the nucleus. In vitro functional analysis demonstrated that in PBMCs of DGF patients, there was a significant KPNA-mediated nuclear translocation of the phosphorylated form of STAT3 (pSTAT3) after short-time stimulation (2 and 5 minutes) with interleukin-6. Conclusions Our study suggests the involvement, immediately before transplantation, of karyopherin-mediated nuclear transport in the onset and development of DGF. Additionally, it reveals that karyopherins could be good candidates as potential DGF predictive clinical biomarkers and targets for pharmacological interventions in renal

  13. Involvement of S6K1 in mitochondria function and structure in HeLa cells.

    PubMed

    Park, Jisoo; Tran, Quangdon; Mun, Kisun; Masuda, Kouhei; Kwon, So Hee; Kim, Seon-Hwan; Kim, Dong-Hoon; Thomas, George; Park, Jongsun

    2016-12-01

    The major biological function of mitochondria is to generate cellular energy through oxidative phosphorylation. Apart from cellular respiration, mitochondria also play a key role in signaling processes, including aging and cancer metabolism. It has been shown that S6K1-knockout mice are resistant to obesity due to enhanced beta-oxidation, with an increased number of large mitochondria. Therefore, in this report, the possible involvement of S6K1 in regulating mitochondria dynamics and function has been investigated in stable lenti-shS6K1-HeLa cells. Interestingly, S6K1-stably depleted HeLa cells showed phenotypical changes in mitochondria morphology. This observation was further confirmed by detailed image analysis of mitochondria shape. Corresponding molecular changes were also observed in these cells, such as the induction of mitochondrial fission proteins (Drp1 and Fis1). Oxygen consumption is elevated in S6K1-depeleted HeLa cells and FL5.12 cells. In addition, S6K1 depletion leads to enhancement of ATP production in cytoplasm and mitochondria. However, the relative ratio of mitochondrial ATP to cytoplasmic ATP is actually decreased in lenti-shS6K1-HeLa cells compared to control cells. Lastly, induction of mitophagy was found in lenti-shS6K1-HeLa cells with corresponding changes of mitochondria shape on electron microscope analysis. Taken together, our results indicate that S6K1 is involved in the regulation of mitochondria morphology and function in HeLa cells. This study will provide novel insights into S6K1 function in mitochondria-mediated cellular signaling.

  14. Involvement of a small GTP binding protein in HIV-1 release

    PubMed Central

    Audoly, Gilles; Popoff, Michel R; Gluschankof, Pablo

    2005-01-01

    Background There is evidence suggesting that actin binding to HIV-1 encoded proteins, or even actin dynamics themselves, might play a key role in virus budding and/or release from the infected cell. A crucial step in the reorganisation of the actin cytoskeleton is the engagement of various different GTP binding proteins. We have thus studied the involvement of GTP-binding proteins in the final steps of the HIV-1 viral replication cycle. Results Our results demonstrate that virus production is abolished when cellular GTP binding proteins involved in actin polymerisation are inhibited with specific toxins. Conclusion We propose a new HIV budding working model whereby Gag interactions with pre-existing endosomal cellular tracks as well as with a yet non identified element of the actin polymerisation pathway are required in order to allow HIV-1 to be released from the infected cell. PMID:16080789

  15. Proteomic Analysis of Differentially Expressed Proteins Involved in Peel Senescence in Harvested Mandarin Fruit.

    PubMed

    Li, Taotao; Zhang, Jingying; Zhu, Hong; Qu, Hongxia; You, Shulin; Duan, Xuewu; Jiang, Yueming

    2016-01-01

    Mandarin (Citrus reticulata), a non-climacteric fruit, is an economically important fruit worldwide. The mechanism underlying senescence of non-climacteric fruit is poorly understood. In this study, a gel-based proteomic study followed by LC-ESI-MS/MS analysis was carried out to investigate the proteomic changes involved in peel senescence in harvested mandarin "Shatangju" fruit stored for 18 days. Over the course of the storage period, the fruit gradually senesced, accompanied by a decreased respiration rate and increased chlorophyll degradation and disruption of membrane integrity. Sixty-three proteins spots that showed significant differences in abundance were identified. The up-regulated proteins were mainly associated with cell wall degradation, lipid degradation, protein degradation, senescence-related transcription factors, and transcription-related proteins. In contrast, most proteins associated with ATP synthesis and scavenging of reactive oxygen species were significantly down-regulated during peel senescence. Three thioredoxin proteins and three Ca(2+) signaling-related proteins were significantly up-regulated during peel senescence. It is suggested that mandarin peel senescence is associated with energy supply efficiency, decreased antioxidant capability, and increased protein and lipid degradation. In addition, activation of Ca(2+) signaling and transcription factors might be involved in cell wall degradation and primary or secondary metabolism.

  16. Proteomic Analysis of Differentially Expressed Proteins Involved in Peel Senescence in Harvested Mandarin Fruit

    PubMed Central

    Li, Taotao; Zhang, Jingying; Zhu, Hong; Qu, Hongxia; You, Shulin; Duan, Xuewu; Jiang, Yueming

    2016-01-01

    Mandarin (Citrus reticulata), a non-climacteric fruit, is an economically important fruit worldwide. The mechanism underlying senescence of non-climacteric fruit is poorly understood. In this study, a gel-based proteomic study followed by LC-ESI-MS/MS analysis was carried out to investigate the proteomic changes involved in peel senescence in harvested mandarin “Shatangju” fruit stored for 18 days. Over the course of the storage period, the fruit gradually senesced, accompanied by a decreased respiration rate and increased chlorophyll degradation and disruption of membrane integrity. Sixty-three proteins spots that showed significant differences in abundance were identified. The up-regulated proteins were mainly associated with cell wall degradation, lipid degradation, protein degradation, senescence-related transcription factors, and transcription-related proteins. In contrast, most proteins associated with ATP synthesis and scavenging of reactive oxygen species were significantly down-regulated during peel senescence. Three thioredoxin proteins and three Ca2+ signaling-related proteins were significantly up-regulated during peel senescence. It is suggested that mandarin peel senescence is associated with energy supply efficiency, decreased antioxidant capability, and increased protein and lipid degradation. In addition, activation of Ca2+ signaling and transcription factors might be involved in cell wall degradation and primary or secondary metabolism. PMID:27303420

  17. Proteins involved in motility and sperm-egg interaction evolve more rapidly in mouse spermatozoa.

    PubMed

    Vicens, Alberto; Lüke, Lena; Roldan, Eduardo R S

    2014-01-01

    Proteomic studies of spermatozoa have identified a large catalog of integral sperm proteins. Rapid evolution of these proteins may underlie adaptive changes of sperm traits involved in different events leading to fertilization, although the selective forces underlying such rapid evolution are not well understood. A variety of selective forces may differentially affect several steps ending in fertilization, thus resulting in a compartmentalized adaptation of sperm proteins. Here we analyzed the evolution of genes associated to various events in the sperm's life, from sperm formation to sperm-egg interaction. Evolutionary analyses were performed on gene sequences from 17 mouse strains whose genomes have been sequenced. Four of these are derived from wild Mus musculus, M. domesticus, M. castaneus and M. spretus. We found a higher proportion of genes exhibiting a signature of positive selection among those related to sperm motility and sperm-egg interaction. Furthermore, sperm proteins involved in sperm-egg interaction exhibited accelerated evolution in comparison to those involved in other events. Thus, we identified a large set of candidate proteins for future comparative analyses of genotype-phenotype associations in spermatozoa of species subjected to different sexual selection pressures. Adaptive evolution of proteins involved in motility could be driven by sperm competition, since this selective force is known to increase the proportion of motile sperm and their swimming velocity. On the other hand, sperm proteins involved in gamete interaction could be coevolving with their egg partners through episodes of sexual selection or sexual conflict resulting in species-specific sperm-egg interactions and barriers preventing interspecies fertilization.

  18. Covalent immobilization of protein onto a functionalized hydrogenated diamond-like carbon substrate.

    PubMed

    Biswas, Hari Shankar; Datta, Jagannath; Chowdhury, D P; Reddy, A V R; Ghosh, Uday Chand; Srivastava, Arvind Kumar; Ray, Nihar Ranjan

    2010-11-16

    Hydrogenated diamond-like carbon (HDLC) has an atomically smooth surface that can be deposited on high-surface area substrata and functionalized with reactive chemical groups, providing an ideal substrate for protein immobilization. A synthetic sequence is described involving deposition and hydrogenation of DLC followed by chemical functionalization. These functional groups are reacted with amines on proteins causing covalent immobilization on contact. Raman measurements confirm the presence of these surface functional groups, and Fourier transform infrared spectroscopy (FTIR) confirms covalent protein immobilization. Atomic force microscopy (AFM) of immobilized proteins is reproducible because proteins do not move as a result of interactions with the AFM probe-tip, thus providing an advantage over mica substrata typically used in AFM studies of protein. HDLC offers many of the same technical advantages as oxidized graphene but also allows for coating large surface areas of biomaterials relevant to the fabrication of medical/biosensor devices.

  19. Lipid droplet-associated proteins (LDAPs) are involved in the compartmentalization of lipophilic compounds in plant cells.

    PubMed

    Gidda, Satinder K; Watt, Samantha; Collins-Silva, Jillian; Kilaru, Aruna; Arondel, Vincent; Yurchenko, Olga; Horn, Patrick J; James, Christopher N; Shintani, David; Ohlrogge, John B; Chapman, Kent D; Mullen, Robert T; Dyer, John M

    2013-11-01

    While lipid droplets have traditionally been considered as inert sites for the storage of triacylglycerols and sterol esters, they are now recognized as dynamic and functionally diverse organelles involved in energy homeostasis, lipid signaling, and stress responses. Unlike most other organelles, lipid droplets are delineated by a half-unit membrane whose protein constituents are poorly understood, except in the specialized case of oleosins, which are associated with seed lipid droplets. Recently, we identified a new class of lipid-droplet associated proteins called LDAPs that localize specifically to the lipid droplet surface within plant cells and share extensive sequence similarity with the small rubber particle proteins (SRPPs) found in rubber-accumulating plants. Here, we provide additional evidence for a role of LDAPs in lipid accumulation in oil-rich fruit tissues, and further explore the functional relationships between LDAPs and SRPPs. In addition, we propose that the larger LDAP/SRPP protein family plays important roles in the compartmentalization of lipophilic compounds, including triacylglycerols and polyisoprenoids, into lipid droplets within plant cells. Potential roles in lipid droplet biogenesis and function of these proteins also are discussed.

  20. The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling

    PubMed Central

    Denker, Annette; Kröhnert, Katharina; Bückers, Johanna; Neher, Erwin; Rizzoli, Silvio O.

    2011-01-01

    Presynaptic nerve terminals contain between several hundred vesicles (for example in small CNS synapses) and several tens of thousands (as in neuromuscular junctions). Although it has long been assumed that such high numbers of vesicles are required to sustain neurotransmission during conditions of high demand, we found that activity in vivo requires the recycling of only a few percent of the vesicles. However, the maintenance of large amounts of reserve vesicles in many evolutionarily distinct species suggests that they are relevant for synaptic function. We suggest here that these vesicles constitute buffers for soluble accessory proteins involved in vesicle recycling, preventing their loss into the axon. Supporting this hypothesis, we found that vesicle clusters contain a large variety of proteins needed for vesicle recycling, but without an obvious function within the clusters. Disrupting the clusters by application of black widow spider venom resulted in the diffusion of numerous soluble proteins into the axons. Prolonged stimulation and ionomycin application had a similar effect, suggesting that calcium influx causes the unbinding of soluble proteins from vesicles. Confirming this hypothesis, we found that isolated synaptic vesicles in vitro sequestered soluble proteins from the cytosol in a process that was inhibited by calcium addition. We conclude that the reserve vesicles support neurotransmission indirectly, ensuring that soluble recycling proteins are delivered upon demand during synaptic activity. PMID:21903923

  1. The microtubule motor protein KIF13A is involved in intracellular trafficking of the Lassa virus matrix protein Z.

    PubMed

    Fehling, Sarah Katharina; Noda, Takeshi; Maisner, Andrea; Lamp, Boris; Conzelmann, Karl-Klaus; Kawaoka, Yoshihiro; Klenk, Hans-Dieter; Garten, Wolfgang; Strecker, Thomas

    2013-02-01

    The small matrix protein Z of arenaviruses has been identified as the main driving force to promote viral particle production at the plasma membrane. Although multiple functions of Z in the arenaviral life cycle have been uncovered, the mechanism of intracellular transport of Z to the site of virus budding is poorly understood and cellular motor proteins that mediate Z trafficking remain to be identified. In the present study, we report that the Z protein of the Old World arenavirus Lassa virus (LASV) interacts with the kinesin family member 13A (KIF13A), a plus-end-directed microtubule-dependent motor protein. Plasmid-driven overexpression of KIF13A results in relocalization of Z to the cell periphery, while functional blockage of endogenous KIF13A by overexpression of a dominant-negative mutant or KIF13A-specific siRNA causes a perinuclearaccumulation and decreased production of both Z-induced virus-like particles and infectious LASV. The interaction of KIF13A with Z proteins from both Old and New World arenaviruses suggests a conserved intracellular transport mechanism. In contrast, the intracellular distribution of the matrix proteins of prototypic members of the paramyxo- and rhabdovirus family is independent of KIF13A. In summary, our studies identify for the first time a molecular motor protein as a critical mediator for intracellular microtubule-dependent transport of arenavirus matrix proteins.

  2. Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles

    NASA Astrophysics Data System (ADS)

    Ballottin, Daniela; Fulaz, Stephanie; Souza, Michele L.; Corio, Paola; Rodrigues, Alexandre G.; Souza, Ana O.; Gaspari, Priscyla M.; Gomes, Alexandre F.; Gozzo, Fábio; Tasic, Ljubica

    2016-06-01

    Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis—isolated as an endophytic fungus from Rizophora mangle—were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon

  3. Nonsymmetric Two-Body Score Function for Protein Fold Recognition:

    NASA Astrophysics Data System (ADS)

    Heo, Muyoung; Cheon, Mookyung; Chang, Iksoo

    The usual two-body score (energy) function to recognize native folds of proteins is Miyazawa-Jernigan (MJ) pairwise-contact function. The pairwise-contact parameters between two amino acids in MJ function are symmetric in a sense that a directional order of amino acids sequence along the backbone of a protein is ignored in constructing score parameters. Here we report that we succeeded in constructing a nonsymmetric two-body score function, capturing a directional order of amino acids sequence, by a perceptron learning and a protein threading. We considered pairs of two adjacent amino acids that are separated by two consecutive peptide bonds with the backbone directionality from the N-terminus to the C-terminus of a protein. We also considered the local environmental character, such as the secondary structures and the hydrophobicity (solvation), of amino acids in protein structures. The score is a corresponding propensity for a directional alignment of these two adjacent amino acids with their local environments. The resulting score function simultaneously recognized native folds of 1006 proteins covering all representative proteins with a homology less than 30% among them. The quality of this score function was validated by a threading test of new distinct 382 proteins with a homology less than 90% among them, and it entailed a high success ratio for recognizing native folds of 364 (95.3%) proteins. It showed a good feasibility of designing protein score functions for protein fold recognition by a perceptron learning and a protein threading.

  4. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

    PubMed Central

    Stenberg, R M; Fortney, J; Barlow, S W; Magrane, B P; Nelson, J A; Ghazal, P

    1990-01-01

    trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression. Images PMID:2157043

  5. Flower development of Phalaenopsis orchid involves functionally divergent SEPALLATA-like genes.

    PubMed

    Pan, Zhao-Jun; Chen, You-Yi; Du, Jian-Syun; Chen, Yun-Yu; Chung, Mei-Chu; Tsai, Wen-Chieh; Wang, Chun-Neng; Chen, Hong-Hwa

    2014-05-01

    The Phalaenopsis orchid produces complex flowers that are commercially valuable, which has promoted the study of its flower development. E-class MADS-box genes, SEPALLATA (SEP), combined with B-, C- and D-class MADS-box genes, are involved in various aspects of plant development, such as floral meristem determination, organ identity, fruit maturation, seed formation and plant architecture. Four SEP-like genes were cloned from Phalaenopsis orchid, and the duplicated PeSEPs were grouped into PeSEP1/3 and PeSEP2/4. All PeSEPs were expressed in all floral organs. PeSEP2 expression was detectable in vegetative tissues. The study of protein-protein interactions suggested that PeSEPs may form higher order complexes with the B-, C-, D-class and AGAMOUS LIKE6-related MADS-box proteins to determine floral organ identity. The tepal became a leaf-like organ when PeSEP3 was silenced by virus-induced silencing, with alterations in epidermis identity and contents of anthocyanin and chlorophyll. Silencing of PeSEP2 had minor effects on the floral phenotype. Silencing of the E-class genes PeSEP2 and PeSEP3 resulted in the downregulation of B-class PeMADS2-6 genes, which indicates an association of PeSEP functions and B-class gene expression. These findings reveal the important roles of PeSEP in Phalaenopsis floral organ formation throughout the developmental process by the formation of various multiple protein complexes.

  6. Computational approaches for rational design of proteins with novel functionalities

    PubMed Central

    Tiwari, Manish Kumar; Singh, Ranjitha; Singh, Raushan Kumar; Kim, In-Won; Lee, Jung-Kul

    2012-01-01

    Proteins are the most multifaceted macromolecules in living systems and have various important functions, including structural, catalytic, sensory, and regulatory functions. Rational design of enzymes is a great challenge to our understanding of protein structure and physical chemistry and has numerous potential applications. Protein design algorithms have been applied to design or engineer proteins that fold, fold faster, catalyze, catalyze faster, signal, and adopt preferred conformational states. The field of de novo protein design, although only a few decades old, is beginning to produce exciting results. Developments in this field are already having a significant impact on biotechnology and chemical biology. The application of powerful computational methods for functional protein designing has recently succeeded at engineering target activities. Here, we review recently reported de novo functional proteins that were developed using various protein design approaches, including rational design, computational optimization, and selection from combinatorial libraries, highlighting recent advances and successes. PMID:24688643

  7. Understanding the functional difference between growth arrest-specific protein 6 and protein S: an evolutionary approach.

    PubMed

    Studer, Romain A; Opperdoes, Fred R; Nicolaes, Gerry A F; Mulder, André B; Mulder, René

    2014-10-01

    Although protein S (PROS1) and growth arrest-specific protein 6 (GAS6) proteins are homologous with a high degree of structural similarity, they are functionally different. The objectives of this study were to identify the evolutionary origins from which these functional differences arose. Bioinformatics methods were used to estimate the evolutionary divergence time and to detect the amino acid residues under functional divergence between GAS6 and PROS1. The properties of these residues were analysed in the light of their three-dimensional structures, such as their stability effects, the identification of electrostatic patches and the identification potential protein-protein interaction. The divergence between GAS6 and PROS1 probably occurred during the whole-genome duplications in vertebrates. A total of 78 amino acid sites were identified to be under functional divergence. One of these sites, Asn463, is involved in N-glycosylation in GAS6, but is mutated in PROS1, preventing this post-translational modification. Sites experiencing functional divergence tend to express a greater diversity of stabilizing/destabilizing effects than sites that do not experience such functional divergence. Three electrostatic patches in the LG1/LG2 domains were found to differ between GAS6 and PROS1. Finally, a surface responsible for protein-protein interactions was identified. These results may help researchers to analyse disease-causing mutations in the light of evolutionary and structural constraints, and link genetic pathology to clinical phenotypes.

  8. HOPS: a novel cAMP-dependent shuttling protein involved in protein synthesis regulation.

    PubMed

    Della Fazia, Maria Agnese; Castelli, Marilena; Bartoli, Daniela; Pieroni, Stefania; Pettirossi, Valentina; Piobbico, Danilo; Viola-Magni, Mariapia; Servillo, Giuseppe

    2005-07-15

    The liver has the ability to autonomously regulate growth and mass. Following partial hepatectomy, hormones, growth factors, cytokines and their coupled signal transduction pathways have been implicated in hepatocyte proliferation. To understand the mechanisms responsible for the proliferative response, we studied liver regeneration by characterization of novel genes that are activated in residual hepatocytes. A regenerating liver cDNA library screening was performed with cDNA-subtracted probes derived from regenerating and normal liver. Here, we describe the biology of Hops (for hepatocyte odd protein shuttling). HOPS is a novel shuttling protein that contains an ubiquitin-like domain, a putative NES and a proline-rich region. HOPS is rapidly exported from the nucleus and is overexpressed during liver regeneration. Evidence shows that cAMP governs HOPS export in hepatocytes of normal and regenerating liver and is mediated via CRM-1. We demonstrate that HOPS binds to elongation factor eEF-1A and interferes in protein synthesis. HOPS overexpression in H-35-hepatoma and 3T3-NIH cells strongly reduces proliferation.

  9. Functional innovation from changes in protein domains and their combinations.

    PubMed

    Lees, Jonathan G; Dawson, Natalie L; Sillitoe, Ian; Orengo, Christine A

    2016-06-01

    Domains are the functional building blocks of proteins. In this work we discuss how domains can contribute to the evolution of new functions. Domains themselves can evolve through various mechanisms, altering their intrinsic function. Domains can also facilitate functional innovations by combining with other domains to make novel proteins. We discuss the mechanisms by which domain and domain combinations support functional innovations. We highlight interesting examples where changes in domain combination promote changes at the domain level.

  10. Hypothesis: NDL proteins function in stress responses by regulating microtubule organization

    PubMed Central

    Khatri, Nisha; Mudgil, Yashwanti

    2015-01-01

    N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants. PMID:26583023

  11. RNA-binding proteins involved in post-transcriptional regulation in bacteria

    PubMed Central

    Van Assche, Elke; Van Puyvelde, Sandra; Vanderleyden, Jos; Steenackers, Hans P.

    2015-01-01

    Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed toward the role of small RNAs (sRNAs) in bacterial post-transcriptional regulation. However, sRNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins (RBPs) play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RBPs, which include (i) adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii) modulating the accessibility of the ribosome binding site of mRNAs, (iii) recruiting and assisting in the interaction of mRNAs with other molecules and (iv) regulating transcription terminator/antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future. PMID:25784899

  12. The functional importance of co-evolving residues in proteins.

    PubMed

    Sandler, Inga; Zigdon, Nitzan; Levy, Efrat; Aharoni, Amir

    2014-02-01

    Computational approaches for detecting co-evolution in proteins allow for the identification of protein-protein interaction networks in different organisms and the assignment of function to under-explored proteins. The detection of co-variation of amino acids within or between proteins, moreover, allows for the discovery of residue-residue contacts and highlights functional residues that can affect the binding affinity, catalytic activity, or substrate specificity of a protein. To explore the functional impact of co-evolutionary changes in proteins, a combined experimental and computational approach must be recruited. Here, we review recent studies that apply computational and experimental tools to obtain novel insight into the structure, function, and evolution of proteins. Specifically, we describe the application of co-evolutionary analysis for predicting high-resolution three-dimensional structures of proteins. In addition, we describe computational approaches followed by experimental analysis for identifying specificity-determining residues in proteins. Finally, we discuss studies addressing the importance of such residues in terms of the functional divergence of proteins, allowing proteins to evolve new functions while avoiding crosstalk with existing cellular pathways or forming reproductive barriers and hence promoting speciation.

  13. Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

    PubMed Central

    Pakula, T M; Caldentey, J; Serrano, M; Gutierrez, C; Hermoso, J M; Salas, M; Bamford, D H

    1990-01-01

    Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication. Images PMID:2251117

  14. Membrane proteins in four acts: function precedes structure determination.

    PubMed

    Cramer, W A; Zakharov, S D; Saif Hasan, S; Zhang, H; Baniulis, D; Zhalnina, M V; Soriano, G M; Sharma, O; Rochet, J C; Ryan, C; Whitelegge, J; Kurisu, G; Yamashita, E

    2011-12-01

    Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) β-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to

  15. Calmodulin and Ca2+/calmodulin-binding proteins are involved in Tetrahymena thermophila phagocytosis.

    PubMed

    Gonda, K; Komatsu, M; Numata, O

    2000-08-01

    The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI. In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.

  16. Involvement of Cyclic Guanosine Monophosphate-Dependent Protein Kinase I in Renal Antifibrotic Effects of Serelaxin

    PubMed Central

    Wetzl, Veronika; Schinner, Elisabeth; Kees, Frieder; Hofmann, Franz; Faerber, Lothar; Schlossmann, Jens

    2016-01-01

    Introduction: Kidney fibrosis has shown to be ameliorated through the involvement of cyclic guanosine monophosphate (cGMP) and its dependent protein kinase I (cGKI). Serelaxin, the recombinant form of human relaxin-II, increases cGMP levels and has shown beneficial effects on kidney function in acute heart failure patients. Antifibrotic properties of serelaxin are supposed to be mediated via relaxin family peptide receptor 1 and subsequently enhanced nitric oxide/cGMP to inhibit transforming growth factor-β (TGF-β) signaling. This study examines the involvement of cGKI in the antifibrotic signaling of serelaxin. Methods and Results: Kidney fibrosis was induced by unilateral ureteral obstruction in wildtype (WT) and cGKI knock-out (KO) mice. After 7 days, renal antifibrotic effects of serelaxin were assessed. Serelaxin treatment for 7 days significantly increased cGMP in the kidney of WT and cGKI-KO. In WT, renal fibrosis was reduced through decreased accumulation of collagen1A1, total collagen, and fibronectin. The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and -9 were positively modulated after treatment. Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased. However, these effects were not observed in cGKI-KO. Conclusion: Antifibrotic renal effects of serelaxin are mediated via cGMP/cGKI to inhibit Smad2- and ERK1-dependent TGF-β signaling and increased PDE5a phosphorylation. PMID:27462268

  17. A Statistical Model of Protein Sequence Similarity and Function Similarity Reveals Overly-Specific Function Predictions

    PubMed Central

    Kolker, Eugene

    2009-01-01

    Background Predicting protein function from primary sequence is an important open problem in modern biology. Not only are there many thousands of proteins of unknown function, current approaches for predicting function must be improved upon. One problem in particular is overly-specific function predictions which we address here with a new statistical model of the relationship between protein sequence similarity and protein function similarity. Methodology Our statistical model is based on sets of proteins with experimentally validated functions and numeric measures of function specificity and function similarity derived from the Gene Ontology. The model predicts the similarity of function between two proteins given their amino acid sequence similarity measured by statistics from the BLAST sequence alignment algorithm. A novel aspect of our model is that it predicts the degree of function similarity shared between two proteins over a continuous range of sequence similarity, facilitating prediction of function with an appropriate level of specificity. Significance Our model shows nearly exact function similarity for proteins with high sequence similarity (bit score >244.7, e-value >1e−62, non-redundant NCBI protein database (NRDB)) and only small likelihood of specific function match for proteins with low sequence similarity (bit score <54.6, e-value <1e−05, NRDB). For sequence similarity ranges in between our annotation model shows an increasing relationship between function similarity and sequence similarity, but with considerable variability. We applied the model to a large set of proteins of unknown function, and predicted functions for thousands of these proteins ranging from general to very specific. We also applied the model to a data set of proteins with previously assigned, specific functions that were electronically based. We show that, on average, these prior function predictions are more specific (quite possibly overly-specific) compared to

  18. Chemical synthesis and biological function of lipidated proteins.

    PubMed

    Yang, Aimin; Zhao, Lei; Wu, Yao-Wen

    2015-01-01

    Lipidated proteins play a key role in many essential biological processes in eukaryotic cells, including signal transduction, membrane trafficking, immune response and pathology. The investigation of the function of lipidated proteins requires access to a reasonable amount of homogenous lipid-modified proteins with defined structures and functional groups. Chemical approaches have provided useful tools to perform such studies. In this review we summarize synthetic methods of lipidated peptides and developments in the chemoselective ligation for the production of lipidated proteins. We introduce the biology of lipidated proteins and highlight the application of synthetic lipidated proteins to tackle important biological questions.

  19. Functional Proteins from Short Peptides: Dayhoff's Hypothesis Turns 50.

    PubMed

    Romero Romero, M Luisa; Rabin, Avigayel; Tawfik, Dan S

    2016-12-23

    First and foremost: Margaret Dayhoff's 1966 hypothesis on the origin of proteins is now an accepted model for the emergence of large, globular, functional proteins from short, simple peptides. However, the fundamental question of how the first protein(s) emerged still stands. The tools and hypotheses pioneered by Dayhoff, and the over 65 million protein sequences and 12 000 structures known today, enable those who follow in her footsteps to address this question.

  20. Involvement of a tissue-specific RNA recognition motif protein in Drosophila spermatogenesis.

    PubMed Central

    Haynes, S R; Cooper, M T; Pype, S; Stolow, D T

    1997-01-01

    RNA binding proteins mediate posttranscriptional regulation of gene expression via their roles in nuclear and cytoplasmic mRNA metabolism. Many of the proteins involved in these processes have a common RNA binding domain, the RNA recognition motif (RRM). We have characterized the Testis-specific RRM protein gene (Tsr), which plays an important role in spermatogenesis in Drosophila melanogaster. Disruption of Tsr led to a dramatic reduction in male fertility due to the production of spermatids with abnormalities in mitochondrial morphogenesis. Tsr is located on the third chromosome at 87F, adjacent to the nuclear pre-mRNA binding protein gene Hrb87F. A 1.7-kb Tsr transcript was expressed exclusively in the male germ line. It encoded a protein containing two RRMs similar to those found in HRB87F as well as a unique C-terminal domain. TSR protein was located in the cytoplasm of spermatocytes and young spermatids but was absent from mature sperm. The cellular proteins expressed in premeiotic primary spermatocytes from Tsr mutant and wild-type males were assessed by two-dimensional gel electrophoresis. Lack of TSR resulted in the premature expression of a few proteins prior to meiosis; this was abolished by a transgenic copy of Tsr. These data demonstrate that TSR negatively regulated the expression of some testis proteins and, in combination with its expression pattern and subcellular localization, suggest that TSR regulates the stability or translatability of some mRNAs during spermatogenesis. PMID:9111341

  1. Functional properties of select edible oilseed proteins.

    PubMed

    Sharma, Girdhari M; Su, Mengna; Joshi, Aditya U; Roux, Kenneth H; Sathe, Shridhar K

    2010-05-12

    Borate saline buffer (0.1 M, pH 8.45) solubilized proteins from almond, Brazil nut, cashew nut, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean seeds were prepared from the corresponding defatted flour. The yield was in the range from 10.6% (macadamia) to 27.4% (almond). The protein content, on a dry weight basis, of the lyophilized preparations ranged from 69.23% (pine nut) to 94.80% (soybean). Isolated proteins from Brazil nut had the lightest and hazelnut the darkest color. Isolated proteins exhibited good solubility in aqueous media. Foaming capacity (<40% overrun) and stability (<1 h) of the isolated proteins were poor to fair. Almond proteins had the highest viscosity among the tested proteins. Oil-holding capacity of the isolated proteins ranged from 2.8 (macadamia) to 7 (soybean) g of oil/g of protein. Least gelation concentrations (% w/v) for almond, Brazil nut, cashew, hazelnut, macadamia, pine nut, pistachio, Spanish peanut, Virginia peanut, and soybean were, respectively, 6, 8, 8, 12, 20, 12, 10, 14, 14, and 16.

  2. Membrane proteins as 14-3-3 clients in functional regulation and intracellular transport.

    PubMed

    Smith, Andrew J; Daut, Jürgen; Schwappach, Blanche

    2011-06-01

    14-3-3 proteins regulate the function and subcellular sorting of membrane proteins. Often, 14-3-3 binding to client proteins requires phosphorylation of the client, but the relevant kinase is unknown in most cases. We summarize current progress in identifying kinases that target membrane proteins with 14-3-3 binding sites and discuss the molecular mechanisms of 14-3-3 action. One of the kinases involved is Akt/PKB, which has recently been shown to activate the 14-3-3-dependent switch in a number of client membrane proteins.

  3. Are Cellulosome Scaffolding Protein CipC and CBM3-Containing Protein HycP, Involved in Adherence of Clostridium cellulolyticum to Cellulose?

    PubMed Central

    Ferdinand, Pierre-Henri; Borne, Romain; Trotter, Valentine; Pagès, Sandrine; Tardif, Chantal; Fierobe, Henri-Pierre; Perret, Stéphanie

    2013-01-01

    Clostridium cellulolyticum, a mesophilic anaerobic bacterium, produces highly active enzymatic complexes called cellulosomes. This strain was already shown to bind to cellulose, however the molecular mechanism(s) involved is not known. In this context we focused on the gene named hycP, encoding a 250-kDa protein of unknown function, containing a Family-3 Carbohydrate Binding Module (CBM3) along with 23 hyaline repeat modules (HYR modules). In the microbial kingdom the gene hycP is only found in C. cellulolyticum and the very close strain recently sequenced Clostridium sp BNL1100. Its presence in C. cellulolyticum guided us to analyze its function and its putative role in adhesion of the cells to cellulose. The CBM3 of HycP was shown to bind to crystalline cellulose and was assigned to the CBM3b subfamily. No hydrolytic activity on cellulose was found with a mini-protein displaying representative domains of HycP. A C. cellulolyticum inactivated hycP mutant strain was constructed, and we found that HycP is neither involved in binding of the cells to cellulose nor that the protein has an obvious role in cell growth on cellulose. We also characterized the role of the cellulosome scaffolding protein CipC in adhesion of C. cellulolyticum to cellulose, since cellulosome scaffolding protein has been proposed to mediate binding of other cellulolytic bacteria to cellulose. A second mutant was constructed, where cipC was inactivated. We unexpectedly found that CipC is only partly involved in binding of C. cellulolyticum to cellulose. Other mechanisms for cellulose adhesion may therefore exist in C. cellulolyticum. In addition, no cellulosomal protuberances were observed at the cellular surface of C. cellulolyticum, what is in contrast to reports from several other cellulosomes producing strains. These findings may suggest that C. cellulolyticum has no dedicated molecular mechanism to aggregate the cellulosomes at the cellular surface. PMID:23935995

  4. The flavivirus capsid protein: Structure, function and perspectives towards drug design.

    PubMed

    Oliveira, Edson R A; Mohana-Borges, Ronaldo; de Alencastro, Ricardo B; Horta, Bruno A C

    2017-01-02

    Flaviviruses, such as dengue and zika viruses, are etiologic agents transmitted to humans mainly by arthropods and are of great epidemiological interest. The flavivirus capsid protein is a structural element required for the viral nucleocapsid assembly that presents the classical function of sheltering the viral genome. After decades of research, many reports have shown its different functionalities and influence over cell normal functioning. The subcellular distribution of this protein, which involves accumulation around lipid droplets and nuclear localization, also corroborates with its multi-functional characteristic. As flavivirus diseases are still in need of global control and in view of the possible key functionalities that the capsid protein promotes over flavivirus biology, novel considerations arise towards anti-flavivirus drug research. This review covers the main aspects concerning structural and functional features of the flavivirus C protein, ultimately, highlighting prospects in drug discovery based on this viral target.

  5. 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.

  6. Under-folded proteins: Conformational ensembles and their roles in protein folding, function, and pathogenesis.

    PubMed

    Uversky, Vladimir N

    2013-11-01

    For decades, protein function was intimately linked to the presence of a unique, aperiodic crystal-like structure in a functional protein. The two only places for conformational ensembles of under-folded (or partially folded) protein forms in this picture were either the end points of the protein denaturation processes or transiently populated folding intermediates. Recent years witnessed dramatic change in this perception and conformational ensembles, which the under-folded proteins are, have moved from the shadow. Accumulated to date data suggest that a protein can exist in at least three global forms-functional and folded, functional and intrinsically disordered (nonfolded), and nonfunctional and misfolded/aggregated. Under-folded protein states are crucial for each of these forms, serving as important folding intermediates of ordered proteins, or as functional states of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs), or as pathology triggers of misfolded proteins. Based on these observations, conformational ensembles of under-folded proteins can be classified as transient (folding and misfolding intermediates) and permanent (IDPs and stable misfolded proteins). Permanently under-folded proteins can further be split into intentionally designed (IDPs and IDPRs) and unintentionally designed (misfolded proteins). Although intrinsic flexibility, dynamics, and pliability are crucial for all under-folded proteins, the different categories of under-foldedness are differently encoded in protein amino acid sequences.

  7. Expression of proteins involved in host plant defense against greenbug infestation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The greenbug, Schizaphis graminum (Rondani), has been recognized as a major pest of small grains, including sorghum and wheat. To understand the molecular mechanisms involved in host plant defense against greenbug aphids, a proteomic analysis of greenbug-induced proteins in the seedlings of sorghum...

  8. Spermidine-Induced Improvement of Reconsolidation of Memory Involves Calcium-Dependent Protein Kinase in Rats

    ERIC Educational Resources Information Center

    Girardi, Bruna Amanda; Ribeiro, Daniela Aymone; Signor, Cristiane; Muller, Michele; Gais, Mayara Ana; Mello, Carlos Fernando; Rubin, Maribel Antonello

    2016-01-01

    In this study, we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by the intrahippocampal administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4-mA footshock as an unconditioned stimulus.…

  9. Crystal structure and functional implications of Pyrococcus furiosus hef helicase domain involved in branched DNA processing.

    PubMed

    Nishino, Tatsuya; Komori, Kayoko; Tsuchiya, Daisuke; Ishino, Yoshizumi; Morikawa, Kosuke

    2005-01-01

    DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus. The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the "thumb" domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family.

  10. Targeting protein function: the expanding toolkit for conditional disruption.

    PubMed

    Campbell, Amy E; Bennett, Daimark

    2016-09-01

    A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.

  11. Targeting protein function: the expanding toolkit for conditional disruption

    PubMed Central

    Campbell, Amy E.; Bennett, Daimark

    2016-01-01

    A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours. PMID:27574023

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

    SciTech Connect

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

    2005-08-01

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

  13. Involvement of the Pepper Antimicrobial Protein CaAMP1 Gene in Broad Spectrum Disease Resistance1[C][OA

    PubMed Central

    Lee, Sung Chul; Hwang, In Sun; Choi, Hyong Woo; Hwang, Byung Kook

    2008-01-01

    Pathogen-inducible antimicrobial defense-related proteins have emerged as key antibiotic peptides and enzymes involved in disease resistance in plants. A novel antimicrobial protein gene, CaAMP1 (for Capsicum annuum ANTIMICROBIAL PROTEIN1), was isolated from pepper (C. annuum) leaves infected with Xanthomonas campestris pv vesicatoria. Expression of the CaAMP1 gene was strongly induced in pepper leaves not only during pathogen infection but also after exposure to abiotic elicitors. The purified recombinant CaAMP1 protein possessed broad-spectrum antimicrobial activity against phytopathogenic bacteria and fungi. CaAMP1:smGFP fusion protein was localized mainly in the external and intercellular regions of onion (Allium cepa) epidermal cells. The virus-induced gene silencing technique and gain-of-function transgenic plants were used to determine the CaAMP1 gene function in plant defense. Silencing of CaAMP1 led to enhanced susceptibility to X. campestris pv vesicatoria and Colletotrichum coccodes infection, accompanied by reduced PATHOGENESIS-RELATED (PR) gene expression. In contrast, overexpression of CaAMP1 in Arabidopsis (Arabidopsis thaliana) conferred broad-spectrum resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora parasitica, and the fungal necrotrophic pathogens Fusarium oxysporum f. sp. matthiolae and Alternaria brassicicola. CaAMP1 overexpression induced the salicylic acid pathway-dependent genes PR1 and PR5 but not the jasmonic acid-dependent defense gene PDF1.2 during P. syringae pv tomato infection. Together, these results suggest that the antimicrobial CaAMP1 protein is involved in broad-spectrum resistance to bacterial and fungal pathogen infection. PMID:18676663

  14. Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level

    PubMed Central

    Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Muñoz-Fuentes, Violeta; Green, Andy J.; Kopuchian, Cecilia; Tubaro, Pablo L.; Alza, Luis; Bulgarella, Mariana; Smith, Matthew M.; Wilson, Robert E.; Fago, Angela; McCracken, Kevin G.; Storz, Jay F.

    2015-01-01

    A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level. PMID:26637114

  15. Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level.

    PubMed

    Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E; Muñoz-Fuentes, Violeta; Green, Andy J; Kopuchian, Cecilia; Tubaro, Pablo L; Alza, Luis; Bulgarella, Mariana; Smith, Matthew M; Wilson, Robert E; Fago, Angela; McCracken, Kevin G; Storz, Jay F

    2015-12-01

    A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level.

  16. Protein function annotation by local binding site surface similarity.

    PubMed

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  17. CML9, a multifunctional Arabidopsis thaliana calmodulin-like protein involved in stress responses and plant growth?

    PubMed Central

    Leba, Louis-Jérôme; Perochon, Alexandre; Cheval, Cécilia; Ranty, Benoit; Galaud, Jean-Philippe; Aldon, Didier

    2012-01-01

    Plants have evolved complex signaling networks to respond to their fluctuating environment and adapt their growth and development. Calcium-dependent signaling pathways play key role in the onset of these adaptive responses. In plant cells, the intracellular calcium transients are triggered by numerous stimuli and it is supposed that the large repertory of calcium sensors present in higher plants could contribute to integrate these signals in physiological responses. Here, we present data on CML9, a calmodulin-like protein that appears to be involved in plant responses to both biotic and abiotic stress. Using a reverse genetic approach based on gain and loss of function mutants, we present here data indicating that this CML might also be involved in root growth control in response to the flagellin, a pathogen-associated molecular pattern (PAMP) also involved in plant immunity. PMID:22899061

  18. Recent approaches in physical modification of protein functionality.

    PubMed

    Mirmoghtadaie, Leila; Shojaee Aliabadi, Saeedeh; Hosseini, Seyede Marzieh

    2016-05-15

    Today, there is a growing demand for novel technologies, such as high hydrostatic pressure, irradiation, ultrasound, filtration, supercritical carbon dioxide, plasma technology, and electrical methods, which are not based on chemicals or heat treatment for modifying ingredient functionality and extending product shelf life. Proteins are essential components in many food processes, and provide various functions in food quality and stability. They can create interfacial films that stabilize emulsions and foams as well as interact to make networks that play key roles in gel and edible film production. These properties of protein are referred to as 'protein functionality', because they can be modified by different processing. The common protein modification (chemical, enzymatic and physical) methods have strong effects on the structure and functionality of food proteins. Furthermore, novel technologies can modify protein structure and functional properties that will be reviewed in this study.

  19. Differential impact of REM sleep deprivation on cytoskeletal proteins of brain regions involved in sleep regulation.

    PubMed

    Rodríguez-Vázquez, Jennifer; Camacho-Arroyo, Ignacio; Velázquez-Moctezuma, Javier

    2012-01-01

    Rapid eye movement (REM) sleep is involved in memory consolidation, which implies synaptic plasticity. This process requires protein synthesis and the reorganization of the neural cytoskeleton. REM sleep deprivation (REMSD) has an impact on some neuronal proteins involved in synaptic plasticity, such as glutamate receptors and postsynaptic density protein 95, but its effects on cytoskeletal proteins is unknown. In this study, the effects of REMSD on the content of the cytoskeletal proteins MAP2 and TAU were analyzed. Adult female rats were submitted to selective REMSD by using the multiple platform technique. After 24, 48 or 72 h of REMSD, rats were decapitated and the following brain areas were dissected: pons, preoptic area, hippocampus and frontal cortex. Protein extraction and Western blot were performed. Results showed an increase in TAU content in the pons, preoptic area and hippocampus after 24 h of REMSD, while in the frontal cortex a significant increase in TAU content was observed after 72 h of REMSD. A TAU content decrease was observed in the hippocampus after 48 h of REMSD. Interestingly, a marked increase in TAU content was observed after 72 h of REMSD. MAP2 content only increased in the preoptic area at 24 h, and in the frontal cortex after 24 and 72 h of REMSD, without significant changes in the pons and hippocampus. These results support the idea that REM sleep plays an important role in the organization of neural cytoskeleton, and that this effect is tissue-specific.

  20. Structural and functional analysis of hypothetical and conserved proteins of Clostridium tetani.

    PubMed

    Enany, Shymaa

    2014-01-01

    The progress in biological technologies has led to rapid accumulation of microbial genomic sequences with a vast number of uncharacterized genes. Proteins encoded by these genes are usually uncharacterized, hypothetical, and/or conserved. In Clostridium tetani (C. tetani), these proteins constitute up to 50% of the expressed proteins. In this regard, understanding the functions and the structures of these proteins is crucially important, particularly in C. tetani, which is a medically important pathogen. Here, we used a variety of bioinformatics tools and databases to analyze 10 hypothetical and conserved proteins in C. tetani. We were able to provide a detailed overview of the functional contributions of some of these proteins in several cellular functions, including (1) evolving antibiotic resistance, (2) interaction with enzymes pathways, and (3) involvement in drug transportation. Among these candidates, we postulated the involvement of one of these hypothetical proteins in the pathogenic activity of tetanus. The structural and functional prediction of these proteins should serve in uncovering and better understanding the function of C. tetani cells to ultimately discover new possible drug targets.

  1. Hantaviral Proteins: Structure, Functions, and Role in Hantavirus Infection

    PubMed Central

    Muyangwa, Musalwa; Martynova, Ekaterina V.; Khaiboullina, Svetlana F.; Morzunov, Sergey P.; Rizvanov, Albert A.

    2015-01-01

    Hantaviruses are the members of the family Bunyaviridae that are naturally maintained in the populations of small mammals, mostly rodents. Most of these viruses can easily infect humans through contact with aerosols or dust generated by contaminated animal waste products. Depending on the particular Hantavirus involved, human infection could result in either hemorrhagic fever with renal syndrome or in Hantavirus cardiopulmonary syndrome. In the past few years, clinical cases of the Hantavirus caused diseases have been on the rise. Understanding structure of the Hantavirus genome and the functions of the key viral proteins are critical for the therapeutic agents’ research. This paper gives a brief overview of the current knowledge on the structure and properties of the Hantavirus nucleoprotein and the glycoproteins. PMID:26640463

  2. Mitogen-activated protein kinases in male reproductive function

    PubMed Central

    Li, Michelle W.M.; Mruk, Dolores D.; Cheng, C. Yan

    2009-01-01

    Recent studies have shown that male reproductive function is modulated via the mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade is involved in numerous male reproductive processes, including spermatogenesis, sperm maturation and activation, capacitation and acrosome reaction, before fertilization of the oocyte. In this review, we discuss the latest findings in this rapidly developing field regarding the role of MAPK in male reproduction in animal models and in human spermatozoa in vitro. This research will facilitate the design of future studies in humans, although much work is needed before this information can be used to manage male infertility and environmental toxicant-induced testicular injury in men, such as blood–testis-barrier disruption. PMID:19303360

  3. Method for printing functional protein microarrays

    NASA Technical Reports Server (NTRS)

    Delehanty, James B.; Ligler, Frances S.

    2003-01-01

    Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.

  4. Proteomic Analysis Reveals Proteins Involved in Seed Imbibition under Salt Stress in Rice

    PubMed Central

    Xu, Enshun; Chen, Mingming; He, Hui; Zhan, Chengfang; Cheng, Yanhao; Zhang, Hongsheng; Wang, Zhoufei

    2017-01-01

    Enhancement of salinity tolerance during seed germination is very important for direct seeding in rice. In this study, the salt-tolerant japonica landrace Jiucaiqing was used to determine the regulators that are involved in seed imbibition under salt stress. Briefly, the comparative proteomic analysis was conducted between dry (0 h) and imbibed (24 h) seeds with 150 mM NaCl. Under salt stress, the uptake of water increased rapidly before 24 h imbibition (Phase I), followed by a plateau of seed imbibition from 24 to 96 h imbibition (Phase II). We identified 14 proteins involved in seed imbibition, in which the majority of these proteins were involved in energy supply and storage protein. The early imbibition process was mediated by protein catabolism; the most of proteins were down-regulated after 24 h imbibition. Eleven genes in salt stress treated seeds were expressed early during the seed imbibition in comparison to control seeds. By comparison, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (BPM), glutelin (GLU2.2 and GLU2.3), glucose-1-phosphate adenylyltransferase large subunit (GAS8), and cupin domain containing protein (CDP3.1 and CDP3.2) were near the regions of quantitative trait loci (QTLs) for seed dormancy, seed reserve utilization, and seed germination in Jiucaiqing. In particular, CDP3.1 was co-located in the region of qIR-3 for imbibition rate, and qGP-3 for germination percentage. The role of CDP3.1 was verified in enhancing seed germination under salt stress using T-DNA mutant. The identified proteins might be applicable for the improvement of seed germination under salt stress in rice. PMID:28105039

  5. Paraoxonase 1 and dietary hyperhomocysteinemia modulate the expression of mouse proteins involved in liver homeostasis.

    PubMed

    Suszyńska-Zajczyk, Joanna; Jakubowski, Hieronim

    2014-01-01

    Homocysteine (Hcy), a product of methionine metabolism, is elevated by the consumption of a high-methionine diet that can cause fatty liver disease. Paraoxonase 1 (Pon1), a hydrolase expressed mainly in the liver and carried in the circulation on high-density lipoprotein, participates in Hcy metabolism. Low Pon1 activity is linked to fatty liver disease. We hypothesize that hyperhomocysteinemia and low Pon1 induce changes in gene expression that could impair liver homeostasis. To test this hypothesis, we analyzed the liver proteome of Pon1(-/-) and Pon1(+/+) mice fed a high methionine diet (1% methionine in the drinking water) for 8 weeks using 2D IEF/SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. We identified seven liver proteins whose expression was significantly altered in Pon1(-/-) mice. In animals fed with a control diet, the expression of three liver proteins involved in lipoprotein metabolism (ApoE), iron metabolism (Ftl), and regulation of nitric oxide generation (Ddah1) was up-regulated by the Pon1(-/-) genotype. In mice fed with a high-methionine diet, expression of four liver proteins was up-regulated and of three proteins was down-regulated by the Pon1(-/-) genotype. The up-regulated proteins are involved in lipoprotein metabolism (ApoE), energy metabolism (Atp5h), oxidative stress response (Prdx2), and nitric oxide regulation (Ddah1). The down-regulated proteins are involved in energy metabolism (Gamt), iron metabolism (Ftl), and catechol metabolism (Comt). Expression of one protein (Ftl) was up-regulated both by the Pon1(-/-) genotype and a high-methionine diet. Our findings suggest that Pon1 interacts with diverse cellular processes - from lipoprotein metabolism, nitric oxide regulation, and energy metabolism to iron transport and antioxidant defenses - that are essential for normal liver homeostasis and modulation of these interactions by a high-methionine diet may contribute to fatty liver disease.

  6. Proteomic Analysis Reveals Proteins Involved in Seed Imbibition under Salt Stress in Rice.

    PubMed

    Xu, Enshun; Chen, Mingming; He, Hui; Zhan, Chengfang; Cheng, Yanhao; Zhang, Hongsheng; Wang, Zhoufei

    2016-01-01

    Enhancement of salinity tolerance during seed germination is very important for direct seeding in rice. In this study, the salt-tolerant japonica landrace Jiucaiqing was used to determine the regulators that are involved in seed imbibition under salt stress. Briefly, the comparative proteomic analysis was conducted between dry (0 h) and imbibed (24 h) seeds with 150 mM NaCl. Under salt stress, the uptake of water increased rapidly before 24 h imbibition (Phase I), followed by a plateau of seed imbibition from 24 to 96 h imbibition (Phase II). We identified 14 proteins involved in seed imbibition, in which the majority of these proteins were involved in energy supply and storage protein. The early imbibition process was mediated by protein catabolism; the most of proteins were down-regulated after 24 h imbibition. Eleven genes in salt stress treated seeds were expressed early during the seed imbibition in comparison to control seeds. By comparison, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (BPM), glutelin (GLU2.2 and GLU2.3), glucose-1-phosphate adenylyltransferase large subunit (GAS8), and cupin domain containing protein (CDP3.1 and CDP3.2) were near the regions of quantitative trait loci (QTLs) for seed dormancy, seed reserve utilization, and seed germination in Jiucaiqing. In particular, CDP3.1 was co-located in the region of qIR-3 for imbibition rate, and qGP-3 for germination percentage. The role of CDP3.1 was verified in enhancing seed germination under salt stress using T-DNA mutant. The identified proteins might be applicable for the improvement of seed germination under salt stress in rice.

  7. Effect of the quality of the interaction data on predicting protein function from protein-protein interactions.

    PubMed

    Ni, Qing-Shan; Wang, Zheng-Zhi; Li, Gang-Guo; Wang, Guang-Yun; Zhao, Ying-Jie

    2009-03-01

    Protein function prediction is an important issue in the post-genomic era. When protein function is deduced from protein interaction data, the traditional methods treat each interaction sample equally, where the qualities of the interaction samples are seldom taken into account. In this paper, we investigate the effect of the quality of protein-protein interaction data on predicting protein function. Moreover, two improved methods, weight neighbour counting method (WNC) and weight chi-square method (WCHI), are proposed by considering the quality of interaction samples with the neighbour counting method (NC) and chi-square method (CHI). Experimental results have shown that the qualities of interaction samples affect the performances of protein function prediction methods seriously. It is also demonstrated that WNC and WCHI methods outperform NC and CHI methods in protein function prediction when example weights are chosen properly.

  8. Emergence of Complexity in Protein Functions and Metabolic Networks

    NASA Technical Reports Server (NTRS)

    Pohorille, Andzej

    2009-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis of very large libraries of random amino acid sequences and subsequently subjecting them to in vitro evolution. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions, important clues have been uncovered. Considerable progress has been also achieved in understanding the origins of membrane proteins. We will address this issue in the example of ion channels - proteins that mediate transport of ions across cell walls. Remarkably, despite overall complexity of these proteins in contemporary cells, their structural motifs are quite simple, with -helices being most common. By combining results of experimental and computer simulation studies on synthetic models and simple, natural channels, I will show that, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during

  9. Insertional Mutagenesis for Genes involved in Otic/Vestibular Development and Function in Xenopus Tropicalis

    NASA Technical Reports Server (NTRS)

    Torrejon, Marcela; Li, Erica; Nguyen, Minh; Winfree, Seth; Wang, Esther; Reinsch, Sigrid; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Sensitivity to gravity is essential for spatial orientation. Consequently, the gravity receptor system is one of the phylogenetically oldest sensory systems, and the special adaptations that enhance sensitivity to gravity are highly conserved. The main goal of this project is to use Xenopus (frog) to identify genes expressed during vestibular and auditory development. These studies will lead a better understanding of the molecular mechanisms involved in vestibular and auditory development and function. We are using a gene-trap approach in Xenopus tropicalis with the green fluorescent protein (GFP) gene as the transgene reporter. GFP expression occurs only when the GFP gene is correctly integrated in actively transcribed genes. Using the GFP as a tag we can easily identify and clone the mutated gene. In addition, we can study the function of the mutated gene by analyzing the defects generated by insertion of the GFP transgene. To date we have tissue specific GFP expression in X. tropicalis including expression in ear, neural tube, kidney, muscle, eyes and nose. Our transgenic animals will soon reach maturity so that we can outcross them and analyze their progeny. Our next goal is to isolate RNA from our transgenics and clone the tagged genes using RACE-PCR. Currently we are optimizing the RACE-PCR method using transgenics with crystallin GFP expression.

  10. Leptin Attenuates the Contractile Function of Adult Rat Cardiomyocytes Involved in Oxidative Stress and Autophagy

    PubMed Central

    Luo, Liu-Jin; Liu, Ying-Ping; Yuan, Xun; Zhang, Gui-Ping; Hou, Ning; Wu, Xiao-Qian; Luo, Jian-Dong; Zhang, Gen-Shui

    2016-01-01

    Background Leptin has been identified as an important protein involved in obesity. As a chronic metabolic disorder, obesity is associated with a high risk of developing cardiovascular and metabolic diseases, including heart failure. The aim of this paper was to investigate the effects and the mechanism of leptin on the contractile function of cardiomyocytes in the adult rat. Methods Isolated adult rat cardiomyocytes were exposed to leptin (1, 10, and 100 nmol/L) for 1 hour. The calcium transients and the contraction of adult rat cardiomyocytes were recorded with SoftEdge MyoCam system. Apocynin, tempol and rapamycin were added respectively, and Western blotting was employed to evaluate the expression of LC3B and Beclin-1. Results The peak shortening and maximal velocity of shortening/relengthening (± dL/dtmax) of cell shortening were significantly decreased, and the time to 50% relengthening was prolonged with leptin perfusion. Leptin also significantly reduced the baseline, peak and time to 50% baseline of calcium transient. Leptin attenuated autophagy as indicated by decreased LC3-II and Beclin-1. All of the abnormalities were significantly attenuated by apocynin, tempol or rapamycin. Conclusions Our results indicated that leptin depressed the intracellular free calcium and myocardial systolic function via increasing oxidative stress and inhibiting autophagy. PMID:27899860

  11. Structure and function of antifreeze proteins.

    PubMed Central

    Davies, Peter L; Baardsnes, Jason; Kuiper, Michael J; Walker, Virginia K

    2002-01-01

    High-resolution three-dimensional structures are now available for four of seven non-homologous fish and insect antifreeze proteins (AFPs). For each of these structures, the ice-binding site of the AFP has been defined by site-directed mutagenesis, and ice etching has indicated that the ice surface is bound by the AFP. A comparison of these extremely diverse ice-binding proteins shows that they have the following attributes in common. The binding sites are relatively flat and engage a substantial proportion of the protein's surface area in ice binding. They are also somewhat hydrophobic -- more so than that portion of the protein exposed to the solvent. Surface-surface complementarity appears to be the key to tight binding in which the contribution of hydrogen bonding seems to be secondary to van der Waals contacts. PMID:12171656

  12. 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.

  13. Bio-basis function neural networks in protein data mining.

    PubMed

    Yang, Zheng Rong; Hamer, Rebecca

    2007-01-01

    Accurately identifying functional sites in proteins is one of the most important topics in bioinformatics and systems biology. In bioinformatics, identifying protease cleavage sites in protein sequences can aid drug/inhibitor design. In systems biology, post-translational protein-protein interaction activity is one of the major components for analyzing signaling pathway activities. Determining functional sites using laboratory experiments are normally time consuming and expensive. Computer programs have therefore been widely used for this kind of task. Mining protein sequence data using computer programs covers two major issues: 1) discovering how amino acid specificity affects functional sites and 2) discovering what amino acid specificity is. Both need a proper coding mechanism prior to using a proper machine learning algorithm. The development of the bio-basis function neural network (BBFNN) has made a new way for protein sequence data mining. The bio-basis function used in BBFNN is biologically sound in well coding biological information in protein sequences, i.e. well measuring the similarity between protein sequences. BBFNN has therefore been outperforming conventional neural networks in many subjects of protein sequence data mining from protease cleavage site prediction to disordered protein identification. This review focuses on the variants of BBFNN and their applications in mining protein sequence data.

  14. Regulation, Signaling, and Physiological Functions of G-Proteins.

    PubMed

    Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

    2016-09-25

    Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators.

  15. Silkmapin of Hyriopsis cumingii, a novel silk-like shell matrix protein involved in nacre formation.

    PubMed

    Liu, Xiaojun; Dong, Shaojian; Jin, Can; Bai, Zhiyi; Wang, Guiling; Li, Jiale

    2015-01-25

    Understanding the role of matrix proteins in nacre formation and biomineralization in mollusks is important for the pearl industry. In this study, the gene encoding the novel Hyriopsis cumingii shell matrix protein silkmapin was characterized. The gene encodes a protein of 30.89kDa in which Gly accounts for 34.41% of the amino acid content, and the C-terminal region binds Ca(2+). Secondary structure prediction indicated a predominantly β-fold and a structure typical of filamentous proteins. Real-time quantitative PCR and in situ hybridization showed that silkmapin was expressed in epithelial cells at the edge and pallial of mantle tissue, indicated that silkmapin play roles in the shell nacreous and prismatic layer formation. Further real-time PCR results indicated an involvement in pearl formation via nucleation of calcium carbonate prior to formation of the nacre.

  16. C11orf83, a Mitochondrial Cardiolipin-Binding Protein Involved in bc1 Complex Assembly and Supercomplex Stabilization

    PubMed Central

    Foti, Michelangelo; Raemy, Etienne; Vaz, Frédéric Maxime; Martinou, Jean-Claude; Bairoch, Amos

    2015-01-01

    Mammalian mitochondria may contain up to 1,500 different proteins, and many of them have neither been confidently identified nor characterized. In this study, we demonstrated that C11orf83, which was lacking experimental characterization, is a mitochondrial inner membrane protein facing the intermembrane space. This protein is specifically associated with the bc1 complex of the electron transport chain and involved in the early stages of its assembly by stabilizing the bc1 core complex. C11orf83 displays some overlapping functions with Cbp4p, a yeast bc1 complex assembly factor. Therefore, we suggest that C11orf83, now called UQCC3, is the functional human equivalent of Cbp4p. In addition, C11orf83 depletion in HeLa cells caused abnormal crista morphology, higher sensitivity to apoptosis, a decreased ATP level due to impaired respiration and subtle, but significant, changes in cardiolipin composition. We showed that C11orf83 binds to cardiolipin by its α-helices 2 and 3 and is involved in the stabilization of bc1 complex-containing supercomplexes, especially the III2/IV supercomplex. We also demonstrated that the OMA1 metalloprotease cleaves C11orf83 in response to mitochondrial depolarization, suggesting a role in the selection of cells with damaged mitochondria for their subsequent elimination by apoptosis, as previously described for OPA1. PMID:25605331

  17. PelC is a Pseudomonas aeruginosa outer membrane lipoprotein of the OMA family of proteins involved in exopolysaccharide transport.

    PubMed

    Vasseur, Perrine; Soscia, Chantal; Voulhoux, Romé; Filloux, Alain

    2007-08-01

    Pseudomonas aeruginosa is a gram-negative bacterium, opportunistic pathogen, which causes severe acute or chronic infections, as is the case with cystic fibrosis patients. Chronic infections are frequently accompanied by the development of the bacterial population into a specialized community called biofilm. The pelA-G gene cluster of P. aeruginosa has been shown to be involved in pellicle production and biofilm formation. The pel genes have been proposed to contribute to the formation of the exopolysaccharide-containing pellicle. However, the function and the subcellular localization of the seven different Pel proteins are poorly understood. Based on bioinformatics analysis, we have previously considered that PelF is a putative glycosyltransferase (GT4 family), whereas PelG is a Wzx-like polysaccharide transporter from the PST family. In this study we have further characterized the PelC protein. We have shown that PelC is an outer membrane lipoprotein. The N-terminal signal peptide of the PelC lipoprotein is sufficient to target the protein into the membranes. However, by constructing various PelC hybrid proteins we also proposed that efficient and functional outer membrane insertion of PelC requires not only the signal peptide and the lipid modification, but also requires the C-terminal domain of PelC. Because the gene encoding the outer membrane lipoprotein PelC is part of a putative gene cluster involved in exopolysaccharide biogenesis, we suggest that PelC is a new member of the outer membrane auxiliary (OMA) family of lipoprotein whose Wza, involved in Escherichia coli capsular polysaccharide transport, is an archetype.

  18. Internal symmetry in protein structures: prevalence, functional relevance and evolution.

    PubMed

    Balaji, Santhanam

    2015-06-01

    Symmetry has been found at various levels of biological organization in the protein structural universe. Numerous evolutionary studies have proposed connections between internal symmetry within protein tertiary structures, quaternary associations and protein functions. Recent computational methods, such as SymD and CE-Symm, facilitate a large-scale detection of internal symmetry in protein structures. Based on the results from these methods, about 20% of SCOP folds, superfamilies and families are estimated to have structures with internal symmetry (Figure 1d). All-β and membrane proteins fold classes contain a relatively high number of unique instances of internal symmetry. In addition to the axis of symmetry, anecdotal evidence suggests that, the region of connection or contact between symmetric units could coincide with functionally relevant sites within a fold. General principles that underlie protein internal symmetry and their connections to protein structural integrity and functions remain to be elucidated.

  19. Structure-based Methods for Computational Protein Functional Site Prediction

    PubMed Central

    Dukka, B KC

    2013-01-01

    Due to the advent of high throughput sequencing techniques and structural genomic projects, the number of gene and protein sequences has been ever increasing. Computational methods to annotate these genes and proteins are even more indispensable. Proteins are important macromolecules and study of the function of proteins is an important problem in structural bioinformatics. This paper discusses a number of methods to predict protein functional site especially focusing on protein ligand binding site prediction. Initially, a short overview is presented on recent advances in methods for selection of homologous sequences. Furthermore, a few recent structural based approaches and sequence-and-structure based approaches for protein functional sites are discussed in details. PMID:24688745

  20. A large-scale evaluation of computational protein function prediction.

    PubMed

    Radivojac, Predrag; Clark, Wyatt T; Oron, Tal Ronnen; Schnoes, Alexandra M; Wittkop, Tobias; Sokolov, Artem; Graim, Kiley; Funk, Christopher; Verspoor, Karin; Ben-Hur, Asa; Pandey, Gaurav; Yunes, Jeffrey M; Talwalkar, Ameet S; Repo, Susanna; Souza, Michael L; Piovesan, Damiano; Casadio, Rita; Wang, Zheng; Cheng, Jianlin; Fang, Hai; Gough, Julian; Koskinen, Patrik; Törönen, Petri; Nokso-Koivisto, Jussi; Holm, Liisa; Cozzetto, Domenico; Buchan, Daniel W A; Bryson, Kevin; Jones, David T; Limaye, Bhakti; Inamdar, Harshal; Datta, Avik; Manjari, Sunitha K; Joshi, Rajendra; Chitale, Meghana; Kihara, Daisuke; Lisewski, Andreas M; Erdin, Serkan; Venner, Eric; Lichtarge, Olivier; Rentzsch, Robert; Yang, Haixuan; Romero, Alfonso E; Bhat, Prajwal; Paccanaro, Alberto; Hamp, Tobias; Kaßner, Rebecca; Seemayer, Stefan; Vicedo, Esmeralda; Schaefer, Christian; Achten, Dominik; Auer, Florian; Boehm, Ariane; Braun, Tatjana; Hecht, Maximilian; Heron, Mark; Hönigschmid, Peter; Hopf, Thomas A; Kaufmann, Stefanie; Kiening, Michael; Krompass, Denis; Landerer, Cedric; Mahlich, Yannick; Roos, Manfred; Björne, Jari; Salakoski, Tapio; Wong, Andrew; Shatkay, Hagit; Gatzmann, Fanny; Sommer, Ingolf; Wass, Mark N; Sternberg, Michael J E; Škunca, Nives; Supek, Fran; Bošnjak, Matko; Panov, Panče; Džeroski, Sašo; Šmuc, Tomislav; Kourmpetis, Yiannis A I; van Dijk, Aalt D J; ter Braak, Cajo J F; Zhou, Yuanpeng; Gong, Qingtian; Dong, Xinran; Tian, Weidong; Falda, Marco; Fontana, Paolo; Lavezzo, Enrico; Di Camillo, Barbara; Toppo, Stefano; Lan, Liang; Djuric, Nemanja; Guo, Yuhong; Vucetic, Slobodan; Bairoch, Amos; Linial, Michal; Babbitt, Patricia C; Brenner, Steven E; Orengo, Christine; Rost, Burkhard; Mooney, Sean D; Friedberg, Iddo

    2013-03-01

    Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report the results from the first large-scale community-based critical assessment of protein function annotation (CAFA) experiment. Fifty-four methods representing the state of the art for protein function prediction were evaluated on a target set of 866 proteins from 11 organisms. Two findings stand out: (i) today's best protein function prediction algorithms substantially outperform widely used first-generation methods, with large gains on all types of targets; and (ii) although the top methods perform well enough to guide experiments, there is considerable need for improvement of currently available tools.

  1. A large-scale evaluation of computational protein function prediction

    PubMed Central

    Radivojac, Predrag; Clark, Wyatt T; Ronnen Oron, Tal; Schnoes, Alexandra M; Wittkop, Tobias; Sokolov, Artem; Graim, Kiley; Funk, Christopher; Verspoor, Karin; Ben-Hur, Asa; Pandey, Gaurav; Yunes, Jeffrey M; Talwalkar, Ameet S; Repo, Susanna; Souza, Michael L; Piovesan, Damiano; Casadio, Rita; Wang, Zheng; Cheng, Jianlin; Fang, Hai; Gough, Julian; Koskinen, Patrik; Törönen, Petri; Nokso-Koivisto, Jussi; Holm, Liisa; Cozzetto, Domenico; Buchan, Daniel W A; Bryson, Kevin; Jones, David T; Limaye, Bhakti; Inamdar, Harshal; Datta, Avik; Manjari, Sunitha K; Joshi, Rajendra; Chitale, Meghana; Kihara, Daisuke; Lisewski, Andreas M; Erdin, Serkan; Venner, Eric; Lichtarge, Olivier; Rentzsch, Robert; Yang, Haixuan; Romero, Alfonso E; Bhat, Prajwal; Paccanaro, Alberto; Hamp, Tobias; Kassner, Rebecca; Seemayer, Stefan; Vicedo, Esmeralda; Schaefer, Christian; Achten, Dominik; Auer, Florian; Böhm, Ariane; Braun, Tatjana; Hecht, Maximilian; Heron, Mark; Hönigschmid, Peter; Hopf, Thomas; Kaufmann, Stefanie; Kiening, Michael; Krompass, Denis; Landerer, Cedric; Mahlich, Yannick; Roos, Manfred; Björne, Jari; Salakoski, Tapio; Wong, Andrew; Shatkay, Hagit; Gatzmann, Fanny; Sommer, Ingolf; Wass, Mark N; Sternberg, Michael J E; Škunca, Nives; Supek, Fran; Bošnjak, Matko; Panov, Panče; Džeroski, Sašo; Šmuc, Tomislav; Kourmpetis, Yiannis A I; van Dijk, Aalt D J; ter Braak, Cajo J F; Zhou, Yuanpeng; Gong, Qingtian; Dong, Xinran; Tian, Weidong; Falda, Marco; Fontana, Paolo; Lavezzo, Enrico; Di Camillo, Barbara; Toppo, Stefano; Lan, Liang; Djuric, Nemanja; Guo, Yuhong; Vucetic, Slobodan; Bairoch, Amos; Linial, Michal; Babbitt, Patricia C; Brenner, Steven E; Orengo, Christine; Rost, Burkhard; Mooney, Sean D; Friedberg, Iddo

    2013-01-01

    Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report the results from the first large-scale community-based Critical Assessment of protein Function Annotation (CAFA) experiment. Fifty-four methods representing the state-of-the-art for protein function prediction were evaluated on a target set of 866 proteins from eleven organisms. Two findings stand out: (i) today’s best protein function prediction algorithms significantly outperformed widely-used first-generation methods, with large gains on all types of targets; and (ii) although the top methods perform well enough to guide experiments, there is significant need for improvement of currently available tools. PMID:23353650

  2. Small GTP-binding proteins of the ras family: a conserved functional mechanism?

    PubMed

    Chardin, P

    1991-04-01

    Mutated ras genes can acquire a transforming potential and are frequently detected in human tumors. The mammalian ras gene family includes at least 35 distinct members that can be divided into three main groups on the basis of their sequence similarity to ras, rho, or rab genes. All these genes encode small GTP-binding proteins. Rho proteins are implicated in actin organization and control of cell shape, probably by interacting with the cytoskeleton and intracellular membranes. Rab proteins are involved in vesicular traffic, and appear to control the translocation of vesicles from donor to acceptor membranes. The precise function of ras proteins is unknown, although the prevailing view is that they act as transducers of mitogenic signals. We propose that ras proteins, by analogy with rho and rab, are involved in the lateral segregation of multi-protein complexes at the plasma membrane, and we suggest how this process may be important for mitogenic signal transduction.

  3. Staphylococcus aureus surface proteins involved in adaptation to oxacillin identified using a novel cell shaving approach.

    PubMed

    Solis, Nestor; Parker, Benjamin L; Kwong, Stephen M; Robinson, Gareth; Firth, Neville; Cordwell, Stuart J

    2014-06-06

    Staphylococcus aureus is a Gram-positive pathogen responsible for a variety of infections, and some strains are resistant to virtually all classes of antibiotics. Cell shaving proteomics using a novel probability scoring algorithm to compare the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted to high levels of oxacillin (APT). APT displayed altered cell morphology compared with COL and increased aggregation in biofilm assays. Increased resistance to β-lactam antibiotics was observed, but adaptation to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150 proteins at a threshold determined by the scoring algorithm. Proteins unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR and SACOL2302. Quantitative RT-PCR showed increased expression of sacol2302 in APT grown with oxacillin (>6-fold compared with COL). Overexpression of sacol2302 in COL to levels consistent with APT (+ oxacillin) did not influence biofilm formation or β-lactam resistance. Proteomics using iTRAQ and LC-MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated elevated APT abundances of LCP proteins, capsule and peptidoglycan biosynthesis proteins, and proteins involved in wall remodelling. Adaptation to oxacillin also induced urease proteins, which maintained culture pH compared to COL. These results show that S. aureus modifies surface architecture in response to antibiotic adaptation.

  4. Evidence against the involvement of ionically bound cell wall proteins in pea epicotyl growth

    NASA Technical Reports Server (NTRS)

    Melan, M. A.; Cosgrove, D. J.

    1988-01-01

    Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.

  5. Identification of glandular (preputial and clitoral) proteins in house rat (Rattus rattus) involved in pheromonal communication.

    PubMed

    Archunan, G; Kamalakkannan, S; Achiraman, S; Rajkumar, R

    2004-10-01

    Proteins (18-20 kDa) belonging to lipocalin family have been reported to act as carriers for ligands binding to pheromones in mouse urine, pig saliva, hamster vaginal fluid and human sweat, that are involved in pheromonal communication. As the preputial gland is a major pheromonal source, the present study was aimed to detect the specific protein bands (around 18-20 kDa) in the preputial and clitoral glands of the house rat, R. rattus. The amount of protein was higher in preputial gland of the male than that of female (clitoral) gland. A 20 kDa protein was noted in male and female glands; however, the intensity of the band was much higher in male than in female. In addition, 70, 60, 35 kDa bands, identified in male preputial gland, were absent in females. The presence of higher concentration of glandular proteins in the male preputial gland suggests that male rats may depend more on these glandular proteins for the maintenance of reproductive and dominance behaviours. The results further suggest that these glandular proteins (20 kDa) may act as a carrier for ligand binding.

  6. BLANNOTATOR: enhanced homology-based function prediction of bacterial proteins

    PubMed Central

    2012-01-01

    Background Automated function prediction has played a central role in determining the biological functions of bacterial proteins. Typically, protein function annotation relies on homology, and function is inferred from other proteins with similar sequences. This approach has become popular in bacterial genomics because it is one of the few methods that is practical for large datasets and because it does not require additional functional genomics experiments. However, the existing solutions produce erroneous predictions in many cases, especially when query sequences have low levels of identity with the annotated source protein. This problem has created a pressing need for improvements in homology-based annotation. Results We present an automated method for the functional annotation of bacterial protein sequences. Based on sequence similarity searches, BLANNOTATOR accurately annotates query sequences with one-line summary descriptions of protein function. It groups sequences identified by BLAST into subsets according to their annotation and bases its prediction on a set of sequences with consistent functional information. We show the results of BLANNOTATOR's performance in sets of bacterial proteins with known functions. We simulated the annotation process for 3090 SWISS-PROT proteins using a database in its state preceding the functional characterisation of the query protein. For this dataset, our method outperformed the five others that we tested, and the improved performance was maintained even in the absence of highly related sequence hits. We further demonstrate the value of our tool by analysing the putative proteome of Lactobacillus crispatus strain ST1. Conclusions BLANNOTATOR is an accurate method for bacterial protein function prediction. It is practical for genome-scale data and does not require pre-existing sequence clustering; thus, this method suits the needs of bacterial genome and metagenome researchers. The method and a web-server are available at

  7. Small-molecule control of protein function through Staudinger reduction

    NASA Astrophysics Data System (ADS)

    Luo, Ji; Liu, Qingyang; Morihiro, Kunihiko; Deiters, Alexander

    2016-11-01

    Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (enhanced green fluorescent protein), protein translocation (nuclear localization sequence), DNA recombination (Cre) and gene editing (Cas9).

  8. Computational design of proteins with novel structure and functions

    NASA Astrophysics Data System (ADS)

    Wei, Yang; Lu-Hua, Lai

    2016-01-01

    Computational design of proteins is a relatively new field, where scientists search the enormous sequence space for sequences that can fold into desired structure and perform desired functions. With the computational approach, proteins can be designed, for example, as regulators of biological processes, novel enzymes, or as biotherapeutics. These approaches not only provide valuable information for understanding of sequence-structure-function relations in proteins, but also hold promise for applications to protein engineering and biomedical research. In this review, we briefly introduce the rationale for computational protein design, then summarize the recent progress in this field, including de novo protein design, enzyme design, and design of protein-protein interactions. Challenges and future prospects of this field are also discussed. Project supported by the National Basic Research Program of China (Grant No. 2015CB910300), the National High Technology Research and Development Program of China (Grant No. 2012AA020308), and the National Natural Science Foundation of China (Grant No. 11021463).

  9. Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C

    PubMed Central

    Nüesch, Jürg P. F.; Dettwiler, Sabine; Corbau, Romuald; Rommelaere, Jean

    1998-01-01

    NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro. PMID:9811734

  10. De novo design of functional proteins: Toward artificial hydrogenases.

    PubMed

    Faiella, Marina; Roy, Anindya; Sommer, Dayn; Ghirlanda, Giovanna

    2013-11-01

    Over the last 25 years, de novo design has proven to be a valid approach to generate novel, well-folded proteins, and most recently, functional proteins. In response to societal needs, this approach is been used increasingly to design functional proteins developed with an eye toward sustainable fuel production. This review surveys recent examples of bioinspired de novo designed peptide based catalysts, focusing in particular on artificial hydrogenases.

  11. PUF proteins: Cellular functions and potential applications.

    PubMed

    Kiani, Seyed Jalal; Taheri, Tahereh; Rafati, Sima; Samimi-Rad, Katayoun

    2016-09-14

    RNA-binding proteins play critical roles in the regulation of gene expression. Among several families of RNA-binding proteins, PUF (Pumilio and FBF) proteins have been the subject of extensive investigations, as they can bind RNA in a sequence-specific manner and they are evolutionarily conserved among a wide range of organisms. The outstanding feature of these proteins is a highly conserved RNA-binding domain, which is known as the Pumilio-homology domain (PUM-HD) that mostly consists of eight tandem repeats. Each repeat recognizes an RNA base with a simple three-letter code that can be programmed in order to change the sequence-specificity of the protein. Using this tailored architecture, researchers have been able to change the specificity of the PUM-HD and target desired transcripts in the cell, even in subcellular compartments. The potential applications of this versatile tool in molecular cell biology seem unbounded and the use of these factors in pharmaceutics might be an interesting field of study in near future.

  12. Protein Carbonylation and Adipocyte Mitochondrial Function*

    PubMed Central

    Curtis, Jessica M.; Hahn, Wendy S.; Stone, Matthew D.; Inda, Jacob J.; Droullard, David J.; Kuzmicic, Jovan P.; Donoghue, Margaret A.; Long, Eric K.; Armien, Anibal G.; Lavandero, Sergio; Arriaga, Edgar; Griffin, Timothy J.; Bernlohr, David A.

    2012-01-01

    Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte. PMID:22822087

  13. 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

  14. Timing Correlations in Proteins Predict Functional Modules and Dynamic Allostery.

    PubMed

    Lin, Milo M

    2016-04-20

    How protein structure encodes functionality is not fully understood. For example, long-range intraprotein communication can occur without measurable conformational change and is often not captured by existing structural correlation functions. It is shown here that important functional information is encoded in the timing of protein motions, rather than motion itself. I introduce the conditional activity function to quantify such timing correlations among the degrees of freedom within proteins. For three proteins, the conditional activities between side-chain dihedral angles were computed using the output of microseconds-long atomistic simulations. The new approach demonstrates that a sparse fraction of side-chain pairs are dynamically correlated over long distances (spanning protein lengths up to 7 nm), in sharp contrast to structural correlations, which are short-ranged (<1 nm). Regions of high self- and inter-side-chain dynamical correlations are found, corresponding to experimentally determined functional modules and allosteric connections, respectively.

  15. Utilization of alkyne bioconjugations to modulate protein function.

    PubMed

    Maza, Johnathan C; Howard, Christina A; Vipani, Megha A; Travis, Christopher R; Young, Douglas D

    2017-01-01

    The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.

  16. A PerR-like protein involved in response to oxidative stress in the extreme bacterium Deinococcus radiodurans

    SciTech Connect

    Liu, Chengzhi; Wang, Liangyan; Li, Tao; Lin, Lin; Dai, Shang; Tian, Bing Hua, Yuejin

    2014-07-18

    Highlights: • We report a novel PerR-like protein of Fur family in D. radiodurans that is not annotated in the current database. • drperR responses to H{sub 2}O{sub 2} and functions as a negative regulator of katE and dps. • We provided implications on how to utilize sequenced genome data and the importance of genome data mining. • This study adds knowledge to complicated regulatory network that responds to ROS stress in D. radiodurans. - Abstract: Response and defense systems against reactive oxygen species (ROS) contribute to the remarkable resistance of Deinococcus radiodurans to oxidative stress induced by oxidants or radiation. However, mechanisms involved in ROS response and defense systems of D. radiodurans are not well understood. Fur family proteins are important in ROS response. Only a single Fur homolog is predicted by sequence similarity in the current D. radiodurans genome database. Our bioinformatics analysis demonstrated an additional guanine nucleotide in the genome of D. radiodurans that is not in the database, leading to the discovery of another Fur homolog DrPerR. Gene disruption mutant of DrPerR showed enhanced resistance to hydrogen peroxide (H{sub 2}O{sub 2}) and increased catalase activity in cell extracts. Real-time PCR results indicated that DrPerR functions as a repressor of the catalase gene katE. Meanwhile, derepression of dps (DNA-binding proteins from starved cells) gene under H{sub 2}O{sub 2} stress by DrPerR point to its regulatory role in metal ions hemostasis. Thus, DrPerR might function as a Fur homolog protein which is involved in ROS response and defense. These results help clarify the complicated regulatory network that responds to ROS stress in D. radiodurans.

  17. Regulator of G Protein Signaling 6 (RGS6) Induces Apoptosis via a Mitochondrial-dependent Pathway Not Involving Its GTPase-activating Protein Activity*

    PubMed Central

    Maity, Biswanath; Yang, Jianqi; Huang, Jie; Askeland, Ryan W.; Bera, Soumen; Fisher, Rory A.

    2011-01-01

    Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for Gα subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (ΔΨm) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ΔΨm was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer. PMID:21041304

  18. Regulator of G protein signaling 6 (RGS6) induces apoptosis via a mitochondrial-dependent pathway not involving its GTPase-activating protein activity.

    PubMed

    Maity, Biswanath; Yang, Jianqi; Huang, Jie; Askeland, Ryan W; Bera, Soumen; Fisher, Rory A

    2011-01-14

    Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for Gα subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (ΔΨ(m)) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ΔΨ(m) was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer.

  19. A survey of computational intelligence techniques in protein function prediction.

    PubMed

    Tiwari, Arvind Kumar; Srivastava, Rajeev

    2014-01-01

    During the past, there was a massive growth of knowledge of unknown proteins with the advancement of high throughput microarray technologies. Protein function prediction is the most challenging problem in bioinformatics. In the past, the homology based approaches were used to predict the protein function, but they failed when a new protein was different from the previous one. Therefore, to alleviate the problems associated with homology based traditional approaches, numerous computational intelligence techniques have been proposed in the recent past. This paper presents a state-of-the-art comprehensive review of various computational intelligence techniques for protein function predictions using sequence, structure, protein-protein interaction network, and gene expression data used in wide areas of applications such as prediction of DNA and RNA binding sites, subcellular localization, enzyme functions, signal peptides, catalytic residues, nuclear/G-protein coupled receptors, membrane proteins, and pathway analysis from gene expression datasets. This paper also summarizes the result obtained by many researchers to solve these problems by using computational intelligence techniques with appropriate datasets to improve the prediction performance. The summary shows that ensemble classifiers and integration of multiple heterogeneous data are useful for