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Sample records for protein complex promotes

  1. Complex architecture of major histocompatibility complex class II promoters: reiterated motifs and conserved protein-protein interactions.

    PubMed Central

    Jabrane-Ferrat, N; Fontes, J D; Boss, J M; Peterlin, B M

    1996-01-01

    The S box (also known as at the H, W, or Z box) is the 5'-most element of the conserved upstream sequences in promoters of major histocompatibility complex class II genes. It is important for their B-cell-specific and interferon gamma-inducible expression. In this study, we demonstrate that the S box represents a duplication of the downstream X box. First, RFX, which is composed of the RFX5-p36 heterodimer that binds to the X box, also binds to the S box and its 5'-flanking sequence. Second, NF-Y, which binds to the Y box and increases interactions between RFX and the X box, also increases the binding of RFX to the S box. Third, RFXs bound to S and X boxes interact with each other in a spatially constrained manner. Finally, we confirmed these protein-protein and protein-DNA interactions by expressing a hybrid RFX5-VP16 protein in cells. We conclude that RFX binds to S and X boxes and that complex interactions between RFX and NF-Y direct B-cell-specific and interferon gamma-inducible expression or major histocompatibility complex class II genes. PMID:8756625

  2. The synaptonemal complex protein, Zip1, promotes the segregation of nonexchange chromosomes at meiosis I

    PubMed Central

    Newnham, Louise; Jordan, Philip; Rockmill, Beth; Roeder, G. Shirleen; Hoffmann, Eva

    2009-01-01

    Crossing over establishes connections between homologous chromosomes that promote their proper segregation at the first meiotic division. However, there exists a backup system to ensure the correct segregation of those chromosome pairs that fail to cross over. We have found that, in budding yeast, a mutation eliminating the synaptonemal complex protein, Zip1, increases the meiosis I nondisjunction rate of nonexchange chromosomes (NECs). The centromeres of NECs become tethered during meiotic prophase, and this tethering is disrupted by the zip1 mutation. Furthermore, the Zip1 protein often colocalizes to the centromeres of the tethered chromosomes, suggesting that Zip1 plays a direct role in holding NECs together. Zip3, a protein involved in the initiation of synaptonemal complex formation, is also important for NEC segregation. In the absence of Zip3, both the tethering of NECs and the localization of Zip1 to centromeres are impaired. A mutation in the MAD3 gene, which encodes a component of the spindle checkpoint, also increases the nondisjunction of NECs. Together, the zip1 and mad3 mutations have an additive effect, suggesting that these proteins act in parallel pathways to promote NEC segregation. We propose that Mad3 promotes the segregation of NECs that are not tethered by Zip1 at their centromeres. PMID:20080752

  3. HIV-1 Tat protein promotes formation of more-processive elongation complexes.

    PubMed Central

    Marciniak, R A; Sharp, P A

    1991-01-01

    The Tat protein of HIV-1 trans-activates transcription in vitro in a cell-free extract of HeLa nuclei. Quantitative analysis of the efficiency of elongation revealed that a majority of the elongation complexes generated by the HIV-1 promoter were not highly processive and terminated within the first 500 nucleotides. Tat trans-activation of transcription from the HIV-1 promoter resulted from an increase in processive character of the elongation complexes. More specifically, the analysis suggests that there exist two classes of elongation complexes initiating from the HIV promoter: a less-processive form and a more-processive form. Addition of purified Tat protein was found to increase the abundance of the more-processive class of elongation complex. The purine nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits transcription in this reaction by decreasing the efficiency of elongation. Surprisingly, stimulation of transcription elongation by Tat was preferentially inhibited by the addition of DRB. Images PMID:1756726

  4. Substrate Binding Promotes Formation of the Skp1-Cul1-Fbxl3 (SCFFbxl3) Protein Complex*

    PubMed Central

    Yumimoto, Kanae; Muneoka, Tetsuya; Tsuboi, Tomohiro; Nakayama, Keiichi I.

    2013-01-01

    The Skp1–Cul1–F-box protein (SCF) complex is one of the most well characterized types of ubiquitin ligase (E3), with the E3 activity of the complex being regulated in part at the level of complex formation. Fbxl3 is an F-box protein that is responsible for the ubiquitylation and consequent degradation of cryptochromes (Crys) and thus regulates oscillation of the circadian clock. Here we show that formation of the SCFFbxl3 complex is regulated by substrate binding in vivo. Fbxl3 did not associate with Skp1 and Cul1 to a substantial extent in transfected mammalian cells. Unexpectedly, however, formation of the SCFFbxl3 complex was markedly promoted by forced expression of its substrate Cry1 in these cells. A mutant form of Fbxl3 that does not bind to Cry1 was unable to form an SCF complex, suggesting that interaction of Cry1 with Fbxl3 is essential for formation of SCFFbxl3. In contrast, recombinant Fbxl3 associated with recombinant Skp1 and Cul1 in vitro even in the absence of recombinant Cry1. Domain-swap analysis revealed that the COOH-terminal leucine-rich repeat domain of Fbxl3 attenuates the interaction of Skp1, suggesting that a yet unknown protein associated with the COOH-terminal domain of Fbxl3 and inhibited SCF complex formation. Our results thus provide important insight into the regulation of both SCF ubiquitin ligase activity and circadian rhythmicity. PMID:24085301

  5. APLF promotes the assembly and activity of non-homologous end joining protein complexes.

    PubMed

    Grundy, Gabrielle J; Rulten, Stuart L; Zeng, Zhihong; Arribas-Bosacoma, Raquel; Iles, Natasha; Manley, Katie; Oliver, Antony; Caldecott, Keith W

    2013-01-01

    Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein-DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway. PMID:23178593

  6. TDP1 promotes assembly of non-homologous end joining protein complexes on DNA.

    PubMed

    Heo, Jinho; Li, Jing; Summerlin, Matthew; Hays, Annette; Katyal, Sachin; McKinnon, Peter J; Nitiss, Karin C; Nitiss, John L; Hanakahi, Leslyn A

    2015-06-01

    The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3'-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ. PMID:25841101

  7. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex

    SciTech Connect

    Nakagawa, Koji; Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie; Asaka, Masahiro; Takeda, Hiroshi; Kobayashi, Masanobu

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer The nuclear protein Artemis physically interacts with AMPK{alpha}2. Black-Right-Pointing-Pointer Artemis co-localizes with AMPK{alpha}2 in the nucleus. Black-Right-Pointing-Pointer Artemis promotes phosphorylation and activation of AMPK. Black-Right-Pointing-Pointer The interaction between AMPK{alpha}2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic {alpha} subunit and regulatory {beta} and {gamma} subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the {alpha}-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPK{alpha}2-binding protein. Artemis was found to co-immunoprecipitate with AMPK{alpha}2, and the co-localization of Artemis with AMPK{alpha}2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK{alpha}2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPK{alpha}2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.

  8. Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4.

    PubMed

    Bullard, Steven A; Seo, Seongjin; Schilling, Birgit; Dyle, Michael C; Dierdorff, Jason M; Ebert, Scott M; DeLau, Austin D; Gibson, Bradford W; Adams, Christopher M

    2016-08-19

    Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level. PMID:27358404

  9. Crystallization and preliminary X-ray analysis of phage Mu activator protein C in a complex with promoter DNA

    SciTech Connect

    Shanmuganatham, Karthik K.; Ravichandran, Manimekalai; Howe, Martha M.; Park, Hee-Won

    2007-07-01

    The isolation and preliminary X-ray analysis of crystals of phage Mu activator protein C bound to promoter DNA are reported. Bacteriophage Mu C protein is an activator of the four Mu late promoters that drive the expression of genes encoding DNA-modification as well as phage head and tail morphogenesis proteins. This report describes the purification and cocrystallization of wild-type and selenomethionine-substituted C protein with a synthetic late promoter P{sub sym}, together with preliminary X-ray diffraction data analysis using SAD phasing. The selenomethionine peak data set was collected from a single crystal which diffracted to 3.1 Å resolution and belonged to space group P4{sub 1} or P4{sub 3}, with unit-cell parameters a = 68.9, c = 187.6 Å and two complexes per asymmetric unit. The structure will reveal the amino acid–DNA interactions and any conformational changes associated with DNA binding.

  10. Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation

    PubMed Central

    Xiong, Hui; Wang, Danling; Chen, Linan; Choo, Yeun Su; Ma, Hong; Tang, Chengyuan; Xia, Kun; Jiang, Wei; Ronai, Ze’ev; Zhuang, Xiaoxi; Zhang, Zhuohua

    2009-01-01

    Mutations in PARKIN, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of Parkinson disease (PD). Although mutations in these genes lead to the same disease state, the functional relationships between them and how their respective disease-associated mutations cause PD are largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed PPD complex) to promote ubiquitination and degradation of Parkin substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human brain lysates. Genetic ablation of either Pink1 or Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well as decreased degradation and increased accumulation of aberrantly expressed Parkin substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat shock–induced misfolded protein. In contrast, PD-pathogenic Parkin and PINK1 mutations showed reduced ability to promote degradation of Parkin substrates. This study identified a functional ubiquitin E3 ligase complex consisting of PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of the complex, which may constitute a mechanism underlying PD pathogenesis. PMID:19229105

  11. A new protein complex promoting the assembly of Rad51 filaments

    PubMed Central

    Sasanuma, Hiroyuki; Tawaramoto, Maki S.; Lao, Jessica P.; Hosaka, Harumi; Sanda, Eri; Suzuki, Mamoru; Yamashita, Eiki; Hunter, Neil; Shinohara, Miki; Nakagawa, Atsushi; Shinohara, Akira

    2015-01-01

    During homologous recombination, eukaryotic RecA homologue Rad51 assembles into a nucleoprotein filament on single-stranded DNA to catalyse homologous pairing and DNA-strand exchange with a homologous template. Rad51 nucleoprotein filaments are highly dynamic and regulated via the coordinated actions of various accessory proteins including Rad51 mediators. Here, we identify a new Rad51 mediator complex. The PCSS complex, comprising budding yeast Psy3, Csm2, Shu1 and Shu2 proteins, binds to recombination sites and is required for Rad51 assembly and function during meiosis. Within the heterotetramer, Psy3-Csm2 constitutes a core sub-complex with DNA-binding activity. In vitro, purified Psy3-Csm2 stabilizes the Rad51–single-stranded DNA complex independently of nucleotide cofactor. The mechanism of Rad51 stabilization is inferred by our high-resolution crystal structure, which reveals Psy3-Csm2 to be a structural mimic of the Rad51-dimer, a fundamental unit of the Rad51-filament. Together, these results reveal a novel molecular mechanism for this class of Rad51-mediators, which includes the human Rad51 paralogues. PMID:23575680

  12. High Fat Diet Enhances β-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting β-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation.

    PubMed

    Maesako, Masato; Uemura, Maiko; Tashiro, Yoshitaka; Sasaki, Kazuki; Watanabe, Kiwamu; Noda, Yasuha; Ueda, Karin; Asada-Utsugi, Megumi; Kubota, Masakazu; Okawa, Katsuya; Ihara, Masafumi; Shimohama, Shun; Uemura, Kengo; Kinoshita, Ayae

    2015-01-01

    Obesity and type 2 diabetes are risk factors of Alzheimer's disease (AD). We reported that a high fat diet (HFD) promotes amyloid precursor protein (APP) cleavage by β-site APP cleaving enzyme 1 (BACE1) without increasing BACE1 levels in APP transgenic mice. However, the detailed mechanism had remained unclear. Here we demonstrate that HFD promotes BACE1/Adaptor protein-2 (AP-2)/clathrin complex formation by increasing AP-2 levels in APP transgenic mice. In Swedish APP overexpressing Chinese hamster ovary (CHO) cells as well as in SH-SY5Y cells, overexpression of AP-2 promoted the formation of BACE1/AP-2/clathrin complex, increasing the level of the soluble form of APP β (sAPPβ). On the other hand, mutant D495R BACE1, which inhibits formation of this trimeric complex, was shown to decrease the level of sAPPβ. Overexpression of AP-2 promoted the internalization of BACE1 from the cell surface, thus reducing the cell surface BACE1 level. As such, we concluded that HFD may induce the formation of the BACE1/AP-2/clathrin complex, which is followed by its transport of BACE1 from the cell surface to the intracellular compartments. These events might be associated with the enhancement of β-site cleavage of APP in APP transgenic mice. Here we present evidence that HFD, by regulation of subcellular trafficking of BACE1, promotes APP cleavage. PMID:26414661

  13. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly

    PubMed Central

    Litovchick, Larisa; Florens, Laurence A.; Swanson, Selene K.; Washburn, Michael P.; DeCaprio, James A.

    2011-01-01

    In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence. PMID:21498570

  14. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly.

    PubMed

    Litovchick, Larisa; Florens, Laurence A; Swanson, Selene K; Washburn, Michael P; DeCaprio, James A

    2011-04-15

    In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence. PMID:21498570

  15. Pax-5 (BSAP) recruits Ets proto-oncogene family proteins to form functional ternary complexes on a B-cell-specific promoter.

    PubMed

    Fitzsimmons, D; Hodsdon, W; Wheat, W; Maira, S M; Wasylyk, B; Hagman, J

    1996-09-01

    The paired box transcription factor Pax-5 (B-cell-specific activator protein) is a key regulator of lineage-specific gene expression and differentiation in B-lymphocytes. We show that Pax-5 functions as a cell type-specific docking protein that facilitates binding of the early B-cell-specific mb-1 promoter by proteins of the Ets proto-oncogene family. Transcriptional activity of the mb-1 promoter in pre-B-cells is critically dependent on binding sites for Pax-5:Ets complexes. Ternary complex assembly requires only the Pax-5 paired box and ETS DNA-binding domains. Mutation of a single base pair in the ternary complex binding site allows for independent binding by Ets proteins but, conversely, inhibits the binding of Pax-5 by itself. Strikingly, the mutation reverses the pattern of complex assembly: Ets proteins recruit Pax-5 to bind the mutated sequence. Recruitment of Net and Elk-1, but not SAP1a, by Pax-5 defines a functional difference between closely related Ets proteins. Replacement of a valine (V68) in the ETS domain of SAP1a by aspartic acid (as found in c-Ets-1, Elk-1, and Net) enhanced ternary complex formation by more than 60-fold. Together, these observations define novel transcription factor interactions that regulate gene expression in B cells. PMID:8804314

  16. Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in Drosophila

    PubMed Central

    Zhao, Shaowei; Geng, Qing; Gao, Yu; Li, Xin; Zhang, Yang; Wang, Zhaohui

    2014-01-01

    In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3′UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage. PMID:25412508

  17. Forkhead transcription factor FoxF1 interacts with Fanconi anemia protein complexes to promote DNA damage response

    PubMed Central

    Pradhan, Arun; Ustiyan, Vladimir; Zhang, Yufang; Kalin, Tanya V.; Kalinichenko, Vladimir V.

    2016-01-01

    Forkhead box F1 (Foxf1) transcription factor is an important regulator of embryonic development but its role in tumor cells remains incompletely understood. While 16 proteins were characterized in Fanconi anemia (FA) core complex, its interactions with cellular transcriptional machinery remain poorly characterized. Here, we identified FoxF1 protein as a novel interacting partner of the FA complex proteins. Using multiple human and mouse tumor cell lines and Foxf1+/− mice we demonstrated that FoxF1 physically binds to and increases stability of FA proteins. FoxF1 co-localizes with FANCD2 in DNA repair foci in cultured cells and tumor tissues obtained from cisplatin-treated mice. In response to DNA damage, FoxF1-deficient tumor cells showed significantly reduced FANCD2 monoubiquitination and FANCM phosphorylation, resulting in impaired formation of DNA repair foci. FoxF1 knockdown caused chromosomal instability, nuclear abnormalities, and increased tumor cell death in response to DNA-damaging agents. Overexpression of FoxF1 in DNA-damaged cells improved stability of FA proteins, decreased chromosomal and nuclear aberrations, restored formation of DNA repair foci and prevented cell death after DNA damage. These findings demonstrate that FoxF1 is a key component of FA complexes and a critical mediator of DNA damage response in tumor cells. PMID:26625197

  18. Upstream stimulatory factor proteins are major components of the glucose response complex of the L-type pyruvate kinase gene promoter.

    PubMed

    Lefrançois-Martinez, A M; Martinez, A; Antoine, B; Raymondjean, M; Kahn, A

    1995-02-10

    L-type pyruvate kinase (L-PK) gene transcription is induced by glucose through its glucose response element (GlRE) composed of two degenerated E boxes able to bind in vitro ubiquitous upstream stimulator factor (USF) proteins. Here we demonstrate in vivo, by transient transfections in hepatoma cells, that (i) native USF proteins synthesized from expression vectors can act as transactivators of the L-PK promoter via the GlRE, stimulating transcription without glucose and, therefore, decreasing the glucose responsiveness of the promoter; (ii) expression of the truncated USF proteins, able to bind the GlRE but devoid of the NH2-terminal activation domain, represses the activation of the L-PK promoter by glucose; and (iii) a similar repression of the glucose effect is observed upon expression of mutant USF proteins devoid of the basic DNA binding domain, able to dimerize with endogenous USF but not to bind the GlRE. We conclude that USF proteins are components of the transcriptional glucose response complex assembled on the L-PK gene promoter. PMID:7852331

  19. MIKC* MADS-Protein Complexes Bind Motifs Enriched in the Proximal Region of Late Pollen-Specific Arabidopsis Promoters[W

    PubMed Central

    Verelst, Wim; Saedler, Heinz; Münster, Thomas

    2007-01-01

    The genome of Arabidopsis (Arabidopsis thaliana) encodes over 100 MADS-domain transcription factors, categorized into five phylogenetic subgroups. Most research efforts have focused on just one of these subgroups (MIKCc), whereas the other four remain largely unexplored. Here, we report on five members of the so-called Mδ or Arabidopsis MIKC* (AtMIKC*) subgroup, which are predominantly expressed during the late stages of pollen development. Very few MADS-box genes function in mature pollen, and from this perspective, the AtMIKC* genes are therefore highly exceptional. We found that the AtMIKC* proteins are able to form multiple heterodimeric complexes in planta, and that these protein complexes exhibit a for the MADS-family unusual and high DNA binding specificity in vitro. Compared to their occurrence in promoters genome wide, AtMIKC* binding sites are strongly overrepresented in the proximal region of late pollen-specific promoters. By combining our experimental data with in silico genomics and pollen transcriptomics approaches, we identified a considerable number of putative direct target genes of the AtMIKC* transcription factor complexes in pollen, many of which have known or proposed functions in pollen tube growth. The expression of several of these predicted targets is altered in mutant pollen in which all AtMIKC* complexes are affected, and in vitro germination of this mutant pollen is severely impaired. Our data therefore suggest that the AtMIKC* protein complexes play an essential role in transcriptional regulation during late pollen development. PMID:17071640

  20. Interactome Analysis of the Human Respiratory Syncytial Virus RNA Polymerase Complex Identifies Protein Chaperones as Important Cofactors That Promote L-Protein Stability and RNA Synthesis

    PubMed Central

    Munday, Diane C.; Wu, Weining; Smith, Nikki; Fix, Jenna; Noton, Sarah Louise; Galloux, Marie; Touzelet, Olivier; Armstrong, Stuart D.; Dawson, Jenna M.; Aljabr, Waleed; Easton, Andrew J.; Rameix-Welti, Marie-Anne; de Oliveira, Andressa Peres; Simabuco, Fernando M.; Ventura, Armando M.; Hughes, David J.; Barr, John N.; Fearns, Rachel; Digard, Paul

    2014-01-01

    ABSTRACT The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is

  1. Self-recognition mechanism of MamA, a magnetosome-associated TPR-containing protein, promotes complex assembly.

    PubMed

    Zeytuni, Natalie; Ozyamak, Ertan; Ben-Harush, Kfir; Davidov, Geula; Levin, Maxim; Gat, Yair; Moyal, Tal; Brik, Ashraf; Komeili, Arash; Zarivach, Raz

    2011-08-16

    The magnetosome, a biomineralizing organelle within magnetotactic bacteria, allows their navigation along geomagnetic fields. Magnetosomes are membrane-bound compartments containing magnetic nanoparticles and organized into a chain within the cell, the assembly and biomineralization of magnetosomes are controlled by magnetosome-associated proteins. Here, we describe the crystal structures of the magnetosome-associated protein, MamA, from Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. MamA folds as a sequential tetra-trico-peptide repeat (TPR) protein with a unique hook-like shape. Analysis of the MamA structures indicates two distinct domains that can undergo conformational changes. Furthermore, structural analysis of seven crystal forms verified that the core of MamA is not affected by crystallization conditions and identified three protein-protein interaction sites, namely a concave site, a convex site, and a putative TPR repeat. Additionally, relying on transmission electron microscopy and size exclusion chromatography, we show that highly stable complexes form upon MamA homooligomerization. Disruption of the MamA putative TPR motif or N-terminal domain led to protein mislocalization in vivo and prevented MamA oligomerization in vitro. We, therefore, propose that MamA self-assembles through its putative TPR motif and its concave site to create a large homooligomeric scaffold which can interact with other magnetosome-associated proteins via the MamA convex site. We discuss the structural basis for TPR homooligomerization that allows the proper function of a prokaryotic organelle. PMID:21784982

  2. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  3. A novel functional interaction between the Sp1-like protein KLF13 and SREBP-Sp1 activation complex underlies regulation of low density lipoprotein receptor promoter function.

    PubMed

    Natesampillai, Sekar; Fernandez-Zapico, Martin E; Urrutia, Raul; Veldhuis, Johannes D

    2006-02-10

    Cholesterol homeostasis is regulated by a family of transcription factors designated sterol regulatory element-binding proteins (SREBPs). Precise control of SREBP-targeted genes requires additional interactions with co-regulatory transcription factors. In the case of the low density lipoprotein receptor (LDLR), SREBP cooperates with the specificity protein Sp1 to activate the promoter. In this report, we describe a novel pathway in LDLR transcriptional regulation distinct from the SREBP-Sp1 activation complex involving the Sp1-like protein Krueppel-like factor 13 (KLF13). Using a combination of RNA interference, electrophoretic mobility shift, chromatin immunoprecipitation, and reporter assays, deletion, and site-directed mutagenesis, we demonstrated that KLF13 mediates repression in a DNA context-selective manner. KLF13 repression of LDLR promoter activity appears to be needed to keep the receptor silent, a state that can be antagonized by Sp1, SREBP, and inhibitors of histone deacetylase activity. Chromatin immunoprecipitation assay confirmed that KLF13 binds proximal LDLR DNA sequences in vivo and that exogenous oxysterol up-regulates such binding. Together these studies identify a novel regulatory pathway in which gene repression by KLF13 must be overcome by the Sp1-SREBP complex to activate the LDLR promoter. Therefore, these data should replace a pre-existent and more simple paradigm that takes into consideration only the induction of the activator proteins Sp1-SREBP as necessary for LDLR promoter drive without including default repression, such as that by KLF13, of the LDLR gene. PMID:16303770

  4. High Expression of PTGR1 Promotes NSCLC Cell Growth via Positive Regulation of Cyclin-Dependent Protein Kinase Complex

    PubMed Central

    Zhou, Weihe; Zhang, Yuefeng; Liu, Yong

    2016-01-01

    Lung cancer has been the most common cancer and the main cause of cancer-related deaths worldwide for several decades. PTGR1 (prostaglandin reductase 1), as a bifunctional enzyme, has been involved in the occurrence and progression of cancer. However, its impact on human lung cancer is rarely reported. In this study, we found that PTGR1 was overexpressed in lung cancer based on the analyses of Oncomine. Moreover, lentivirus-mediated shRNA knockdown of PTGR1 reduced cell viability in human lung carcinoma cells 95D and A549 by MTT and colony formation assay. PTGR1 depletion led to G2/M phase cell cycle arrest and increased the proportion of apoptotic cells in 95D cells by flow cytometry. Furthermore, silencing PTGR1 in 95D cells resulted in decreased levels of cyclin-dependent protein kinase complex (CDK1, CDK2, cyclin A2, and cyclin B1) by western blotting and then PTGR1 is positively correlated with cyclin-dependent protein by using the data mining of the Oncomine database. Therefore, our findings suggest that PTGR1 may play a role in lung carcinogenesis through regulating cell proliferation and is a potential new therapeutic strategy for lung cancer. PMID:27429979

  5. Capping Protein Increases the Rate of Actin-based Motility by Promoting Filament Nucleation by the Arp2/3 Complex

    PubMed Central

    Akin, Orkun; Mullins, R. Dyche

    2008-01-01

    Summary Capping protein is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between capping protein and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex, and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of capping protein and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility. PMID:18510928

  6. A Specific Form of Phospho Protein Phosphatase 2 Regulates Anaphase-promoting Complex/Cyclosome Association with Spindle Poles

    PubMed Central

    Ban, Kenneth H.

    2010-01-01

    In early mitosis, the END (Emi1/NuMA/Dynein-dynactin) network anchors the anaphase-promoting complex/cyclosome (APC/C) to the mitotic spindle and poles. Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity. Emi1 binds directly to hypophosphorylated APC/C, linking the APC/C to the spindle via NuMA. However, whether the phosphorylation state of the APC/C is important for its association with the spindle and what kinases and phosphatases are necessary for regulating this event remain unknown. Here, we describe the regulation of APC/C-mitotic spindle pole association by phosphorylation. We find that only hypophosphorylated APC/C associates with microtubule asters, suggesting that phosphatases are important. Indeed, a specific form of PPP2 (CA/R1A/R2B) binds APC/C, and PPP2 activity is necessary for Cdc27 dephosphorylation. Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle. Consistently, inhibition of cyclin B/Cdk1 activity increased APC/C binding to microtubules. Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation. PMID:20089842

  7. Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

    PubMed Central

    Jin, Han; Zhang, Kai; Qiao, Chunyan; Yuan, Anliang; Li, Daowei; Zhao, Liang; Shi, Ce; Xu, Xiaowei; Ni, Shilei; Zheng, Changyu; Liu, Xiaohua; Yang, Bai; Sun, Hongchen

    2014-01-01

    Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI–al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration. PMID:24855355

  8. Crystallization and X-ray analysis of the transcription-activator protein C1 of bacteriophage P22 in complex with the PRE promoter element.

    PubMed

    Mondal, Avisek; Chattopadhyaya, Rajagopal; Datta, Ajit Bikram; Parrack, Pradeep

    2015-10-01

    The transcription-activator protein C1 of the temperate phage P22 of Salmonella typhimurium plays a key role in the lytic versus lysogenic switch of the phage. A homotetramer of 92-residue polypeptides, C1 binds to an approximate direct repeat similar to the transcription activator CII of coliphage λ. Despite this and several other similarities, including 57% sequence identity to coliphage CII, many biochemical observations on P22 C1 cannot be explained based on the structure of CII. To understand the molecular basis of these differences, C1 was overexpressed and purified and subjected to crystallization trials. Although no successful hits were obtained for the apoprotein, crystals could be obtained when the protein was subjected to crystallization trials in complex with a 23-mer promoter DNA fragment (PRE). These crystals diffracted very well at the home source, allowing the collection of a 2.2 Å resolution data set. The C1-DNA crystals belonged to space group P21, with unit-cell parameters a = 87.27, b = 93.58, c = 111.16 Å, β = 94.51°. Solvent-content analysis suggests that the asymmetric unit contains three tetramer-DNA complexes. The three-dimensional structure is expected to shed light on the mechanism of activation by C1 and the molecular basis of its specificity. PMID:26457520

  9. The Spo12 protein of Saccharomyces cerevisiae: a regulator of mitotic exit whose cell cycle-dependent degradation is mediated by the anaphase-promoting complex.

    PubMed Central

    Shah, R; Jensen, S; Frenz, L M; Johnson, A L; Johnston, L H

    2001-01-01

    The Spo12 protein plays a regulatory role in two of the most fundamental processes of biology, mitosis and meiosis, and yet its biochemical function remains elusive. In this study we concentrate on the genetic and biochemical analysis of its mitotic function. Since high-copy SPO12 is able to suppress a wide variety of mitotic exit mutants, all of which arrest with high Clb-Cdc28 activity, we speculated whether SPO12 is able to facilitate exit from mitosis when overexpressed by antagonizing mitotic kinase activity. We show, however, that Spo12 is not a potent regulator of Clb-Cdc28 activity and can function independently of either the cyclin-dependent kinase inhibitor (CDKi), Sic1, or the anaphase-promoting complex (APC) regulator, Hct1. Spo12 protein level is regulated by the APC and the protein is degraded in G1 by an Hct1-dependent mechanism. We also demonstrate that in addition to localizing to the nucleus Spo12 is a nucleolar protein. We propose a model where overexpression of Spo12 may lead to the delocalization of a small amount of Cdc14 from the nucleolus, resulting in a sufficient lowering of mitotic kinase levels to facilitate mitotic exit. Finally, site-directed mutagenesis of highly conserved residues in the Spo12 protein sequence abolishes both its mitotic suppressor activity as well as its meiotic function. This result is the first indication that Spo12 may carry out the same biochemical function in mitosis as it does in meiosis. PMID:11729145

  10. Head-specific gene expression in Hydra: Complexity of DNA– protein interactions at the promoter of ks1 is inversely correlated to the head activation potential

    PubMed Central

    Endl, Ingrid; Lohmann, Jan U.; Bosch, Thomas C. G.

    1999-01-01

    To gain insight into the molecular mechanisms that direct position-dependent gene expression in the simple and evolutionarily old metazoan Hydra, we have examined DNA–protein interactions in the 1.5-kb cis regulatory region of the head-specific gene ks1. In vitro footprinting and gel-retardation techniques have been used to map the location of all protein-binding sites. To our surprise, we found substantially more proteins binding to ks1 promoter elements in nuclear extract from basal (gastric) than from apical (head- and tentacle-formation zone) cells. One of these proteins is the homeobox protein Cnox-2. In the head regeneration-deficient mutant reg-16, an increased level of nuclear protein binds to ks1 promoter elements. Treatment of polyps with the ks1-inducing phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in reduced binding of nuclear proteins to the ks1 cis regulatory region. As activation of ks1 transcription is correlated with the absence of nuclear proteins binding to the ks1 promoter, we propose that the majority of these proteins act as transcriptional repressors. In this view, the gradient of head activation along the Hydra body axis is caused by a decreasing amount of inhibitory factors, rather than an increasing amount of activators, toward the head. Thus, inhibitory mechanisms might have played a crucial role in regulating position-dependent gene activation during early metazoan evolution. PMID:9990043

  11. Systematic Characterization of Human Protein Complexes Identifies Chromosome Segregation Proteins

    PubMed Central

    Hutchins, James R.A.; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M.; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A.; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A.; Peters, Jan-Michael

    2010-01-01

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference (RNAi) screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization and tandem affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex (APC/C) and the γ-tubulin ring complex (γ-TuRC), large complexes which are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high throughput follow-up analyses of phenotypic screens in mammalian cells. PMID:20360068

  12. Systematic analysis of human protein complexes identifies chromosome segregation proteins.

    PubMed

    Hutchins, James R A; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A; Peters, Jan-Michael

    2010-04-30

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells. PMID:20360068

  13. Initiation complex structure and promoter proofreading.

    PubMed

    Liu, Xin; Bushnell, David A; Silva, Daniel-Adriano; Huang, Xuhui; Kornberg, Roger D

    2011-07-29

    The initiation of transcription by RNA polymerase II is a multistage process. X-ray crystal structures of transcription complexes containing short RNAs reveal three structural states: one with 2- and 3-nucleotide RNAs, in which only the 3'-end of the RNA is detectable; a second state with 4- and 5-nucleotide RNAs, with an RNA-DNA hybrid in a grossly distorted conformation; and a third state with RNAs of 6 nucleotides and longer, essentially the same as a stable elongating complex. The transition from the first to the second state correlates with a markedly reduced frequency of abortive initiation. The transition from the second to the third state correlates with partial "bubble collapse" and promoter escape. Polymerase structure is permissive for abortive initiation, thereby setting a lower limit on polymerase-promoter complex lifetime and allowing the dissociation of nonspecific complexes. Abortive initiation may be viewed as promoter proofreading, and the structural transitions as checkpoints for promoter control. PMID:21798951

  14. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

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

    2005-05-01

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

  15. Complex assembly on the human CYP17 promoter

    PubMed Central

    Sewer, Marion B.; Jagarlapudi, Srinath

    2009-01-01

    Optimal steroid hormone biosynthesis occurs via the integration of multiple regulatory processes, one of which entails a coordinate increase in the transcription of all genes required for steroidogenesis. In the human adrenal cortex adrenocorticotropin (ACTH) activates a signaling cascade that promotes the dynamic assembly of protein complexes on the promoters of steroidogenic genes. For CYP17, multiple transcription factors, including steroidogenic factor-1 (SF-1), GATA-6, and sterol regulatory binding protein 1 (SREBP1), are recruited to the promoter during activated transcription. The ability of these factors to increase CYP17 mRNA expression requires the formation of higher order coregulatory complexes, many of which contain enzymatic activities that post-translationally modify both the transcription factors and histones. We discuss the mechanisms by which transcription factors and coregulatory proteins regulate CYP17 transcription and summarize the role of kinases, phosphatases, acetyltransferases, and histone deacetylases in controlling CYP17 mRNA expression. PMID:19007851

  16. Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin gamma2-chain gene promoter by hepatocyte growth factor (scatter factor).

    PubMed Central

    Olsen, J; Lefebvre, O; Fritsch, C; Troelsen, J T; Orian-Rousseau, V; Kedinger, M; Simon-Assmann, P

    2000-01-01

    Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF. PMID:10749670

  17. Algorithmic complexity of a protein

    NASA Astrophysics Data System (ADS)

    Dewey, T. Gregory

    1996-07-01

    The information contained in a protein's amino acid sequence dictates its three-dimensional structure. To quantitate the transfer of information that occurs in the protein folding process, the Kolmogorov information entropy or algorithmic complexity of the protein structure is investigated. The algorithmic complexity of an object provides a means of quantitating its information content. Recent results have indicated that the algorithmic complexity of microstates of certain statistical mechanical systems can be estimated from the thermodynamic entropy. In the present work, it is shown that the algorithmic complexity of a protein is given by its configurational entropy. Using this result, a quantitative estimate of the information content of a protein's structure is made and is compared to the information content of the sequence. Additionally, the mutual information between sequence and structure is determined. It is seen that virtually all the information contained in the protein structure is shared with the sequence.

  18. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  19. The budding yeast Cdc48(Shp1) complex promotes cell cycle progression by positive regulation of protein phosphatase 1 (Glc7).

    PubMed

    Böhm, Stefanie; Buchberger, Alexander

    2013-01-01

    The conserved, ubiquitin-selective AAA ATPase Cdc48 regulates numerous cellular processes including protein quality control, DNA repair and the cell cycle. Cdc48 function is tightly controlled by a multitude of cofactors mediating substrate specificity and processing. The UBX domain protein Shp1 is a bona fide substrate-recruiting cofactor of Cdc48 in the budding yeast S. cerevisiae. Even though Shp1 has been proposed to be a positive regulator of Glc7, the catalytic subunit of protein phosphatase 1 in S. cerevisiae, its cellular functions in complex with Cdc48 remain largely unknown. Here we show that deletion of the SHP1 gene results in severe growth defects and a cell cycle delay at the metaphase to anaphase transition caused by reduced Glc7 activity. Using an engineered Cdc48 binding-deficient variant of Shp1, we establish the Cdc48(Shp1) complex as a critical regulator of mitotic Glc7 activity. We demonstrate that shp1 mutants possess a perturbed balance of Glc7 phosphatase and Ipl1 (Aurora B) kinase activities and show that hyper-phosphorylation of the kinetochore protein Dam1, a key mitotic substrate of Glc7 and Ipl1, is a critical defect in shp1. We also show for the first time a physical interaction between Glc7 and Shp1 in vivo. Whereas loss of Shp1 does not significantly affect Glc7 protein levels or localization, it causes reduced binding of the activator protein Glc8 to Glc7. Our data suggest that the Cdc48(Shp1) complex controls Glc7 activity by regulating its interaction with Glc8 and possibly further regulatory subunits. PMID:23418575

  20. p53 can repress transcription of cell cycle genes through a p21WAF1/CIP1-dependent switch from MMB to DREAM protein complex binding at CHR promoter elements

    PubMed Central

    Quaas, Marianne; Müller, Gerd A.; Engeland, Kurt

    2012-01-01

    The tumor suppressor p53 plays an important role in cell cycle arrest by downregulating transcription. Many genes repressed by p53 code for proteins with functions in G₂/M. A large portion of these genes is controlled by cell cycle-dependent elements (CDE) and cell cycle genes homology regions (CHR) in their promoters. Cyclin B2 is an example of such a gene, with a function at the transition from G₂ to mitosis. We find that p53-dependent downregulation of cyclin B2 promoter activity is dependent on an intact CHR element. In the presence of high levels of p53 or p21WAF1/CIP1, protein binding to the CHR switches from MMB to DREAM complex by shifting MuvB core-associated proteins from B-Myb to E2F4/DP1/p130. The results suggest a model for p53-dependent transcriptional repression by which p53 directly activates p21WAF1/CIP1. The inhibitor then prevents further phosphorylation of p130 by cyclin-dependent kinases. The presence of hypophosphorylated pocket proteins shifts the equilibrium for complex formation from MMB to DREAM. In the case of promoters that do not hold CDE or E2F elements, binding of DREAM and MMB solely relies on a CHR site. Thus, p53 can repress target genes indirectly through CHR elements. PMID:23187802

  1. Promoting Transfer by Grounding Complex Systems Principles

    ERIC Educational Resources Information Center

    Goldstone, Robert L.; Wilensky, Uri

    2008-01-01

    Understanding scientific phenomena in terms of complex systems principles is both scientifically and pedagogically important. Situations from different disciplines of science are often governed by the same principle, and so promoting knowledge transfer across disciplines makes valuable cross-fertilization and scientific unification possible.…

  2. Abscisic Acid Promotion of Arbuscular Mycorrhizal Colonization Requires a Component of the PROTEIN PHOSPHATASE 2A Complex1[W][OPEN

    PubMed Central

    Charpentier, Myriam; Sun, Jongho; Wen, Jiangqi; Mysore, Kirankumar S.; Oldroyd, Giles E.D.

    2014-01-01

    Legumes can establish intracellular interactions with symbiotic microbes to enhance their fitness, including the interaction with arbuscular mycorrhizal (AM) fungi. AM fungi colonize root epidermal cells to gain access to the root cortex, and this requires the recognition by the host plant of fungus-made mycorrhizal factors. Genetic dissection has revealed the symbiosis signaling pathway that allows the recognition of AM fungi, but the downstream processes that are required to promote fungal infection are poorly understood. Abscisic acid (ABA) has been shown to promote arbuscule formation in tomato (Solanum lycopersicum). Here, we show that ABA modulates the establishment of the AM symbiosis in Medicago truncatula by promoting fungal colonization at low concentrations and impairing it at high concentrations. We show that the positive regulation of AM colonization via ABA requires a PROTEIN PHOSPHATASE 2A (PP2A) holoenzyme subunit, PP2AB′1. Mutations in PP2AB′1 cause reduced levels of AM colonization that cannot be rescued with permissive ABA application. The action of PP2AB′1 in response to ABA is unlinked to the generation of calcium oscillations, as the pp2aB′1 mutant displays a normal calcium response. This contrasts with the application of high concentrations of ABA that impairs mycorrhizal factor-induced calcium oscillations, suggesting different modes of action of ABA on the AM symbiosis. Our work reveals that ABA functions at multiple levels to regulate the AM symbiosis and that a PP2A phosphatase is required for the ABA promotion of AM colonization. PMID:25293963

  3. Binding Efficiency of Protein-Protein Complexes

    PubMed Central

    Day, Eric S.; Cote, Shaun M.; Whitty, Adrian

    2012-01-01

    We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPI), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for the large receptor TNFR1 binding to its ligands TNFα (KD = 1.4 ± 0.4 nM) and lymphotoxin-α (KD = 50 ± 10 nM), and also for the small receptor Fn14 binding to TWEAK (KD = 70 ± 10 nM). We additionally assembled data for all other TNF/TNFR family complexes for which reliable single site binding affinities have been reported. We used these values to calculate the binding efficiency – defined as binding energy per Å2 of surface area buried at the contact interface – for the nine of these complexes for which co-crystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinity values. The results show that the most efficient PPI complexes generate ~20 cal.mol−1/Å2 of binding energy. A minimum contact area of ~500 Å2 is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of co-localizing two proteins from 1 M solution. The most compact and efficient TNF/TNFR complex was BAFF/BR3, which achieved ~80% of the maximum achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method to predict how large a natural or engineered contact interface must be to achieve a given level of binding affinity. PMID:23088250

  4. Drosophila homeodomain-interacting protein kinase inhibits the Skp1-Cul1-F-box E3 ligase complex to dually promote Wingless and Hedgehog signaling.

    PubMed

    Swarup, Sharan; Verheyen, Esther M

    2011-06-14

    Drosophila Homeodomain-interacting protein kinase (Hipk) has been shown to regulate in vivo, the stability of Armadillo, the transcriptional effector of Wingless signaling. The Wingless pathway culminates in the stabilization of Armadillo that, in the absence of signaling, is sequentially phosphorylated, polyubiquitinated and degraded. Loss-of-function clones for hipk result in reduced stabilized Armadillo, whereas overexpression of hipk elevates Armadillo levels to promote Wingless-responsive target gene expression. Here, we show that overexpression of hipk can suppress the effects of negative regulators of Armadillo to prevent its degradation in the wing imaginal disc. Hipk acts to stabilize Armadillo by impeding the function of the E3 ubiquitin ligase Skp1-Cul1-F-box (SCF)(Slimb), thereby inhibiting Armadillo ubiquitination and subsequent degradation. Vertebrate Hipk2 displays a similar ability to prevent β-catenin ubiquitination in a functionally conserved mechanism. We find that Hipk's ability to inhibit SCF(Slimb)-mediated ubiquitination is not restricted to Armadillo and extends to other substrates of SCF(Slimb), including the Hedgehog signaling effector Ci. Thus, similar to casein kinase 1 and glycogen synthase kinase 3, Hipk dually regulates both Wingless and Hedgehog signaling by controlling the stability of their respective signaling effectors, but it is the first kinase to our knowledge identified that promotes the stability of both Armadillo and Ci. PMID:21628596

  5. Stabilized β-Catenin Functions through TCF/LEF Proteins and the Notch/RBP-Jκ Complex To Promote Proliferation and Suppress Differentiation of Neural Precursor Cells▿

    PubMed Central

    Shimizu, Takeshi; Kagawa, Tetsushi; Inoue, Toshihiro; Nonaka, Aya; Takada, Shinji; Aburatani, Hiroyuki; Taga, Tetsuya

    2008-01-01

    The proliferation and differentiation of neural precursor cells are mutually exclusive during brain development. Despite its importance for precursor cell self renewal, the molecular linkage between these two events has remained unclear. Fibroblast growth factor 2 (FGF2) promotes neural precursor cell proliferation and concurrently inhibits their differentiation, suggesting a cross talk between proliferation and differentiation signaling pathways downstream of the FGF receptor. We demonstrate that FGF2 signaling through phosphatidylinositol 3 kinase activation inactivates glycogen synthase kinase 3β (GSK3β) and leads to the accumulation of β-catenin in a manner different from that in the Wnt canonical pathway. The nuclear accumulated β-catenin leads to cell proliferation by activating LEF/TCF transcription factors and concurrently inhibits neuronal differentiation by potentiating the Notch1-RBP-Jκ signaling pathway. β-Catenin and the Notch1 intracellular domain form a molecular complex with the promoter region of the antineurogenic hes1 gene, allowing its expression. This signaling interplay is especially essential for neural stem cell maintenance, since the misexpression of dominant-active GSK3β completely inhibits the self renewal of neurosphere-forming stem cells and prompts their neuronal differentiation. Thus, the GSK3β/β-catenin signaling axis regulated by FGF and Wnt signals plays a pivotal role in the maintenance of neural stem/precursor cells by linking the cell proliferation to the inhibition of differentiation. PMID:18852283

  6. Molecular architecture and mechanism of the anaphase-promoting complex

    PubMed Central

    Yang, Jing; McLaughlin, Stephen H.; Barford, David

    2015-01-01

    The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of G1. The APC/C is an unusually large multimeric cullin-RING ligase. Its activity is strictly dependent on regulatory coactivator subunits that promote APC/C – substrate interactions and stimulate its catalytic reaction. Because the structures of many APC/C subunits and their organization within the assembly are unknown, the molecular basis for these processes is poorly understood. Here, from a cryo-EM reconstruction of a human APC/C-coactivator-substrate complex at 7.4 Å resolution, we have determined the complete secondary structural architecture of the complex. With this information we identified protein folds for structurally uncharacterized subunits, and the definitive location of all 20 APC/C subunits within the 1.2 MDa assembly. Comparison with apo APC/C shows that coactivator promotes a profound allosteric transition involving displacement of the cullin-RING catalytic subunits relative to the degron recognition module of coactivator and Apc10. This transition is accompanied by increased flexibility of the cullin-RING subunits and enhanced affinity for UbcH10~ubiquitin, changes which may contribute to coactivator-mediated stimulation of APC/C E3 ligase activity. PMID:25043029

  7. Dye-promoted precipitation of serum proteins. Mechanism and application.

    PubMed

    Birkenmeier, G; Kopperschläger, G

    1991-11-01

    Immobilized dyes have been used primarily for purification of nucleotide dependent enzymes and proteins from plasma and other sources. Due to their low costs, high protein binding capacity and resistance to degradation dyes bear the potential as ligand for affinity separation of proteins on a large scale. In this paper dyes have been used for precipitation of proteins. Using albumin, prealbumin, alpha 1-acid glycoprotein and immunoglobulin G as model proteins we could demonstrate that dye-promoted precipitation depends on several factors which include the structure of the dye, the pH of the solution, the dye/protein molar ratio and the intrinsic properties of the proteins. It revealed that most of the dyes tested were endowed with the precipitating potential. The efficacy of precipitation was found to increase with the complexity of the dye structure. However, the amount of a dye required for total precipitation was found to be different for a given protein. Electrostatic as well as hydrophobic forces are involved in the mechanism of precipitation. It was demonstrated that by optimizing the conditions, mixtures of proteins can be resolved by dye-promoted precipitation. The high sensitivity of the reaction offers the possibility of using this method for rapid concentration of very diluted protein solutions. PMID:1367693

  8. GECluster: a novel protein complex prediction method

    PubMed Central

    Su, Lingtao; Liu, Guixia; Wang, Han; Tian, Yuan; Zhou, Zhihui; Han, Liang; Yan, Lun

    2014-01-01

    Identification of protein complexes is of great importance in the understanding of cellular organization and functions. Traditional computational protein complex prediction methods mainly rely on the topology of protein–protein interaction (PPI) networks but seldom take biological information of proteins (such as Gene Ontology (GO)) into consideration. Meanwhile, the environment relevant analysis of protein complex evolution has been poorly studied, partly due to the lack of high-precision protein complex datasets. In this paper, a combined PPI network is introduced to predict protein complexes which integrate both GO and expression value of relevant protein-coding genes. A novel protein complex prediction method GECluster (Gene Expression Cluster) was proposed based on a seed node expansion strategy, in which a combined PPI network was utilized. GECluster was applied to a training combined PPI network and it predicted more credible complexes than peer methods. The results indicate that using a combined PPI network can efficiently improve protein complex prediction accuracy. In order to study protein complex evolution within cells due to changes in the living environment surrounding cells, GECluster was applied to seven combined PPI networks constructed using the data of a test set including yeast response to stress throughout a wine fermentation process. Our results showed that with the rise of alcohol concentration, protein complexes within yeast cells gradually evolve from one state to another. Besides this, the number of core and attachment proteins within a protein complex both changed significantly. PMID:26019559

  9. The novel complement inhibitor human CUB and Sushi multiple domains 1 (CSMD1) protein promotes factor I-mediated degradation of C4b and C3b and inhibits the membrane attack complex assembly.

    PubMed

    Escudero-Esparza, Astrid; Kalchishkova, Nikolina; Kurbasic, Emila; Jiang, Wen G; Blom, Anna M

    2013-12-01

    CUB and Sushi multiple domains 1 (CSMD1) is a transmembrane protein containing 15 consecutive complement control protein (CCP) domains, which are characteristic for complement inhibitors. We expressed a membrane-bound fragment of human CSMD1 composed of the 15 C-terminal CCP domains and demonstrated that it inhibits deposition of C3b by the classical pathway on the surface of Chinese hamster ovary cells by 70% at 6% serum and of C9 (component of membrane attack complex) by 90% at 1.25% serum. Furthermore, this fragment of CSMD1 served as a cofactor to factor I-mediated degradation of C3b. In all functional assays performed, well-characterized complement inhibitors were used as positive controls, whereas Coxsackie adenovirus receptor, a protein with no effect on complement, was a negative control. Moreover, attenuation of expression in human T47 breast cancer cells that express endogenous CSMD1 significantly increased C3b deposition on these cells by 45% at 8% serum compared with that for the controls. Furthermore, by expressing a soluble 17-21 CCP fragment of CSMD1, we found that CSMD1 inhibits complement by promoting factor I-mediated C4b/C3b degradation and inhibition of MAC assembly at the level of C7. Our results revealed a novel complement inhibitor for the classical and lectin pathways. PMID:23964079

  10. Redox-Promoting Protein Motions in Rubredoxin

    SciTech Connect

    Myles, Dean A A; He, Junhong; Meilleur, Flora; Weiss, Kevin L; Agarwal, Pratul K; Borreguero Calvo, Jose M; Barthes, Mariette; Brown, Craig; Herwig, Kenneth W

    2011-01-01

    Proteins are dynamic objects, constantly undergoing conformational fluctuations, yet the linkage between internal protein motion and function is widely debated. This manuscript reports on the characterization of temperature-activated collective and individual atomic motions of oxidized rubredoxin, a small 53 residue protein from thermophilic Pyrococcus furiosus (RdPf), by neutron scattering and computational simulations. The changes in motion have been explored in connection to their role in promoting reduction of the Fe+3 ion which is responsible for the electron transfer function of RdPf. Just above the dynamical transition temperature of 220 K which marks the onset of significant anharmonic motions of the protein, the computer simulations show both a significant reorientation of the average electrostatic force experienced by the Fe+3 ion and a dramatic rise in its strength. At higher temperatures, additional anharmonic modes become activated which dominate the electrostatic fluctuations experienced by the ion. At 360 K, close to the optimal growth temperature of Pyrococcus furiosus, computer simulations show that three anharmonic modes involving two conserved residues located at the protein active site (Ile7 and Ile40) give rise to the majority of the electrostatic fluctuations experienced by the Fe+3 ion and include displacements which allow solvent access to the ion. The low-frequency, high amplitude motions of these residues at low temperatures may be precursors of the high temperature, anharmonic motions necessary for protein function.

  11. Structure of a bacterial RNA polymerase holoenzyme open promoter complex

    SciTech Connect

    Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; Landick, Robert; Darst, Seth A.

    2015-09-08

    Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.

  12. Structure of a bacterial RNA polymerase holoenzyme open promoter complex

    DOE PAGESBeta

    Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; Landick, Robert; Darst, Seth A.

    2015-09-08

    Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.« less

  13. Structure of a bacterial RNA polymerase holoenzyme open promoter complex

    PubMed Central

    Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; Landick, Robert; Darst, Seth A

    2015-01-01

    Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the −10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation. DOI: http://dx.doi.org/10.7554/eLife.08504.001 PMID:26349032

  14. Structure Prediction of Protein Complexes

    NASA Astrophysics Data System (ADS)

    Pierce, Brian; Weng, Zhiping

    Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.

  15. Immunoisolation of Protein Complexes from Xenopus

    PubMed Central

    Conlon, Frank L.; Miteva, Yana; Kaltenbrun, Erin; Waldron, Lauren; Greco, Todd M.; Cristea, Ileana M.

    2013-01-01

    The immunoaffinity isolation of protein complexes is an essential technique for the purification and concentration of protein complexes from cells and tissues. In this chapter we present the methodologies for the purification of proteins and protein complexes from Xenopus laev is and Xenopus tropical is. Specific to this protocol are the techniques for the cryolysis of Xenopus cells and tissues, a procedure that limits contamination from yolk proteins while preserving endogenous protein complexes, the methodologies for immunoaffinity purification of proteins using magnetic beads, and the protocols for western blot analysis. In addition, the procedures in this chapter can be extended to use with proteomic analysis of protein complexes as presented in the following chapter. PMID:22956099

  16. Transport proteins promoting Escherichia coli pathogenesis

    PubMed Central

    Tang, Fengyi; Saier, Milton H.

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies. PMID:24747185

  17. Protein camouflage in cytochrome c-calixarene complexes

    NASA Astrophysics Data System (ADS)

    McGovern, Róise E.; Fernandes, Humberto; Khan, Amir R.; Power, Nicholas P.; Crowley, Peter B.

    2012-07-01

    Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host-guest interactions have been published. Although there is growing interest in protein-calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein-calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein-protein interactions, with potential applications in generating assemblies and promoting crystallization.

  18. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  19. Investigation of a protein complex network

    NASA Astrophysics Data System (ADS)

    Mashaghi, A. R.; Ramezanpour, A.; Karimipour, V.

    2004-09-01

    The budding yeast Saccharomyces cerevisiae is the first eukaryote whose genome has been completely sequenced. It is also the first eukaryotic cell whose proteome (the set of all proteins) and interactome (the network of all mutual interactions between proteins) has been analyzed. In this paper we study the structure of the yeast protein complex network in which weighted edges between complexes represent the number of shared proteins. It is found that the network of protein complexes is a small world network with scale free behavior for many of its distributions. However we find that there are no strong correlations between the weights and degrees of neighboring complexes. To reveal non-random features of the network we also compare it with a null model in which the complexes randomly select their proteins. Finally we propose a simple evolutionary model based on duplication and divergence of proteins.

  20. Trapping mammalian protein complexes in viral particles

    PubMed Central

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

    2016-01-01

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

  1. Improved method for protein complex detection using bottleneck proteins

    PubMed Central

    2013-01-01

    Background Detecting protein complexes is one of essential and fundamental tasks in understanding various biological functions or processes. Therefore accurate identification of protein complexes is indispensable. Methods For more accurate detection of protein complexes, we propose an algorithm which detects dense protein sub-networks of which proteins share closely located bottleneck proteins. The proposed algorithm is capable of finding protein complexes which allow overlapping with each other. Results We applied our algorithm to several PPI (Protein-Protein Interaction) networks of Saccharomyces cerevisiae and Homo sapiens, and validated our results using public databases of protein complexes. The prediction accuracy was even more improved over our previous work which used also bottleneck information of the PPI network, but showed limitation when predicting small-sized protein complex detection. Conclusions Our algorithm resulted in overlapping protein complexes with significantly improved F1 score over existing algorithms. This result comes from high recall due to effective network search, as well as high precision due to proper use of bottleneck information during the network search. PMID:23566214

  2. Metabolic Adaptation and Protein Complexes in Prokaryotes

    PubMed Central

    Krüger, Beate; Liang, Chunguang; Prell, Florian; Fieselmann, Astrid; Moya, Andres; Schuster, Stefan; Völker, Uwe; Dandekar, Thomas

    2012-01-01

    Protein complexes are classified and have been charted in several large-scale screening studies in prokaryotes. These complexes are organized in a factory-like fashion to optimize protein production and metabolism. Central components are conserved between different prokaryotes; major complexes involve carbohydrate, amino acid, fatty acid and nucleotide metabolism. Metabolic adaptation changes protein complexes according to environmental conditions. Protein modification depends on specific modifying enzymes. Proteins such as trigger enzymes display condition-dependent adaptation to different functions by participating in several complexes. Several bacterial pathogens adapt rapidly to intracellular survival with concomitant changes in protein complexes in central metabolism and optimize utilization of their favorite available nutrient source. Regulation optimizes protein costs. Master regulators lead to up- and downregulation in specific subnetworks and all involved complexes. Long protein half-life and low level expression detaches protein levels from gene expression levels. However, under optimal growth conditions, metabolite fluxes through central carbohydrate pathways correlate well with gene expression. In a system-wide view, major metabolic changes lead to rapid adaptation of complexes and feedback or feedforward regulation. Finally, prokaryotic enzyme complexes are involved in crowding and substrate channeling. This depends on detailed structural interactions and is verified for specific effects by experiments and simulations. PMID:24957769

  3. Predicting Physical Interactions between Protein Complexes*

    PubMed Central

    Clancy, Trevor; Rødland, Einar Andreas; Nygard, Ståle; Hovig, Eivind

    2013-01-01

    Protein complexes enact most biochemical functions in the cell. Dynamic interactions between protein complexes are frequent in many cellular processes. As they are often of a transient nature, they may be difficult to detect using current genome-wide screens. Here, we describe a method to computationally predict physical interactions between protein complexes, applied to both humans and yeast. We integrated manually curated protein complexes and physical protein interaction networks, and we designed a statistical method to identify pairs of protein complexes where the number of protein interactions between a complex pair is due to an actual physical interaction between the complexes. An evaluation against manually curated physical complex-complex interactions in yeast revealed that 50% of these interactions could be predicted in this manner. A community network analysis of the highest scoring pairs revealed a biologically sensible organization of physical complex-complex interactions in the cell. Such analyses of proteomes may serve as a guide to the discovery of novel functional cellular relationships. PMID:23438732

  4. Autophagy promotes DNA-protein crosslink clearance.

    PubMed

    Mu, Haibo; Liu, Qianjin; Niu, Hong; Wang, Dongdong; Tang, Jiangjiang; Duan, Jinyou

    2016-02-01

    Toxic DNA-protein crosslinks (DPCs) can result from exposure to radiation or chemotherapeutic agents. DPCs can also accumulate during aging or stress. However, the cellular mechanisms underlying clearance of DPCs remain largely unknown. Here, we have identified an important role of autophagy in the processing of DPCs induced by three representative agents: formaldehyde, a chemical used widely in industry; UV light; and camptothecin, a cytotoxic anticancer drug. Autophagy inhibitors, 3-methyladenine (3-MA) or chloroquine (CQ), promoted the accumulation of DPCs in damaged cells and injured organs. siRNA-mediated silencing of Atg5 or Atg7, two essential components for the formation of the autophagosome, gave similar results. In contrast, the autophagy inducer rapamycin (RAP) attenuated DPCs in vitro and in vivo. Our findings reveal the importance of autophagy in controlling the level of DPCs, and may open up a new avenue for understanding the formation and clearance of this detrimental DNA adduct. PMID:26921017

  5. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  6. Proteins as paradigms of complex systems.

    SciTech Connect

    Fenimore, P. W.; Frauenfelder, Hans,; Young, R. D.

    2003-03-26

    The science of complexity has moved to center stage within the past few decades. Complex systems range from glasses to the immune system and the brain. Glasses are too simple to possess all aspects of complexity; brains are too complex to expose common concepts and laws of complexity. Proteins, however, are systems where many concepts and laws of complexity can be explored experimentally, theoretically, and computationally. Such studies have elucidated crucial aspects. The energy landscape has emerged as one central concept; it describes the free energy of a system as a function of temperature and the coordinates of all relevant atoms. A second concept is that of fluctuations. Without fluctuations, proteins would be dead and life impossible. A third concept is slaving. Proteins are not isolated systems; they are embedded in cells and membranes. Slaving arises when the fluctuations in the surroundings of a protein dominate many of the motions of the protein proper.

  7. Complex Reconstitution from Individual Protein Modules.

    PubMed

    Basquin, Jérôme; Taschner, Michael; Lorentzen, Esben

    2016-01-01

    Cellular function relies on protein complexes that work as nano-machines. The structure and function of protein complexes is an outcome of the specific combination of protein subunits, or modules, within the complex. A major focus of molecular biology is thus to understand how protein subunits assemble to form complexes with distinct biological function. To this end, in vitro reconstitution of complexes from individual subunits to study their assembly, structure and activity is of central importance. With purified individual subunits and sub-modules at hand one can systematically dissect the hierarchical assembly of larger complexes using direct protein-protein interaction assays. Furthermore, activity assays can be carried out with individual subunits or smaller sub-complexes and compared to those of the fully assembled complex to precisely map functional sites and provide a molecular basis for in vivo observations. In this chapter we review methods for protein complex assembly from individual subunits and provide examples of advantages and potential pitfalls to this approach. PMID:27165333

  8. Protein Complex Purification by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  9. Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes.

    PubMed

    Angerer, Heike

    2015-01-01

    In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria. PMID:25686363

  10. Rep provides a second motor at the replisome to promote duplication of protein-bound DNA.

    PubMed

    Guy, Colin P; Atkinson, John; Gupta, Milind K; Mahdi, Akeel A; Gwynn, Emma J; Rudolph, Christian J; Moon, Peter B; van Knippenberg, Ingeborg C; Cadman, Chris J; Dillingham, Mark S; Lloyd, Robert G; McGlynn, Peter

    2009-11-25

    Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA. PMID:19941825

  11. Mycobacterial RNA polymerase forms unstable open promoter complexes that are stabilized by CarD

    PubMed Central

    Davis, Elizabeth; Chen, James; Leon, Katherine; Darst, Seth A.; Campbell, Elizabeth A.

    2015-01-01

    Escherichia coli has served as the archetypal organism on which the overwhelming majority of biochemical characterizations of bacterial RNA polymerase (RNAP) have been focused; the properties of E. coli RNAP have been accepted as generally representative for all bacterial RNAPs. Here, we directly compare the initiation properties of a mycobacterial transcription system with E. coli RNAP on two different promoters. The detailed characterizations include abortive transcription assays, RNAP/promoter complex stability assays and DNAse I and KMnO4 footprinting. Based on footprinting, we find that promoter complexes formed by E. coli and mycobacterial RNAPs use very similar protein/DNA interactions and generate the same transcription bubbles. However, we find that the open promoter complexes formed by E. coli RNAP on the two promoters tested are highly stable and essentially irreversible (with lifetimes much greater than 1 h), while the open promoter complexes on the same two promoters formed by mycobacterial RNAP are very unstable (lifetimes of about 2 min or less) and readily reversible. We show here that CarD, an essential mycobacterial transcription activator that is not found in E. coli, stabilizes the mycobacterial RNAP/open promoter complexes considerably by preventing transcription bubble collapse. PMID:25510492

  12. Drosophila IAP antagonists form multimeric complexes to promote cell death

    PubMed Central

    Sandu, Cristinel; Ryoo, Hyung Don

    2010-01-01

    Apoptosis is a specific form of cell death that is important for normal development and tissue homeostasis. Caspases are critical executioners of apoptosis, and living cells prevent their inappropriate activation through inhibitor of apoptosis proteins (IAPs). In Drosophila, caspase activation depends on the IAP antagonists, Reaper (Rpr), Head involution defective (Hid), and Grim. These proteins share a common motif to bind Drosophila IAP1 (DIAP1) and have partially redundant functions. We now show that IAP antagonists physically interact with each other. Rpr is able to self-associate and also binds to Hid and Grim. We have defined the domain involved in self-association and demonstrate that it is critical for cell-killing activity in vivo. In addition, we show that Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo. Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis. Our results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis. PMID:20837774

  13. Promoters and proteins from Clostridium thermocellum and uses thereof

    DOEpatents

    Wu, J. H. David; Newcomb, Michael

    2012-11-13

    The present invention relates to an inducible and a high expression nucleic acid promoter isolated from Clostridium thermocellum. These promoters are useful for directing expression of a protein or polypeptide encoded by a nucleic acid molecule operably associated with the nucleic acid promoters. The present invention also relates to nucleic acid constructs including the C. thermocellum promoters, and expression vectors and hosts containing such nucleic acid constructs. The present invention also relates to protein isolated from Clostridium thermocellum, including a repressor protein. The present invention also provides methods of using the isolated promoters and proteins from Clostridium thermocellum, including methods for directing inducible in vitro and in vivo expression of a protein or polypeptide in a host, and methods of producing ethanol from a cellulosic biomass.

  14. A secretory kinase complex regulates extracellular protein phosphorylation.

    PubMed

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. PMID:25789606

  15. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    PubMed

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  16. Basal core promoters control the equilibrium between negative cofactor 2 and preinitiation complexes in human cells

    PubMed Central

    2010-01-01

    Background The general transcription factor TFIIB and its antagonist negative cofactor 2 (NC2) are hallmarks of RNA polymerase II (RNAPII) transcription. Both factors bind TATA box-binding protein (TBP) at promoters in a mutually exclusive manner. Dissociation of NC2 is thought to be followed by TFIIB association and subsequent preinitiation complex formation. TFIIB dissociates upon RNAPII promoter clearance, thereby providing a specific measure for steady-state preinitiation complex levels. As yet, genome-scale promoter mapping of human TFIIB has not been reported. It thus remains elusive how human core promoters contribute to preinitiation complex formation in vivo. Results We compare target genes of TFIIB and NC2 in human B cells and analyze associated core promoter architectures. TFIIB occupancy is positively correlated with gene expression, with the vast majority of promoters being GC-rich and lacking defined core promoter elements. TATA elements, but not the previously in vitro defined TFIIB recognition elements, are enriched in some 4 to 5% of the genes. NC2 binds to a highly related target gene set. Nonetheless, subpopulations show strong variations in factor ratios: whereas high TFIIB/NC2 ratios select for promoters with focused start sites and conserved core elements, high NC2/TFIIB ratios correlate to multiple start-site promoters lacking defined core elements. Conclusions TFIIB and NC2 are global players that occupy active genes. Preinitiation complex formation is independent of core elements at the majority of genes. TATA and TATA-like elements dictate TFIIB occupancy at a subset of genes. Biochemical data support a model in which preinitiation complex but not TBP-NC2 complex formation is regulated. PMID:20230619

  17. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    PubMed

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. PMID:27053724

  18. Adaptable Lipid Matrix Promotes Protein-Protein Association in Membranes.

    PubMed

    Kuznetsov, Andrey S; Polyansky, Anton A; Fleck, Markus; Volynsky, Pavel E; Efremov, Roman G

    2015-09-01

    The cell membrane is "stuffed" with proteins, whose transmembrane (TM) helical domains spontaneously associate to form functionally active complexes. For a number of membrane receptors, a modulation of TM domains' oligomerization has been shown to contribute to the development of severe pathological states, thus calling for detailed studies of the atomistic aspects of the process. Despite considerable progress achieved so far, several crucial questions still remain: How do the helices recognize each other in the membrane? What is the driving force of their association? Here, we assess the dimerization free energy of TM helices along with a careful consideration of the interplay between the structure and dynamics of protein and lipids using atomistic molecular dynamics simulations in the hydrated lipid bilayer for three different model systems - TM fragments of glycophorin A, polyalanine and polyleucine peptides. We observe that the membrane driven association of TM helices exhibits a prominent entropic character, which depends on the peptide sequence. Thus, a single TM peptide of a given composition induces strong and characteristic perturbations in the hydrophobic core of the bilayer, which may facilitate the initial "communication" between TM helices even at the distances of 20-30 Å. Upon tight helix-helix association, the immobilized lipids accommodate near the peripheral surfaces of the dimer, thus disturbing the packing of the surrounding. The dimerization free energy of the modeled peptides corresponds to the strength of their interactions with lipids inside the membrane being the lowest for glycophorin A and similarly higher for both homopolymers. We propose that the ability to accommodate lipid tails determines the dimerization strength of TM peptides and that the lipid matrix directly governs their association. PMID:26575933

  19. Protein-protein interaction networks (PPI) and complex diseases

    PubMed Central

    Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

    2014-01-01

    The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

  20. Solid-phase preparation of protein complexes.

    PubMed

    Pengo, Paolo; Veggiani, Gianluca; Rattanamanee, Kwanchai; Gallotta, Andrea; Beneduce, Luca; Fassina, Giorgio

    2010-01-01

    Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies. PMID:21038355

  1. Rapid purification of protein complexes from mammalian cells

    PubMed Central

    Medina, Dan; Moskowitz, Neal; Khan, Subarna; Christopher, Scott; Germino, Joseph

    2000-01-01

    The evaluation of the protein binding partner(s) of biologically important proteins is currently an area of intense research, especially since the development of the yeast two-hybrid assay. However, not all protein–protein interactions uncovered by this assay are biologically relevant and another confirmatory assay must be performed. Ideally, this assay should be rapid, versatile and performed under conditions which mimic the ‘normal’ physiological state as closely as possible. Towards this goal, we have constructed two eukaryotic expression vectors that facilitate the purification of a protein of interest, along with any associated proteins, from mammalian cells. These vectors incorporate the following features: (i) a tetracycline-responsive promoter so that the level of protein production can be regulated; (ii) an N-terminal glutathione S-transferase tag or a triple repeat of the HA1 epitope, to facilitate purification of the protein either by glutathione affinity chromatography or immunoprecipitation, respectively, followed by a multiple cloning site; (iii) the gene for the enhanced green fluorescent protein (for detection of the presence of the fusion protein and subcellular localization); (iv) a puromycin marker for the selection of stable transformants; (v) a truncated EBNA protein and oriP sequence for episomal replication of the vector. These latter two features permit expansion of small cultures of transfected cells under puromycin selection, thereby increasing the amount of tagged protein that can be purified. We show that these vectors can be used to direct the doxycycline-inducible expresssion of tagged proteins and to recover tagged CIP1–p21 protein complexes from HeLa cells. Furthermore, we show that these tagged p21-purified complexes contain both cyclin A and Cdk2, which are known to interact with p21, but not β-actin. PMID:10871384

  2. Redox-Promoting Protein Motions in Rubredoxin

    SciTech Connect

    Borreguero Calvo, Jose M; He, Junhong; Meilleur, Flora; Weiss, Kevin L; Myles, Dean A A; Herwig, Kenneth W; Agarwal, Pratul K

    2011-01-01

    Proteins are dynamic objects, constantly undergoing conformational fluctuations, yet the linkage between internal protein motion and function is widely debated. This study reports on the characterization of temperature-activated collective and individual atomic motions of oxidized rubredoxin, a small 53 residue protein from thermophilic Pyrococcus furiosus (RdPf). Computational modeling allows detailed investigations of protein motions as a function of temperature, and neutron scattering experiments are used to compare to computational results. Just above the dynamical transition temperature which marks the onset of significant anharmonic motions of the protein, the computational simulations show both a significant reorientation of the average electrostatic force experienced by the coordinated Fe{sup 3+} ion and a dramatic rise in its strength. At higher temperatures, additional anharmonic modes become activated and dominate the electrostatic fluctuations experienced by the ion. At 360 K, close to the optimal growth temperature of P. furiosus, simulations show that three anharmonic modes including motions of two conserved residues located at the protein active site (Ile7 and Ile40) give rise to the majority of the electrostatic fluctuations experienced by the Fe{sup 3+} ion. The motions of these residues undergo displacements which may facilitate solvent access to the ion.

  3. Solvation dynamics in a protein surfactant complex

    NASA Astrophysics Data System (ADS)

    Dutta, Partha; Sen, Pratik; Halder, Arnab; Mukherjee, Saptarshi; Sen, Sobhan; Bhattacharyya, Kankan

    2003-08-01

    Solvation dynamics in the denatured state of a protein, lysozyme (denatured by sodium dodecyl sulfate, SDS) is markedly slower than that in the native state. For coumarin 153 bound to lysozyme, the average solvation time, < τs> is 330 ps. In the lysozyme-SDS complex, the solvation dynamics is markedly slower with < τs>=7250 ps. On addition of dithiothreitol (DTT) to the lysozyme-SDS complex, when the di-sulfide bonds are destroyed, < τs> is found to be 1140 ps. The slow dynamics in the denatured protein is attributed to the polymer chain dynamics and the exchange of bound and free water molecules.

  4. Immobilization of two organometallic complexes into a single cage to construct protein-based microcompartments.

    PubMed

    Maity, Basudev; Fukumori, Kazuki; Abe, Satoshi; Ueno, Takafumi

    2016-04-01

    Natural protein-based microcompartments containing multiple enzymes promote cascade reactions within cells. We use the apo-ferritin protein cage to mimic such biocompartments by immobilizing two organometallic Ir and Pd complexes into the single protein cage. Precise locations of the metals and their accumulation mechanism were studied by X-ray crystallography. PMID:27021005

  5. Peroxisome protein import: a complex journey

    PubMed Central

    Baker, Alison; Hogg, Thomas Lanyon; Warriner, Stuart L.

    2016-01-01

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor–cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. PMID:27284042

  6. Metal complexes as "protein surface mimetics".

    PubMed

    Hewitt, Sarah H; Wilson, Andrew J

    2016-07-28

    A key challenge in chemical biology is to identify small molecule regulators for every single protein. However, protein surfaces are notoriously difficult to recognise with synthetic molecules, often having large flat surfaces that are poorly matched to traditional small molecules. In the surface mimetic approach, a supramolecular scaffold is used to project recognition groups in such a manner as to make multivalent non-covalent contacts over a large area of protein surface. Metal based supramolecular scaffolds offer unique advantages over conventional organic molecules for protein binding, including greater stereochemical and geometrical diversity conferred through the metal centre and the potential for direct assessment of binding properties and even visualisation in cells without recourse to further functionalisation. This feature article will highlight the current state of the art in protein surface recognition using metal complexes as surface mimetics. PMID:27353704

  7. On protein abundance distributions in complex mixtures

    PubMed Central

    2013-01-01

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

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

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data. PMID:27165321

  9. Assembly reflects evolution of protein complexes.

    PubMed

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

    2008-06-26

    A homomer is formed by self-interacting copies of a protein unit. This is functionally important, as in allostery, and structurally crucial because mis-assembly of homomers is implicated in disease. Homomers are widespread, with 50-70% of proteins with a known quaternary state assembling into such structures. Despite their prevalence, their role in the evolution of cellular machinery and the potential for their use in the design of new molecular machines, little is known about the mechanisms that drive formation of homomers at the level of evolution and assembly in the cell. Here we present an analysis of over 5,000 unique atomic structures and show that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity. Where quaternary structure is not conserved among the members of a protein family, a detailed investigation revealed well-defined evolutionary pathways by which proteins transit between different quaternary structure types. Furthermore, we show by perturbing subunit interfaces within complexes and by mass spectrometry analysis, that the (dis)assembly pathway mimics the evolutionary pathway. These data represent a molecular analogy to Haeckel's evolutionary paradigm of embryonic development, where an intermediate in the assembly of a complex represents a form that appeared in its own evolutionary history. Our model of self-assembly allows reliable prediction of evolution and assembly of a complex solely from its crystal structure. PMID:18563089

  10. Over-producing soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system.

    PubMed

    Zeng, Jumei; Zhang, Lei; Li, Yuqing; Wang, Yi; Wang, Mingchao; Duan, Xin; He, Zheng-Guo

    2010-01-01

    Many proteins exert their functions through a protein complex and protein-protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein-protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein-protein interaction of interest in a wide range of biological organisms. PMID:19747546

  11. Structure of promoter-bound TFIID and model of human pre-initiation complex assembly.

    PubMed

    Louder, Robert K; He, Yuan; López-Blanco, José Ramón; Fang, Jie; Chacón, Pablo; Nogales, Eva

    2016-03-31

    The general transcription factor IID (TFIID) plays a central role in the initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-initiation complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1-13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP-TATA complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation. PMID:27007846

  12. Hydrohalogenative aromatization of multiynes promoted by ruthenium alkylidene complexes.

    PubMed

    Karmakar, Rajdip; Wang, Kung-Pern; Yun, Sang Young; Mamidipalli, Phani; Lee, Daesung

    2016-06-01

    A new functionalization method of arynes promoted by a novel catalytic role of the Grubbs-type ruthenium alkylidene complex is described. Through a sequence of aryne formation followed by their halo-functionalization, various bis-1,3-diynes were directly converted to functionalized aryl chlorides, bromides and iodides in good yields in the presence of a catalytic amount of a ruthenium alkylidene complex and halogenated hydrocarbons such as CH2Cl2, CHCl3, CH2Br2, and CH2I2. The utility of this novel transformation is demonstrated by a formal synthesis of herbindole B. PMID:27145857

  13. Characterization of the human granulocyte-macrophage colony-stimulating factor gene promoter: an AP1 complex and an Sp1-related complex transactivate the promoter activity that is suppressed by a YY1 complex.

    PubMed Central

    Ye, J; Zhang, X; Dong, Z

    1996-01-01

    It is well documented that a repeated CATT element in the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter is required for promoter activity. However, the transcription factors that are able to transactivate this enhancer element remain unidentified. Recently, we have found that nuclear factor YY1 can interact with the enhancer element. Here, we report that in addition to YY1, two other nuclear factors have been identified in the DNA-protein complexes formed by the CATT oligonucleotide and the Jurkat T-cell nuclear protein. One of these factors is AP1, and the other one is an Sp1-related protein. Results from transient transfection of Jurkat T cells have revealed that formation of both AP1 and the Sp1-related complex is required for the full enhancer activity of the CATT element. This result is supported by cotransfection of a c-jun expression vector and mutational analysis of the AP1 site or the Sp1-related protein binding site. In contrast, formation of the YY1 complex suppresses enhancer activity, since deletion of the YY1 complex induces an augmentation of the enhancer activity and overexpression of YY1 results in an attenuation of the enhancer activity. Results from the mechanism study have revealed that YY1 is able to inhibit transactivation mediated by either AP1 or the Sp1-related protein, and YY1 suppressive activity is DNA binding dependent. Taken together, these data support the ideas that AP1 and the Sp1-related nuclear protein are required for transactivation of the human GM-CSF gene promoter and that YY1 can suppress transactivation of the promoter even under inducible conditions. PMID:8524292

  14. Enhancer-promoter interactions are encoded by complex genomic signatures on looping chromatin.

    PubMed

    Whalen, Sean; Truty, Rebecca M; Pollard, Katherine S

    2016-05-01

    Discriminating the gene target of a distal regulatory element from other nearby transcribed genes is a challenging problem with the potential to illuminate the causal underpinnings of complex diseases. We present TargetFinder, a computational method that reconstructs regulatory landscapes from diverse features along the genome. The resulting models accurately predict individual enhancer-promoter interactions across multiple cell lines with a false discovery rate up to 15 times smaller than that obtained using the closest gene. By evaluating the genomic features driving this accuracy, we uncover interactions between structural proteins, transcription factors, epigenetic modifications, and transcription that together distinguish interacting from non-interacting enhancer-promoter pairs. Most of this signature is not proximal to the enhancers and promoters but instead decorates the looping DNA. We conclude that complex but consistent combinations of marks on the one-dimensional genome encode the three-dimensional structure of fine-scale regulatory interactions. PMID:27064255

  15. Enhancer-promoter interactions are encoded by complex genomic signatures on looping chromatin

    PubMed Central

    Whalen, Sean; Truty, Rebecca M.; Pollard, Katherine S.

    2016-01-01

    Discriminating the gene target of a distal regulatory element from other nearby transcribed genes is a challenging problem with the potential to illuminate the causal underpinnings of complex diseases. We present TargetFinder, a computational method that reconstructs regulatory landscapes from genomic features along the genome. The resulting models accurately predict individual enhancer-promoter interactions across diverse cell lines with a false discovery rate up to fifteen times smaller than using the closest gene. By evaluating the genomic features driving this accuracy, we uncover interactions between structural proteins, transcription factors, epigenetic modifications, and transcription that together distinguish interacting from non-interacting enhancer-promoter pairs. Most of this signature is not proximal to the enhancers and promoters, but instead decorates the looping DNA. We conclude that complex but consistent combinations of marks on the one-dimensional genome encode the three-dimensional structure of fine-scale regulatory interactions. PMID:27064255

  16. Dynamic interactions of proteins in complex networks

    SciTech Connect

    Appella, E.; Anderson, C.

    2009-10-01

    Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact with the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing

  17. Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits

    PubMed Central

    Verheyen, Toon; Görnemann, Janina; Verbinnen, Iris; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-01-01

    Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits. PMID:25990731

  18. Radiolysis of DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Běgusová, Marie; Gillard, Nathalie; Sy, Denise; Castaing, Bertrand; Charlier, Michel; Spotheim-Maurizot, Melanie

    2005-02-01

    We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK.

  19. Human Mitochondrial Transcription Initiation Complexes Have Similar Topology on the Light and Heavy Strand Promoters.

    PubMed

    Morozov, Yaroslav I; Temiakov, Dmitry

    2016-06-24

    Transcription is a highly regulated process in all domains of life. In human mitochondria, transcription of the circular genome involves only two promoters, called light strand promoter (LSP) and heavy strand promoter (HSP), located in the opposite DNA strands. Initiation of transcription occurs upon sequential assembly of an initiation complex that includes mitochondrial RNA polymerase (mtRNAP) and the initiation factors mitochondrial transcription factor A (TFAM) and TFB2M. It has been recently suggested that the transcription initiation factor TFAM binds to HSP and LSP in opposite directions, implying that the mechanisms of transcription initiation are drastically dissimilar at these promoters. In contrast, we found that binding of TFAM to HSP and the subsequent recruitment of mtRNAP results in a pre-initiation complex that is remarkably similar in topology and properties to that formed at the LSP promoter. Our data suggest that assembly of the pre-initiation complexes on LSP and HSP brings these transcription units in close proximity, providing an opportunity for regulatory proteins to simultaneously control transcription initiation in both mtDNA strands. PMID:27226527

  20. PCDq: human protein complex database with quality index which summarizes different levels of evidences of protein complexes predicted from H-Invitational protein-protein interactions integrative dataset

    PubMed Central

    2012-01-01

    Background Proteins interact with other proteins or biomolecules in complexes to perform cellular functions. Existing protein-protein interaction (PPI) databases and protein complex databases for human proteins are not organized to provide protein complex information or facilitate the discovery of novel subunits. Data integration of PPIs focused specifically on protein complexes, subunits, and their functions. Predicted candidate complexes or subunits are also important for experimental biologists. Description Based on integrated PPI data and literature, we have developed a human protein complex database with a complex quality index (PCDq), which includes both known and predicted complexes and subunits. We integrated six PPI data (BIND, DIP, MINT, HPRD, IntAct, and GNP_Y2H), and predicted human protein complexes by finding densely connected regions in the PPI networks. They were curated with the literature so that missing proteins were complemented and some complexes were merged, resulting in 1,264 complexes comprising 9,268 proteins with 32,198 PPIs. The evidence level of each subunit was assigned as a categorical variable. This indicated whether it was a known subunit, and a specific function was inferable from sequence or network analysis. To summarize the categories of all the subunits in a complex, we devised a complex quality index (CQI) and assigned it to each complex. We examined the proportion of consistency of Gene Ontology (GO) terms among protein subunits of a complex. Next, we compared the expression profiles of the corresponding genes and found that many proteins in larger complexes tend to be expressed cooperatively at the transcript level. The proportion of duplicated genes in a complex was evaluated. Finally, we identified 78 hypothetical proteins that were annotated as subunits of 82 complexes, which included known complexes. Of these hypothetical proteins, after our prediction had been made, four were reported to be actual subunits of the

  1. Cold shock domain proteins repress transcription from the GM-CSF promoter.

    PubMed Central

    Coles, L S; Diamond, P; Occhiodoro, F; Vadas, M A; Shannon, M F

    1996-01-01

    The human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter binds a sequence-specific single-strand DNA binding protein termed NF-GMb. We previously demonstrated that the NF-GMb binding sites were required for repression of tumor necrosis factor-alpha (TNF-alpha) induction of the proximal GM-CSF promoter sequences in fibroblasts. We now describe the isolation of two different cDNA clones that encode cold shock domain (CSD) proteins with NF-GMb binding characteristics. One is identical to the previously reported CSD protein dbpB and the other is a previously unreported variant of the dbpA CSD factor. This is the first report of CSD factors binding to a cytokine gene. Nuclear NF-GMb and expressed CSD proteins have the same binding specificity for the GM-CSF promoter and other CSD binding sites. We present evidence that CSD factors are components of the nuclear NF-GMb complex. We also demonstrate that overexpression of the CSD proteins leads to complete repression of the proximal GM-CSF promoter containing the NF-GMb/CSD binding sites. Surprisingly, we show that CSD overexpression can also directly repress a region of the promoter which apparently lacks NF-GMb/CSD binding sites. NF-GMb/CSD factors may hence be acting by two different mechanisms. We discuss the potential importance of CSD factors in maintaining strict regulation of the GM-CSF gene. PMID:8710501

  2. STAG3-mediated stabilization of REC8 cohesin complexes promotes chromosome synapsis during meiosis.

    PubMed

    Fukuda, Tomoyuki; Fukuda, Nanaho; Agostinho, Ana; Hernández-Hernández, Abrahan; Kouznetsova, Anna; Höög, Christer

    2014-06-01

    Cohesion between sister chromatids in mitotic and meiotic cells is promoted by a ring-shaped protein structure, the cohesin complex. The cohesin core complex is composed of four subunits, including two structural maintenance of chromosome (SMC) proteins, one α-kleisin protein, and one SA protein. Meiotic cells express both mitotic and meiosis-specific cohesin core subunits, generating cohesin complexes with different subunit composition and possibly separate meiotic functions. Here, we have analyzed the in vivo function of STAG3, a vertebrate meiosis-specific SA protein. Mice with a hypomorphic allele of Stag3, which display a severely reduced level of STAG3, are viable but infertile. We show that meiocytes in homozygous mutant Stag3 mice display chromosome axis compaction, aberrant synapsis, impaired recombination and developmental arrest. We find that the three different α-kleisins present in meiotic cells show different dosage-dependent requirements for STAG3 and that STAG3-REC8 cohesin complexes have a critical role in supporting meiotic chromosome structure and functions. PMID:24797475

  3. A role for TOR complex 2 signaling in promoting autophagy.

    PubMed

    Vlahakis, Ariadne; Powers, Ted

    2014-01-01

    The conserved target of rapamycin (TOR) kinase is a central regulator of cell growth in response to nutrient availability. TOR forms 2 structurally and functionally distinct complexes, TORC1 and TORC2, and negatively regulates autophagy via TORC1. Here we demonstrate TOR also operates independently through the TORC2 signaling pathway to promote autophagy upon amino acid limitation. Under these conditions, TORC2, through its downstream target kinase Ypk1, inhibits the Ca(2+)- and Cmd1/calmodulin-dependent phosphatase, calcineurin, to enable the activation of the amino acid-sensing EIF2S1/eIF2α kinase, Gcn2, and promote autophagy. Thus TORC2 signaling regulates autophagy in a pathway distinct from TORC1 to provide a tunable response to the cellular metabolic state. PMID:25426890

  4. Curcumin inhibits HIV-1 by promoting Tat protein degradation.

    PubMed

    Ali, Amjad; Banerjea, Akhil C

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  5. Curcumin inhibits HIV-1 by promoting Tat protein degradation

    PubMed Central

    Ali, Amjad; Banerjea, Akhil C.

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  6. Proteomic Analysis Identifies an NADPH Oxidase 1 (Nox1)-Mediated Role for Actin-Related Protein 2/3 Complex Subunit 2 (ARPC2) in Promoting Smooth Muscle Cell Migration

    PubMed Central

    Al Ghouleh, Imad; Rodríguez, Andrés; Pagano, Patrick J.; Csányi, Gábor

    2013-01-01

    A variety of vascular pathologies, including hypertension, restenosis and atherosclerosis, are characterized by vascular smooth muscle cell (VSMC) hypertrophy and migration. NADPH oxidase 1 (Nox1) plays a pivotal role in these phenotypes via distinct downstream signaling. However, the mediators differentiating these distinct phenotypes and their precise role in vascular disease are still not clear. The present study was designed to identify novel targets of VSMC Nox1 signaling using 2D Differential In-Gel Electrophoresis and Mass Spectrometry (2D-DIGE/MS). VSMC treatment with scrambled (Scrmb) or Nox1 siRNA and incubation with the oxidant hydrogen peroxide (H2O2; 50 μM, 3 h) followed by 2D-DIGE/MS on cell lysates identified 10 target proteins. Among these proteins, actin-related protein 2/3 complex subunit 2 (ARPC2) with no previous link to Nox isozymes, H2O2, or other reactive oxygen species (ROS), was identified and postulated to play an intermediary role in VSMC migration. Western blot confirmed that Nox1 mediates H2O2-induced ARPC2 expression in VSMC. Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H2O2-treated VSMC. Additionally, wound-healing “scratch” assay confirmed that H2O2 stimulates VSMC migration via Nox1. Importantly, gene silencing of ARPC2 suppressed H2O2-stimulated VSMC migration. These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target. PMID:24152438

  7. The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms

    PubMed Central

    Jungfleisch, Jennifer; Chowdhury, Ashis; Alves-Rodrigues, Isabel; Tharun, Sundaresan; Díez, Juana

    2015-01-01

    The Lsm1-7-Pat1 complex binds to the 3′ end of cellular mRNAs and promotes 3′ end protection and 5′–3′ decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes. PMID:26092942

  8. Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication.

    PubMed

    Kodani, Andrew; Yu, Timothy W; Johnson, Jeffrey R; Jayaraman, Divya; Johnson, Tasha L; Al-Gazali, Lihadh; Sztriha, Lāszló; Partlow, Jennifer N; Kim, Hanjun; Krup, Alexis L; Dammermann, Alexander; Krogan, Nevan J; Walsh, Christopher A; Reiter, Jeremy F

    2015-01-01

    Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication. PMID:26297806

  9. Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication

    PubMed Central

    Kodani, Andrew; Yu, Timothy W; Johnson, Jeffrey R; Jayaraman, Divya; Johnson, Tasha L; Al-Gazali, Lihadh; Sztriha, Lāszló; Partlow, Jennifer N; Kim, Hanjun; Krup, Alexis L; Dammermann, Alexander; Krogan, Nevan J; Walsh, Christopher A; Reiter, Jeremy F

    2015-01-01

    Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication. DOI: http://dx.doi.org/10.7554/eLife.07519.001 PMID:26297806

  10. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001 PMID:25789606

  11. A Least Square Method Based Model for Identifying Protein Complexes in Protein-Protein Interaction Network

    PubMed Central

    Dai, Qiguo; Guo, Maozu; Guo, Yingjie; Liu, Xiaoyan; Liu, Yang; Teng, Zhixia

    2014-01-01

    Protein complex formed by a group of physical interacting proteins plays a crucial role in cell activities. Great effort has been made to computationally identify protein complexes from protein-protein interaction (PPI) network. However, the accuracy of the prediction is still far from being satisfactory, because the topological structures of protein complexes in the PPI network are too complicated. This paper proposes a novel optimization framework to detect complexes from PPI network, named PLSMC. The method is on the basis of the fact that if two proteins are in a common complex, they are likely to be interacting. PLSMC employs this relation to determine complexes by a penalized least squares method. PLSMC is applied to several public yeast PPI networks, and compared with several state-of-the-art methods. The results indicate that PLSMC outperforms other methods. In particular, complexes predicted by PLSMC can match known complexes with a higher accuracy than other methods. Furthermore, the predicted complexes have high functional homogeneity. PMID:25405206

  12. 193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology.

    PubMed

    Morrison, Lindsay J; Brodbelt, Jennifer S

    2016-08-31

    Protein-protein interfaces and architecture are critical to the function of multiprotein complexes. Mass spectrometry-based techniques have emerged as powerful strategies for characterization of protein complexes, particularly for heterogeneous mixtures of structures. In the present study, activation and dissociation of three tetrameric protein complexes (streptavidin, transthyretin, and hemoglobin) in the gas phase was undertaken by 193 nm ultraviolet photodissociation (UVPD) for the characterization of higher order structure. High pulse energy UVPD resulted in the production of dimers and low charged monomers exhibiting symmetrical charge partitioning among the subunits (the so-called symmetrical dissociation pathways), consistent with the subunit organization of the complexes. In addition, UVPD promoted backbone cleavages of the monomeric subunits, the abundances of which corresponded to the more flexible loop regions of the proteins. PMID:27480400

  13. Quality Control of a Cytoplasmic Protein Complex

    PubMed Central

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

    2015-01-01

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

  14. Isolation of a human nuclear complex that may promote oncogenic translocations

    SciTech Connect

    Eisen, A.

    1994-09-01

    Specific chromosomal translocations can lead to the activation of oncogenes and the formation of characteristic malignancies. The DNA sequences flanking translocation break points may be recombinogenic and promote these genomic rearrangements. Indeed, the t(14;18) seen in most human follicular lymphomas occurs adjacent to octanucleotides that resemble the recombinogenic Chi (5{prime}-GCTGGTGG-3{prime}) sequence. These Chi-like sequences are likely to be targets for nuclear proteins that cleave superhelical chromosomal DNA and re-ligate non-homologous chromosome ends. A complex of human nuclear proteins with these properties has now been isolated and the component proteins identified. The complex consists of three human DNA repair proteins. The three proteins co-purify from HeLa nuclear extracts in a single step by Chi-sequence DNA affinity chromatography. The Chi-binding protein was identified as the DNA repair enzyme poly (ADP-ribose) polymerase (PARP). Sequence specificity is a newly described property for this DNA-binding protein. Furthermore, the DNA-dependent enzymatic activity of PARP is enhanced by the presence of a Chi sequence. PARP associates with a stable heterodimer consisting of DNA ligase I and its putative inhibitor. The complex leads to rearrangements of DNA in the presence of an exact Chi sequence. Together, these proteins will cleave and re-ligate superhelical plasmid DNA with an exact Chi sequence to form end-to-end linear multimers. The concerted activities of this complex may account for the t(14;18) chromosomal rearrangement adjacent to Chi-like sequences frequently seen in various tumors.

  15. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation.

    PubMed

    Shindo, Yutaka; Komatsu, Hirokazu; Hotta, Kohji; Ariga, Katsuhiko; Oka, Kotaro

    2016-01-01

    Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells. PMID:27374857

  16. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

    PubMed Central

    Shindo, Yutaka; Komatsu, Hirokazu; Hotta, Kohji; Ariga, Katsuhiko; Oka, Kotaro

    2016-01-01

    Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells. PMID:27374857

  17. Functional Redundancy of Paralogs of an Anaphase Promoting Complex/Cyclosome Subunit in Caenorhabditis elegans Meiosis

    PubMed Central

    Stein, Kathryn K.; Nesmith, Jessica E.; Ross, Benjamin D.; Golden, Andy

    2010-01-01

    The anaphase promoting complex/cyclosome (APC/C) mediates the metaphase-to-anaphase transition by instructing the ubiquitination and turnover of key proteins at this stage of the cell cycle. We have recovered a gain-of-function allele in an APC5 subunit of the anaphase promoting complex/cyclosome. This finding led us to investigate further the role of APC5 in Caenorhabditis elegans, which contains two APC5 paralogs. We have shown that these two paralogs, such-1 and gfi-3, are coexpressed in the germline but have nonoverlapping expression patterns in other tissues. Depletion of such-1 or gfi-3 alone does not have a notable effect on the meiotic divisions; however, codepletion of these two factors results in meiotic arrest. In sum, the two C. elegans APC5 paralogs have a redundant function during the meiotic divisions. PMID:20944012

  18. The genomic sequence of the murine major vault protein and its promoter.

    PubMed

    Mossink, Marieke; van Zon, Arend; Fränzel-Luiten, Erna; Schoester, Martijn; Scheffer, George L; Scheper, Rik J; Sonneveld, Pieter; Wiemer, Erik A C

    2002-07-10

    Vaults are ribonucleoproteins of unknown function, consisting of three different proteins and multiple copies of small untranslated RNA molecules. One of the protein subunits has been identified as TEP1, a protein that is also associated with the telomerase complex. Another protein appears to contain a functional PARP domain and is hence called VPARP. The third protein, major vault protein (MVP), is believed to make up 70% of the total mass of the vault complex and to be responsible for the typical barrel-shaped structure of vaults. We have isolated the murine MVP cDNA and compared the amino acid sequence with MVP from other species. Over 90% of sequence identity was found between mouse, human and rat, and a considerable degree of identity between mouse and MVPs from lower eukaryotes. We also found that the genomic structure of the murine MVP gene closely resembles the organization of the human MVP gene, both consisting of 15 exons of which most have exactly the same size. Finally we have isolated a genomic region upstream (and partially overlapping) the first untranslated exon, that displayed promoter activity in a luciferase reporter assay. Furthermore, we showed that the sequences from the first exon together with the 5'-end of the first intron enhance the promoter activity, implying the presence of essential promoter elements in this region. Alignment of the murine promoter region with the homologous sequences of the human gene revealed an identity of 58%. The apparent presence of conserved promoter elements suggests a similar regulation of human and murine MVP expression. PMID:12234684

  19. Hedgehog induces formation of PKA-Smoothened complexes to promote Smoothened phosphorylation and pathway activation

    PubMed Central

    Li, Shuang; Ma, Guoqiang; Wang, Bing; Jiang, Jin

    2015-01-01

    Hedgehog (Hh) is a secreted glycoprotein that binds its receptor Patched to activate the G protein-coupled receptor-like protein Smoothened (Smo). In Drosophila, protein kinase A (PKA) phosphorylates and activates Smo in cells stimulated with Hh. In unstimulated cells, PKA phosphorylates and inhibits the transcription factor Cubitus interruptus (Ci). Here, we found that in cells exposed to Hh, the catalytic subunit of PKA (PKAc) bound to the juxtamembrane region of the C terminus of Smo. PKA-mediated phosphorylation of Smo further enhanced its association with PKAc to form stable kinase-substrate complexes that promoted the PKA-mediated trans-phosphorylation of Smo dimers. We identified multiple basic residues in the C-terminus of Smo that were required for interaction with PKAc, Smo phosphorylation, and Hh pathway activation. Hh induced a switch from the association of PKAc with a cytosolic complex of Ci and the kinesin-like protein Costal2 (Cos2) to a membrane-bound Smo-Cos2 complex. Thus, our study uncovers a previously uncharacterized mechanism for regulation of PKA activity and demonstrates that the signal-regulated formation of kinase-substrate complexes plays a central role in Hh signal transduction. PMID:24985345

  20. Building a pseudo-atomic model of the anaphase-promoting complex

    SciTech Connect

    Kulkarni, Kiran; Zhang, Ziguo; Chang, Leifu; Yang, Jing; Fonseca, Paula C. A. da; Barford, David

    2013-11-01

    This article describes an example of molecular replacement in which atomic models are used to interpret electron-density maps determined using single-particle electron-microscopy data. The anaphase-promoting complex (APC/C) is a large E3 ubiquitin ligase that regulates progression through specific stages of the cell cycle by coordinating the ubiquitin-dependent degradation of cell-cycle regulatory proteins. Depending on the species, the active form of the APC/C consists of 14–15 different proteins that assemble into a 20-subunit complex with a mass of approximately 1.3 MDa. A hybrid approach of single-particle electron microscopy and protein crystallography of individual APC/C subunits has been applied to generate pseudo-atomic models of various functional states of the complex. Three approaches for assigning regions of the EM-derived APC/C density map to specific APC/C subunits are described. This information was used to dock atomic models of APC/C subunits, determined either by protein crystallography or homology modelling, to specific regions of the APC/C EM map, allowing the generation of a pseudo-atomic model corresponding to 80% of the entire complex.

  1. Functional analysis of bipartite begomovirus coat protein promoter sequences

    SciTech Connect

    Lacatus, Gabriela; Sunter, Garry

    2008-06-20

    We demonstrate that the AL2 gene of Cabbage leaf curl virus (CaLCuV) activates the CP promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for Tomato golden mosaic virus (TGMV). Binding studies indicate that sequences mediating repression and activation of the TGMV and CaLCuV CP promoter specifically bind different nuclear factors common to Nicotiana benthamiana, spinach and tomato. However, chromatin immunoprecipitation demonstrates that TGMV AL2 can interact with both sequences independently. Binding of nuclear protein(s) from different crop species to viral sequences conserved in both bipartite and monopartite begomoviruses, including TGMV, CaLCuV, Pepper golden mosaic virus and Tomato yellow leaf curl virus suggests that bipartite begomoviruses bind common host factors to regulate the CP promoter. This is consistent with a model in which AL2 interacts with different components of the cellular transcription machinery that bind viral sequences important for repression and activation of begomovirus CP promoters.

  2. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking.

    PubMed

    Heo, Lim; Lee, Hasup; Seok, Chaok

    2016-01-01

    Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex. PMID:27535582

  3. GalaxyRefineComplex: Refinement of protein-protein complex model structures driven by interface repacking

    PubMed Central

    Heo, Lim; Lee, Hasup; Seok, Chaok

    2016-01-01

    Protein-protein docking methods have been widely used to gain an atomic-level understanding of protein interactions. However, docking methods that employ low-resolution energy functions are popular because of computational efficiency. Low-resolution docking tends to generate protein complex structures that are not fully optimized. GalaxyRefineComplex takes such low-resolution docking structures and refines them to improve model accuracy in terms of both interface contact and inter-protein orientation. This refinement method allows flexibility at the protein interface and in the overall docking structure to capture conformational changes that occur upon binding. Symmetric refinement is also provided for symmetric homo-complexes. This method was validated by refining models produced by available docking programs, including ZDOCK and M-ZDOCK, and was successfully applied to CAPRI targets in a blind fashion. An example of using the refinement method with an existing docking method for ligand binding mode prediction of a drug target is also presented. A web server that implements the method is freely available at http://galaxy.seoklab.org/refinecomplex. PMID:27535582

  4. Cellular Prion Protein Promotes Brucella Infection into Macrophages

    PubMed Central

    Watarai, Masahisa; Kim, Suk; Erdenebaatar, Janchivdorj; Makino, Sou-ichi; Horiuchi, Motohiro; Shirahata, Toshikazu; Sakaguchi, Suehiro; Katamine, Shigeru

    2003-01-01

    The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex–associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection. PMID:12847134

  5. Probing nanoparticle effect in protein-surfactant complexes

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Aswal, V. K.; Kohlbrecher, J.

    2015-06-01

    SANS experiments have been carried to probe the role of anionic silica nanoparticles in the anionic BSA protein-cationic DTAB surfactant complexes. In protein-surfactant complex, surfactant molecules aggregate to form micelle-like clusters along the unfolded polypeptide chains of the protein. The nanoparticle aggregation mediated by oppositely charged protein-surfactant complex coexists with the free protein-surfactant complexes in the nanoparticle-protein-surfactant system. There is rearrangement of micelles in adsorbed protein-surfactant complex on nanoparticles in leading to their (nanoparticle) aggregation. On the other hand, the unfolding of protein in free protein-surfactant complex is found to be significantly enhanced in presence of nanoparticles.

  6. Formation and fate of a complete 31-protein RNA polymerase II transcription preinitiation complex.

    PubMed

    Murakami, Kenji; Calero, Guillermo; Brown, Christopher R; Liu, Xin; Davis, Ralph E; Boeger, Hinrich; Kornberg, Roger D

    2013-03-01

    Whereas individual RNA polymerase II (pol II)-general transcription factor (GTF) complexes are unstable, an assembly of pol II with six GTFs and promoter DNA could be isolated in abundant homogeneous form. The resulting complete pol II transcription preinitiation complex (PIC) contained equimolar amounts of all 31 protein components. An intermediate in assembly, consisting of four GTFs and promoter DNA, could be isolated and supplemented with the remaining components for formation of the PIC. Nuclease digestion and psoralen cross-linking mapped the PIC between positions -70 and -9, centered on the TATA box. Addition of ATP to the PIC resulted in quantitative conversion to an open complex, which retained all 31 proteins, contrary to expectation from previous studies. Addition of the remaining NTPs resulted in run-off transcription, with an efficiency that was promoter-dependent and was as great as 17.5% with the promoters tested. PMID:23303183

  7. Enhanced green fluorescent protein expression in Pleurotus ostreatus for in vivo analysis of fungal laccase promoters.

    PubMed

    Amore, Antonella; Honda, Yoichi; Faraco, Vincenza

    2012-10-01

    The laccase family of Pleurotus ostreatus has been widely characterized, and studies of the genes coding for laccase isoenzymes in P. ostreatus have so far led to the identification of four different genes and the corresponding cDNAs, poxc, pox1, poxa1b and poxa3. Analyses of P. ostreatus laccase promoters poxc, pox1, poxa1b and poxa3 have allowed identification of several putative response elements, and sequences of metal-responsive elements involved in the formation of complexes with fungal proteins have been identified in poxc and poxa1b promoters. In this work, development of a system for in vivo analysis of P. ostreatus laccase promoter poxc by enhanced green fluorescent protein expression is performed, based on a poly ethylene glycol-mediated procedure for fungal transformation. A quantitative measurement of fluorescence expressed in P. ostreatus transformants is hereby reported for the first time for this fungus. PMID:22893518

  8. Characterization of Protein Complexes and Subcomplexes in Protein-Protein Interaction Databases

    PubMed Central

    Zaki, Nazar; Mohamed, Elfadil A.; Mora, Antonio

    2015-01-01

    The identification and characterization of protein complexes implicated in protein-protein interaction data are crucial to the understanding of the molecular events under normal and abnormal physiological conditions. This paper provides a novel characterization of subcomplexes in protein interaction databases, stressing definition and representation issues, quantification, biological validation, network metrics, motifs, modularity, and gene ontology (GO) terms. The paper introduces the concept of “nested group” as a way to represent subcomplexes and estimates that around 15% of those nested group with the higher Jaccard index may be a result of data artifacts in protein interaction databases, while a number of them can be found in biologically important modular structures or dynamic structures. We also found that network centralities, enrichment in essential proteins, GO terms related to regulation, imperfect 5-clique motifs, and higher GO homogeneity can be used to identify proteins in nested complexes. PMID:25722891

  9. Identification and analysis of multi-protein complexes in placenta.

    PubMed

    Wang, Fuqiang; Wang, Ling; Xu, Zhiyang; Liang, Gaolin

    2013-01-01

    Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders. PMID:23638173

  10. Adenovirus E3/19K Promotes Evasion of NK Cell Recognition by Intracellular Sequestration of the NKG2D Ligands Major Histocompatibility Complex Class I Chain-Related Proteins A and B▿

    PubMed Central

    McSharry, Brian P.; Burgert, Hans-Gerhard; Owen, Douglas P.; Stanton, Richard J.; Prod'homme, Virginie; Sester, Martina; Koebernick, Katja; Groh, Veronika; Spies, Thomas; Cox, Steven; Little, Ann-Margaret; Wang, Eddie C. Y.; Tomasec, Peter; Wilkinson, Gavin W. G.

    2008-01-01

    The adenovirus (Ad) early transcription unit 3 (E3) encodes multiple immunosubversive functions that are presumed to facilitate the establishment and persistence of infection. Indeed, the capacity of E3/19K to inhibit transport of HLA class I (HLA-I) to the cell surface, thereby preventing peptide presentation to CD8+ T cells, has long been recognized as a paradigm for viral immune evasion. However, HLA-I downregulation has the potential to render Ad-infected cells vulnerable to natural killer (NK) cell recognition. Furthermore, expression of the immediate-early Ad gene E1A is associated with efficient induction of ligands for the key NK cell-activating receptor NKG2D. Here we show that while infection with wild-type Ad enhances synthesis of the NKG2D ligands, major histocompatibility complex class I chain-related proteins A and B (MICA and MICB), their expression on the cell surface is actively suppressed. Both MICA and MICB are retained within the endoplasmic reticulum as immature endoglycosidase H-sensitive forms. By analyzing a range of cell lines and viruses carrying mutated versions of the E3 gene region, E3/19K was identified as the gene responsible for this activity. The structural requirements within E3/19K necessary to sequester MICA/B and HLA-I are similar. In functional assays, deletion of E3/19K rendered Ad-infected cells more sensitive to NK cell recognition. We report the first NK evasion function in the Adenoviridae and describe a novel function for E3/19K. Thus, E3/19K has a dual function: inhibition of T-cell recognition and NK cell activation. PMID:18287244

  11. Neonatal Fc receptor promotes immune complex-mediated glomerular disease.

    PubMed

    Olaru, Florina; Luo, Wentian; Suleiman, Hani; St John, Patricia L; Ge, Linna; Mezo, Adam R; Shaw, Andrey S; Abrahamson, Dale R; Miner, Jeffrey H; Borza, Dorin-Bogdan

    2014-05-01

    The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG. PMID:24357670

  12. Stalled transcription complexes promote DNA repair at a distance

    PubMed Central

    Haines, Nia M.; Kim, Young-In T.; Smith, Abigail J.; Savery, Nigel J.

    2014-01-01

    Transcription-coupled nucleotide excision repair (TCR) accelerates the removal of noncoding lesions from the template strand of active genes, and hence contributes to genome-wide variations in mutation frequency. Current models for TCR suppose that a lesion must cause RNA polymerase (RNAP) to stall if it is to be a substrate for accelerated repair. We have examined the substrate requirements for TCR using a system in which transcription stalling and damage location can be uncoupled. We show that Mfd-dependent TCR in bacteria involves the formation of a damage search complex that can detect lesions downstream of a stalled RNAP, and that the strand specificity of the accelerated repair pathway is independent of the requirement for a lesion to stall RNAP. We also show that an ops (operon polarity suppressor) transcription pause site, which causes backtracking of RNAP, can promote the repair of downstream lesions when those lesions do not themselves cause the polymerase to stall. Our findings indicate that the transcription-repair coupling factor Mfd, which is an ATP-dependent superfamily 2 helicase that binds to RNAP, continues to translocate along DNA after RNAP has been displaced until a lesion in the template strand is located. The discovery that pause sites can promote the repair of nonstalling lesions suggests that TCR pathways may play a wider role in modulating mutation frequencies in different parts of the genome than has previously been suspected. PMID:24554077

  13. Responses of two protein-protein complexes to solvent stress: does water play a role at the interface?

    PubMed Central

    Kornblatt, J A; Kornblatt, M J; Hoa, G H; Mauk, A G

    1993-01-01

    We have analyzed the stability of the cytochrome c-cytochrome b5 and cytochrome c-cytochrome c oxidase complexes as a function of solvent stress. High concentrations of glycerol were used to displace the two equilibria. Glycerol promotes complex formation between cytochrome c and cytochrome b5 but inhibits that between cytochrome c and cytochrome c oxidase. The results with cytochrome b5 and cytochrome c were expected; the association of this complex is largely entropy driven. Our interpretation is that the cytochrome c-cytochrome b5 complex excludes water. The results with the cytochrome c oxidase and cytochrome c couple were not expected. We interpret them to mean that either glycerol is binding to the oxidase, thereby displacing the cytochrome c, or that water is required at this protein-protein interface. A requirement for substantial quantities of water at the interface of some protein complexes is logical but has been reported only once. PMID:8241386

  14. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation.

    PubMed

    Chen, Chao; Cui, Zhenling; Song, Xiangfei; Liu, Ya-Jun; Cui, Qiu; Feng, Yingang

    2016-03-01

    Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions, which are the most efficient system for the degradation of lignocellulosic resources in nature. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins, Clocl_1298 and Clocl_1862, contain a dockerin module, which suggests that they are components of the cellulosome. Bacterial expansin-like proteins do not have hydrolytic activities, but can facilitate the degradation of cellulosic biomass via synergistic effects with cellulases. In this study, the synergistic effect of the expansin-like proteins with both native and designer cellulosomes was investigated. The free expansin-like proteins, including expansin-like domains of Clocl_1298 and Clocl_1862, as well as a well-studied bacterial expansin-like protein BsEXLX1 from Bacillus subtilis, promoted the cellulose degradation by native cellulosomes, indicating the cellulosomal expansin-like proteins have the synergistic function. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. These results indicated that non-catalytic expansin-like proteins in the cellulosome can enhance the activity of the cellulosome in lignocellulose degradation. The involvement of functional expansin-like proteins in the cellulosome also implies new physiological functions of bacterial expansin-like proteins and cellulosomes. PMID:26521249

  15. HKC: an algorithm to predict protein complexes in protein-protein interaction networks.

    PubMed

    Wang, Xiaomin; Wang, Zhengzhi; Ye, Jun

    2011-01-01

    With the availability of more and more genome-scale protein-protein interaction (PPI) networks, research interests gradually shift to Systematic Analysis on these large data sets. A key topic is to predict protein complexes in PPI networks by identifying clusters that are densely connected within themselves but sparsely connected with the rest of the network. In this paper, we present a new topology-based algorithm, HKC, to detect protein complexes in genome-scale PPI networks. HKC mainly uses the concepts of highest k-core and cohesion to predict protein complexes by identifying overlapping clusters. The experiments on two data sets and two benchmarks show that our algorithm has relatively high F-measure and exhibits better performance compared with some other methods. PMID:22174556

  16. Engineering of complex protein sialylation in plants.

    PubMed

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-08-23

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  17. Engineering of complex protein sialylation in plants

    PubMed Central

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-01-01

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  18. Activated protein C prevents inflammation yet stimulates angiogenesis to promote cutaneous wound healing.

    PubMed

    Jackson, Christopher J; Xue, Meilang; Thompson, Patrick; Davey, Ross A; Whitmont, Kaley; Smith, Susan; Buisson-Legendre, Nathalie; Sztynda, Tamara; Furphy, Louise J; Cooper, Alan; Sambrook, Philip; March, Lyn

    2005-01-01

    Activated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation, and has more recently been shown to be a potent anti-inflammatory mediator. Using cultured human cells, we show here that APC up-regulates the angiogenic promoters matrix metalloproteinase-2 in skin fibroblasts and umbilical vein endothelial cells, vascular endothelial growth factor in keratinocytes and fibroblasts, and monocyte chemoattractant protein-1 in fibroblasts. In the chick embryo chorioallantoic membrane assay, APC promoted the granulation/remodeling phases of wound healing by markedly stimulating angiogenesis as well as promoting reepithelialization. In a full-thickness rat skin-healing model, a single topical application of APC enhanced wound healing compared to saline control. APC-treated wounds had markedly more blood vessels on day 7 and a significantly lower infiltration of neutrophils at days 4 and 7. The broad spectrum matrix metallo-proteinase, GM6001, prevented the ability of APC to promote wound healing. In summary, our results show that APC promotes cutaneous wound healing via a complex mechanism involving stimulation of angiogenesis and inhibition of inflammation. These unique properties of APC make it an attractive therapeutic agent to promote the healing of chronic wounds. PMID:15953048

  19. The Adenovirus L4 33-Kilodalton Protein Binds to Intragenic Sequences of the Major Late Promoter Required for Late Phase-Specific Stimulation of Transcription▿

    PubMed Central

    Ali, Humayra; LeRoy, Gary; Bridge, Gemma; Flint, S. J.

    2007-01-01

    The adenovirus late IVa2 protein is required for maximally efficient transcription from the viral major late (ML) promoter, and hence, the synthesis of the majority of viral late proteins. This protein is a sequence-specific DNA-binding protein that also promotes the assembly of progeny virus particles. Previous studies have established that a IVa2 protein dimer (DEF-B) binds specifically to an intragenic ML promoter sequence necessary for late phase-specific stimulation of ML transcription. However, activation of transcription from the ML promoter correlates with binding of at least one additional infected-cell-specific protein, termed DEF-A, to the promoter. Using an assay for the DNA-binding activity of DEF-A, we identified the unknown protein by using conventional purification methods, purification of FLAG-tagged IVa2-protein-containing complexes, and transient synthesis of viral late proteins. The results of these experiments established that the viral L4 33-kDa protein is the only component of DEF-A: the IVa2 and L4 33-kDa proteins are necessary and sufficient for formation of all previously described complexes in the intragenic control region of the ML promoter. Furthermore, the L4 33-kDa protein binds to the promoter with the specificity characteristic of DEF-A and stimulates transcription from the ML promoter in transient-expression assays. PMID:17093188

  20. Detecting Overlapping Protein Complexes by Rough-Fuzzy Clustering in Protein-Protein Interaction Networks

    PubMed Central

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks. PMID:24642838

  1. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    PubMed

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks. PMID:24642838

  2. Strand invasion promoted by recombination protein of coliphage

    NASA Astrophysics Data System (ADS)

    Rybalchenko, Nataliya; Golub, Efim I.; Bi, Baoyuan; Radding, Charles M.

    2004-12-01

    Studies of phage in vivo have indicated that its own recombination enzymes, protein and exonuclease, are capable of catalyzing two dissimilar pathways of homologous recombination that are widely distributed in nature: single-strand annealing and strand invasion. The former is an enzymatic splicing of overlapping ends of broken homologous DNA molecules, whereas the latter is characterized by the formation of a three-stranded synaptic intermediate and subsequent strand exchange. Previous studies in vitro have shown that protein has annealing activity, and that exonuclease, acting on branched substrates, can produce a perfect splice that requires only ligation for completion. The present study shows that protein can initiate strand invasion in vitro, as evidenced both by the formation of displacement loops (D-loops) in superhelical DNA and by strand exchange between colinear single-stranded and double-stranded molecules. Thus, protein can catalyze steps that are central to both strand annealing and strand invasion pathways of recombination. These observations add protein to a set of diverse proteins that appear to promote recognition of homology by a unitary mechanism governed by the intrinsic dynamic properties of base pairs in DNA. genetic recombination | phage λ

  3. The yeast ubiquitin protease, Ubp3p, promotes protein stability.

    PubMed Central

    Brew, Christine T; Huffaker, Tim C

    2002-01-01

    Stu1p is a microtubule-associated protein required for spindle assembly. In this article we show that the temperature-sensitive stu1-5 allele is synthetically lethal in combination with ubp3, gim1-gim5, and kem1 mutations. The primary focus of this article is on the stu1-5 ubp3 interaction. Ubp3 is a deubiquitination enzyme and a member of a large family of cysteine proteases that cleave ubiquitin moieties from protein substrates. UBP3 is the only one of 16 UBP genes in yeast whose loss is synthetically lethal with stu1-5. Stu1p levels in stu1-5 cells are several-fold lower than the levels in wild-type cells and the stu1-5 temperature sensitivity can be rescued by additional copies of stu1-5. These results indicate that the primary effect of the stu1-5 mutation is to make the protein less stable. The levels of Stu1p are even lower in ubp3Delta stu1-5 cells, suggesting that Ubp3p plays a role in promoting protein stability. We also found that ubp3Delta produces growth defects in combination with mutations in other genes that decrease protein stability. Overall, these data support the idea that Ubp3p has a general role in the reversal of protein ubiquitination. PMID:12454057

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

    PubMed

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

    2005-01-01

    While recent technological advances have made available large datasets of experimentally-detected pairwise protein-protein interactions, there is still a lack of experimentally-determined protein complex data. To make up for this lack of protein complex data, we explore the mining of existing protein interaction graphs for protein complexes. This paper proposes a novel graph mining algorithm to detect the dense neighborhoods (highly connected regions) in an interaction graph which may correspond to protein complexes. Our algorithm first locates local cliques for each graph vertex (protein) and then merge the detected local cliques according to their affinity to form maximal dense regions. We present experimental results with yeast protein interaction data to demonstrate the effectiveness of our proposed method. Compared with other existing techniques, our predicted complexes can match or overlap significantly better with the known protein complexes in the MIPS benchmark database. Novel protein complexes were also predicted to help biologists in their search for new protein complexes. PMID:16901108

  5. Comparison of ADH3 promoter with commonly used promoters for recombinant protein production in Pichia pastoris.

    PubMed

    Karaoglan, Mert; Karaoglan, Fidan Erden; Inan, Mehmet

    2016-05-01

    Recombinant protein production under the control of the PADH3 was compared with Pichia pastoris PAOX1 and PGAP. The single-copy-clones expressing Aspergillus niger xylanase (XylB) gene with the three different promoters were tested in shake flask and 5 L fed-batch fermentation processes. Recombinant protein production with PADH3, PAOX1 and PGAP were initiated by addition of ethanol, methanol and glucose, respectively in the culture medium. The fermentation process was carried out for 72 h at 30 °C, pH 5 and 30% dissolved oxygen. Extracellular protein production yield for PADH3 (3725 U/mL) was higher than for PAOX1 (2095 U/mL) and PGAP (580 U/mL) at fermentor scale under the conditions tested. These results show that the PADH3 promoter is a promising tool for large scale production of recombinant proteins and can be an alternative to the PAOX1 and PGAP. PMID:26835836

  6. Evaluation of the Saccharomyces cerevisiae ADH2 promoter for protein synthesis.

    PubMed

    Lee, K Michael; DaSilva, Nancy A

    2005-04-30

    The Saccharomyces cerevisiae ADH2 promoter (P(ADH2)) is repressed several hundred-fold in the presence of glucose; transcription is initiated once the glucose in the medium is exhausted. The promoter can thus be utilized for effective regulation of recombinant gene expression in S. cerevisiae without the addition of an inducer. To evaluate this promoter in the absence of plasmid copy number and stability variations, the P(ADH2)-lacZ cassette was integrated into the yeast chromosomes. The effects of medium composition, glucose concentration and cultivation time on promoter derepression and expression level were investigated. Maximum protein activity was obtained after 48 h of growth in complex YPD medium containing 1% glucose. The widely used S. cerevisiae GAL1 and CUP1 promoters both require the addition of an inducer [galactose and copper(II) ion, respectively] before regulated genes will be expressed. The strengths of these three different promoters were compared for cells containing one copy of an integrated lacZ gene under their control. The ADH2 promoter was superior for all induction strategies investigated. PMID:15849781

  7. Polycomb complex protein BMI-1 promotes invasion and metastasis of pancreatic cancer stem cells by activating PI3K/AKT signaling, an ex vivo, in vitro, and in vivo study

    PubMed Central

    Wang, Min-Cong; Jiao, Min; Wu, Tao; Jing, Li; Cui, Jie; Guo, Hui; Tian, Tao; Ruan, Zhi-ping; Wei, Yong-Chang; Jiang, Li-Li; Sun, Hai-Feng; Huang, Lan-Xuan; Nan, Ke-Jun; Li, Chun-Li

    2016-01-01

    Cancer stem cell theory indicates cancer stem cells are the key to promote tumor invasion and metastasis. Studies showed that BMI-1 could promote self-renew, differentiation and tumor formation of CSCs and invasion/metastasis of human cancer. However, whether BMI-1 could regulate invasion and metastasis ability of CSCs is still unclear. In our study, we found that up-regulated expression of BMI-1 was associated with tumor invasion, metastasis and poor survival of pancreatic cancer patients. CD133+ cells were obtained by using magnetic cell sorting and identified of CSCs properties such as self-renew, multi-differentiation and tumor formation ability. Then, we found that BMI-1 expression was up-regulated in pancreatic cancer stem cells. Knockdown of BMI-1 expression attenuated invasion ability of pancreatic cancer stem cells in Transwell system and liver metastasis capacity in nude mice which were injected CSCs through the caudal vein. We are the first to reveal that BMI-1 could promote invasion and metastasis ability of pancreatic cancer stem cells. Finally, we identified that BMI-1 expression activating PI3K/AKT singing pathway by negative regulating PTEN was the main mechanism of promoting invasion and metastasis ability of pancreatic CSCs. In summary, our findings indicate that BMI-1 could be used as the therapeutic target to inhibiting CSCs-mediated pancreatic cancer metastasis. PMID:26840020

  8. Structural basis for the subunit assembly of the anaphase-promoting complex.

    PubMed

    Schreiber, Anne; Stengel, Florian; Zhang, Ziguo; Enchev, Radoslav I; Kong, Eric H; Morris, Edward P; Robinson, Carol V; da Fonseca, Paula C A; Barford, David

    2011-02-10

    The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates. PMID:21307936

  9. Bacterial promoter repression by DNA looping without protein-protein binding competition.

    PubMed

    Becker, Nicole A; Greiner, Alexander M; Peters, Justin P; Maher, L James

    2014-05-01

    The Escherichia coli lactose operon provides a paradigm for understanding gene control by DNA looping where the lac repressor (LacI) protein competes with RNA polymerase for DNA binding. Not all promoter loops involve direct competition between repressor and RNA polymerase. This raises the possibility that positioning a promoter within a tightly constrained DNA loop is repressive per se, an idea that has previously only been considered in vitro. Here, we engineer living E. coli bacteria to measure repression due to promoter positioning within such a tightly constrained DNA loop in the absence of protein-protein binding competition. We show that promoters held within such DNA loops are repressed ∼100-fold, with up to an additional ∼10-fold repression (∼1000-fold total) dependent on topological positioning of the promoter on the inner or outer face of the DNA loop. Chromatin immunoprecipitation data suggest that repression involves inhibition of both RNA polymerase initiation and elongation. These in vivo results show that gene repression can result from tightly looping promoter DNA even in the absence of direct competition between repressor and RNA polymerase binding. PMID:24598256

  10. Magnetic Resonance Access to Transiently Formed Protein Complexes**

    PubMed Central

    Sára, Tomáš; Schwarz, Thomas C; Kurzbach, Dennis; Wunderlich, Christoph H; Kreutz, Christoph; Konrat, Robert

    2014-01-01

    Protein–protein interactions are of utmost importance to an understanding of biological phenomena since non-covalent and therefore reversible couplings between basic proteins leads to the formation of complex regulatory and adaptive molecular systems. Such systems are capable of maintaining their integrity and respond to external stimuli, processes intimately related to living organisms. These interactions, however, span a wide range of dissociation constants, from sub-nanomolar affinities in tight complexes to high-micromolar or even millimolar affinities in weak, transiently formed protein complexes. Herein, we demonstrate how novel NMR and EPR techniques can be used for the characterization of weak protein–protein (ligand) complexes. Applications to intrinsically disordered proteins and transiently formed protein complexes illustrate the potential of these novel techniques to study hitherto unobserved (and unobservable) higher-order structures of proteins. PMID:25050230

  11. Interplay between chaperones and protein disorder promotes the evolution of protein networks.

    PubMed

    Pechmann, Sebastian; Frydman, Judith

    2014-06-01

    Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular

  12. Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks

    PubMed Central

    Pechmann, Sebastian; Frydman, Judith

    2014-01-01

    Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular

  13. Spatial Regulation of the Spindle Assembly Checkpoint and Anaphase Promoting Complex in Aspergillus nidulans

    PubMed Central

    Edgerton, Heather; Paolillo, Vitoria; Oakley, Berl R.

    2014-01-01

    Summary The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of γ-tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time-lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co-localize at the spindle pole body. SAC activity is, thus, spatially regulated in A. nidulans. Likewise, Cdc20, an activator of the anaphase promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the γ-tubulin mutation mipAD159 causes a nuclear-specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2. PMID:25417844

  14. Translating complex science into life-course health promoting strategies.

    PubMed

    Buttriss, Judith L

    2011-02-01

    These days, we are bombarded with nutrition information from diverse sources and of varying quality. There has been a dramatic increase in communication channels, including more television channels with airtime to fill, and the emergence of the Internet and 'new media' such as social networking sites. Part of this culture is to deliver ever changing and novel angles. The background 'noise' that this creates can make delivery of evidence-based advice about healthy eating that generally carries less novelty value, a huge challenge. This paper illustrates ways in which complex scientific information can be translated into meaningful health promoting strategies that can be applied across the life course. The examples used are nutrition in the context of healthy ageing, communicating the concept of energy density in the context of satiety, healthy hydration, health effects of probiotics and resources for use by teachers in the classroom. This selection of examples demonstrates the processes adopted at the British Nutrition Foundation to identify the evidence base for a particular topic and then to communicate this information to various target audiences. The British Nutrition Foundation's approach typically starts with preparation of a detailed review of the evidence, often with the involvement of external expertise, followed by peer review. For much of this work conventional science communication routes are used, but use is also made of the Internet and various forms of new media. PMID:21208499

  15. Translocation and activation of protein kinase C by the plasma cell tumor-promoting alkane pristane.

    PubMed

    Janz, S; Gawrisch, K; Lester, D S

    1995-02-01

    Pristane (2,6,10,14-tetramethylpentadecane) is a C19-isoalkane that promotes the development of plasmacytomas in genetically susceptible BALB/c mice. Similarities between the effects of pristane and protein kinase C (PKC)-activating phorbol esters suggested that the tumor promoting activity of pristane might involve the activation of PKC. Here we show that up to 5 mol% of pristane can be homogeneously incorporated into phosphatidylcholine/phosphatidylserine bilayers. Membrane-incorporated pristane partially activated PKC and increased phorbol ester binding to the bilayer by more than 50%. Pristane (50 microM) delivered as an inclusion complex with beta-cyclodextrin to promyelocytic HL-60 leukemia cells induced a partial long-term translocation of PKC to the cell membrane. This was accompanied by differentiation of HL-60 cells into macrophage-like cells. It is concluded that activation of PKC may comprise an important aspect of the tumor promoting potential of pristane. PMID:7834620

  16. An overview of the structures of protein-DNA complexes

    PubMed Central

    Luscombe, Nicholas M; Austin, Susan E; Berman , Helen M; Thornton, Janet M

    2000-01-01

    On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes. PMID:11104519

  17. Histone H2B Ubiquitination Promotes the Function of the Anaphase-Promoting Complex/Cyclosome in Schizosaccharomyces pombe

    PubMed Central

    Elmore, Zachary C.; Beckley, Janel R.; Chen, Jun-Song; Gould, Kathleen L.

    2014-01-01

    Ubiquitination and deubiquitination of proteins are reciprocal events involved in many cellular processes, including the cell cycle. During mitosis, the metaphase to anaphase transition is regulated by the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C). Although the E3 ubiquitin ligase function of the APC/C has been well characterized, it is not clear whether deubiquitinating enzymes (DUBs) play a role in reversing APC/C substrate ubiquitination. Here we performed a genetic screen to determine what DUB, if any, antagonizes the function of the APC/C in the fission yeast Schizosaccharomyces pombe. We found that deletion of ubp8, encoding the Spt-Ada-Gcn5-Acetyl transferase (SAGA) complex associated DUB, suppressed temperature-sensitive phenotypes of APC/C mutants cut9-665, lid1-6, cut4-533, and slp1-362. Our analysis revealed that Ubp8 antagonizes APC/C function in a mechanism independent of the spindle assembly checkpoint and proteasome activity. Notably, suppression of APC/C mutants was linked to loss of Ubp8 catalytic activity and required histone H2B ubiquitination. On the basis of these data, we conclude that Ubp8 antagonizes APC/C function indirectly by modulating H2B ubiquitination status. PMID:24948786

  18. Structure of an APC3-APC16 complex: Insights into assembly of the Anaphase Promoting Complex/Cyclosome

    PubMed Central

    Yamaguchi, Masaya; Yu, Shanshan; Qiao, Renping; Weissmann, Florian; Miller, Darcie J.; VanderLinden, Ryan; Brown, Nicholas G.; Frye, Jeremiah J.; Peters, Jan-Michael; Schulman, Brenda A.

    2015-01-01

    The Anaphase Promoting Complex/Cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-twofold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3’s TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7, and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3’s loop and IR-tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer, and together with biochemistry and prior data explain how APC16 recruits APC7 to APC3, show how APC3’s C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR-tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C. PMID:25490258

  19. The enamel protein amelotin is a promoter of hydroxyapatite mineralization.

    PubMed

    Abbarin, Nastaran; San Miguel, Symone; Holcroft, James; Iwasaki, Kengo; Ganss, Bernhard

    2015-05-01

    Amelotin (AMTN) is a recently discovered protein that is specifically expressed during the maturation stage of dental enamel formation. It is localized at the interface between the enamel surface and the apical surface of ameloblasts. AMTN knock-out mice have hypomineralized enamel, whereas transgenic mice overexpressing AMTN have a compact but disorganized enamel hydroxyapatite (HA) microstructure, indicating a possible involvement of AMTN in regulating HA mineralization directly. In this study, we demonstrated that recombinant human (rh) AMTN dissolved in a metastable buffer system, based on light scattering measurements, promotes HA precipitation. The mineral precipitates were characterized by scanning and transmission electron microscopy and electron diffraction. Colloidal gold immunolabeling of AMTN in the mineral deposits showed that protein molecules were associated with HA crystals. The binding affinity of rh-AMTN to HA was found to be comparable to that of amelogenin, the major protein of the forming enamel matrix. Overexpression of AMTN in mouse calvaria cells also increased the formation of calcium deposits in the culture medium. Overexpression of AMTN during the secretory stage of enamel formation in vivo resulted in rapid and uncontrolled enamel mineralization. Site-specific mutagenesis of the potential serine phosphorylation motif SSEEL reduced the in vitro mineral precipitation to less than 25%, revealing that this motif is important for the HA mineralizing function of the protein. A synthetic short peptide containing the SSEEL motif was only able to facilitate mineralization in its phosphorylated form ((P)S(P) SEEL), indicating that this motif is necessary but not sufficient for the mineralizing properties of AMTN. These findings demonstrate that AMTN has a direct influence on biomineralization by promoting HA mineralization and suggest a critical role for AMTN in the formation of the compact aprismatic enamel surface layer during the maturation

  20. Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans.

    PubMed

    McClendon, T Brooke; Sullivan, Meghan R; Bernstein, Kara A; Yanowitz, Judith L

    2016-05-01

    Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1 Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1 We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability. PMID:26936927

  1. Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

    PubMed Central

    Rehman, Asma; Archbold, Julia K.; Hu, Shu-Hong; Norwood, Suzanne J.; Collins, Brett M.; Martin, Jennifer L.

    2014-01-01

    Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins. PMID:25485130

  2. Multi-protein delivery by nanodiamonds promotes bone formation.

    PubMed

    Moore, L; Gatica, M; Kim, H; Osawa, E; Ho, D

    2013-11-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE(®) for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation. PMID:24045646

  3. Tumor promotion by caspase-resistant retinoblastoma protein

    PubMed Central

    Borges, Helena L.; Bird, Jeff; Wasson, Katherine; Cardiff, Robert D.; Varki, Nissi; Eckmann, Lars; Wang, Jean Y. J.

    2005-01-01

    The retinoblastoma (RB) protein regulates cell proliferation and cell death. RB is cleaved by caspase during apoptosis. A mutation of the caspase-cleavage site in the RB C terminus has been made in the mouse Rb-1 locus; the resulting Rb-MI mice are resistant to endotoxin-induced apoptosis in the intestine. The Rb-MI mice do not exhibit increased tumor incidence, because the MI mutation does not disrupt the Rb tumor suppressor function. In this study, we show that Rb-MI can promote the formation of colonic adenomas in the p53-null genetic background. Consistent with this tumor phenotype, Rb-MI reduces colorectal epithelial apoptosis and ulceration caused by dextran sulfate sodium. By contrast, Rb-MI does not affect the lymphoma phenotype of p53-null mice, in keeping with its inability to protect thymocytes and splenocytes from apoptosis. The Rb-MI protein is expressed and phosphorylated in the tumors, thereby inactivating its growth suppression function. These results suggest that RB tumor suppressor function, i.e., inhibition of proliferation, is inactivated by phosphorylation, whereas RB tumor promoting function, i.e., inhibition of apoptosis, is inactivated by caspase cleavage. PMID:16227443

  4. Multi-protein Delivery by Nanodiamonds Promotes Bone Formation

    PubMed Central

    Moore, L.; Gatica, M.; Kim, H.; Osawa, E.; Ho, D.

    2013-01-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE® for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation. PMID:24045646

  5. The multifaceted roles of intrinsic disorder in protein complexes.

    PubMed

    Uversky, Vladimir N

    2015-09-14

    Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) are important constituents of many protein complexes, playing various structural, functional, and regulatory roles. In such disorder-based protein complexes, functional disorder is used both internally (for assembly, movement, and functional regulation of the different parts of a given complex) and externally (for interactions of a complex with its external regulators). In complex assembly, IDPs/IDPRs serve as the molecular glue that cements complexes or as highly flexible scaffolds. Disorder defines the order of complex assembly and the ability of a protein to be involved in polyvalent interactions. It is at the heart of various binding mechanisms and interaction modes ascribed to IDPs. Disorder in protein complexes is related to multifarious applications of induced folding and induced functional unfolding, or defines the entropic chain activities, such as stochastic machines and binding rheostats. This review opens a FEBS Letters Special Issue on Dynamics, Flexibility, and Intrinsic Disorder in protein assemblies and represents a brief overview of intricate roles played by IDPs and IDPRs in various aspects of protein complexes. PMID:26073257

  6. Chlorophyll-Protein Complexes of the Cyanophyte, Nostoc sp. 1

    PubMed Central

    Rusckowski, Mary; Zilinskas, Barbara A.

    1980-01-01

    Four chlorophyll-protein complexes have been resolved from the cyanophyte, Nostoc sp., by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis at 4 C. Complexes solubilized by SDS from Spinacia oleracea were run for comparison. As has been well documented, the P700-chlorophyll a-protein complex from the higher plant and blue-green algal samples are similar, and the light-harvesting pigment protein complex is present only in the former. Most noteworthy are two closely migrating chlorophyll proteins in Nostoc sp. which have approximately the same mobility as a single chlorophyll-protein band resolvable from spinach. The absorption maximum of the complex from spinach is at 667 nanometers, and those of the two complexes from Nostoc sp. are at 667 and 669 nanometers; the fluorescence emission maximum at −196 C is at 685 nanometers, and the 735 nanometer fluorescence peak, characteristic of the P700-chlorophyll a-protein complex, is absent. The apoproteins of these new complexes from Nostoc sp. and spinach are in the kilodalton range. It appears that at least one of these two chlorophyll-protein complexes from Nostoc sp. compares with those recently described by others from higher plants and green algae as likely photosystem II complexes, perhaps containing P680, although no photochemical data are yet available. Images PMID:16661198

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

    PubMed Central

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

    2013-01-01

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

  8. Advances in protein complex analysis using mass spectrometry.

    PubMed

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

    2005-02-15

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

  9. Protein-protein binding affinities by pulse proteolysis: application to TEM-1/BLIP protein complexes.

    PubMed

    Hanes, Melinda S; Ratcliff, Kathleen; Marqusee, Susan; Handel, Tracy M

    2010-10-01

    Efficient methods for quantifying dissociation constants have become increasingly important for high-throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein-ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein-protein complex involving the β-lactamase TEM-1 and various β-lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C(m), of TEM-1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein-protein complexes. From a small set (n = 4) of TEM-1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC(m) was observed. From this "calibration curve," accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis-derived ΔC(m) values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high-throughput mutagenesis binding studies. PMID:20669180

  10. p31comet promotes disassembly of the mitotic checkpoint complex in an ATP-dependent process

    PubMed Central

    Teichner, Adar; Eytan, Esther; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Dumin, Elena; Gromis, Jonathan; Hershko, Avram

    2011-01-01

    Accurate segregation of chromosomes in mitosis is ensured by a surveillance mechanism called the mitotic (or spindle assembly) checkpoint. It prevents sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle through their kinetochores. The checkpoint acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. The activity of APC/C is inhibited by a mitotic checkpoint complex (MCC), composed of the APC/C activator Cdc20 bound to the checkpoint proteins MAD2, BubR1, and Bub3. When all kinetochores acquire bipolar attachment the checkpoint is inactivated, but the mechanisms of checkpoint inactivation are not understood. We have previously observed that hydrolyzable ATP is required for exit from checkpoint-arrested state. In this investigation we examined the possibility that ATP hydrolysis in exit from checkpoint is linked to the action of the Mad2-binding protein p31comet in this process. It is known that p31comet prevents the formation of a Mad2 dimer that it thought to be important for turning on the mitotic checkpoint. This explains how p31comet blocks the activation of the checkpoint but not how it promotes its inactivation. Using extracts from checkpoint-arrested cells and MCC isolated from such extracts, we now show that p31comet causes the disassembly of MCC and that this process requires β,γ-hydrolyzable ATP. Although p31comet binds to Mad2, it promotes the dissociation of Cdc20 from BubR1 in MCC. PMID:21300909

  11. Antifreeze Proteins in Winter Rye Leaves Form Oligomeric Complexes1

    PubMed Central

    Yu, Xiao-Ming; Griffith, Marilyn

    1999-01-01

    Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides. PMID:10198095

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

    PubMed

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

    2006-03-30

    Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology. PMID:16554755

  13. Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry*

    PubMed Central

    Kaake, Robyn M.; Wang, Xiaorong; Huang, Lan

    2010-01-01

    Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand how protein complexes work together in cells to maintain cell viability and normal homeostasis. Affinity purification coupled with quantitative mass spectrometry has become the primary method for studying in vivo protein interactions of protein complexes and whole organism proteomes. Recent developments in sample preparation and affinity purification strategies allow the capture, identification, and quantification of protein interactions of protein complexes that are stable, dynamic, transient, and/or weak. Current efforts have mainly focused on generating reliable, reproducible, and high confidence protein interaction data sets for functional characterization. The availability of increasing amounts of information on protein interactions in eukaryotic systems and new bioinformatics tools allow functional analysis of quantitative protein interaction data to unravel the biological significance of the identified protein interactions. Existing studies in this area have laid a solid foundation toward generating a complete map of in vivo protein interaction networks of protein complexes in cells or tissues. PMID:20445003

  14. Design and characterization of complex protein films

    NASA Astrophysics Data System (ADS)

    Bui, Holt P.

    Once a biomaterial is implanted into biological system, a layer of protein is immediately deposited on the surface of that material. The newly formed protein film will dictate how the implanted material will interact with the surrounding biological environment and lead to either the acceptance or rejection of the biomaterial. One method to enhance performance involves the activation the surface of the biomaterial with one or more proteins to direct specific interactions with the host environment. The focus of my dissertation was to develop and characterize model biomaterials surfaces that are activated with one or more proteins to help understand how the protein films may affect biological processes and a biomaterial's performance. One model system consisted of a patterned film of two proteins on a gold surface. Characterization of this protein pattern indicated that patterning protein films with a focused ion beam produced protein patterns with high biological contrast and high spatial control. The second model protein film involved the adsorption of fibronectin on surfaces with different surface energies. The characterization of the adsorbed fibronectin films suggest that fibronectin adsorbed on a hydrophilic surface is in an orientation that projects hydrophilic amino acid residues towards surface of the protein and dehydration causes reorientation to project hydrophobic amino acids towards the surface. In contrast, fibronectin is adsorbed onto a hydrophobic surface in a manner that resulted in dehydration and denaturation during the adsorption process. The last model protein film studied in this work consisted of fibronectin patterned in a manner so that the film consisted of spatially controlled domains of fibronectin adsorbed onto a hydrophilic surface as well as a hydrophobic surface. Lateral characterization of this pattern demonstrated a difference in secondary structure of fibronectin adsorbed on the two domains with varying surface energies.

  15. Protein promoting vibrations: Subpicosecond enzyme dynamics and catalysis

    NASA Astrophysics Data System (ADS)

    Mincer, Joshua S.

    The coupling of protein dynamics to enzymatic reactions is explored, specifically with reference to subpicosecond quasioscillatory motions, protein promoting vibrations (PPVS), that dynamically modulate the reaction potential energy surface on the same timescale as the reaction barrier passage itself. Computational algorithms to study these motions are presented. The first identifies a PPV in a specific enzymatic reaction, if it exists. The second identifies protein residues, the motions of which drive the PPV. Application to the enzyme horse liver alcohol dehydrogenase (HLADH) explains experimental mutagenesis studies and makes predictions for the many residues as yet not studied. PPV contribution to rate enhancement in HLADH is considered in light of these studies. The reaction rate theory developed by Antoniou and Schwartz is generalized to include an electron coordinate, allowing for the modeling of enzymes that catalyze proton-coupled electron transfer (PCET) reactions. Model system calculations yield kinetic parameters and primary HID kinetic isotope effects (KIEs) in qualitative agreement with experiment and allow for insights into KIE temperature dependence as well as KIE masking when these reactions are coupled to a PPV. Finally, PPV coupling to bond cleavage reactions in general is investigated in the specific context of PPV coupling to reaction center electron density in the enzyme purine nucleoside phosphorylase.

  16. Dopamine signaling promotes the xenobiotic stress response and protein homeostasis.

    PubMed

    Joshi, Kishore K; Matlack, Tarmie L; Rongo, Christopher

    2016-09-01

    Multicellular organisms encounter environmental conditions that adversely affect protein homeostasis (proteostasis), including extreme temperatures, toxins, and pathogens. It is unclear how they use sensory signaling to detect adverse conditions and then activate stress response pathways so as to offset potential damage. Here, we show that dopaminergic mechanosensory neurons in C. elegans release the neurohormone dopamine to promote proteostasis in epithelia. Signaling through the DA receptor DOP-1 activates the expression of xenobiotic stress response genes involved in pathogenic resistance and toxin removal, and these genes are required for the removal of unstable proteins in epithelia. Exposure to a bacterial pathogen (Pseudomonas aeruginosa) results in elevated removal of unstable proteins in epithelia, and this enhancement requires DA signaling. In the absence of DA signaling, nematodes show increased sensitivity to pathogenic bacteria and heat-shock stress. Our results suggest that dopaminergic sensory neurons, in addition to slowing down locomotion upon sensing a potential bacterial feeding source, also signal to frontline epithelia to activate the xenobiotic stress response so as to maintain proteostasis and prepare for possible infection. PMID:27261197

  17. Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL.

    PubMed

    Money, T; Jones, T; Dixon, R; Austin, S

    1999-08-01

    In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation. PMID:10419940

  18. Multi-LZerD: Multiple protein docking for asymmetric complexes

    PubMed Central

    Esquivel-Rodríguez, Juan; Yang, Yifeng David; Kihara, Daisuke

    2012-01-01

    The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Here, we have developed a novel computational multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on eleven hetero-multimeric complexes resulted in near native conformations for all but one of them (a root mean square deviation smaller than 2.5Å). We also show that our method copes with unbound docking cases well, outperforming the methodology that can be directly compared to our approach. Multi-LZerD was able to predict near native structures for multimeric complexes of various topologies. PMID:22488467

  19. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. PMID:26659058

  20. LINC complex proteins in cardiac structure, function, and disease

    PubMed Central

    Stroud, Matthew J; Banerjee, Indroneal; Lowe, Jennifer; Chen, Ju

    2014-01-01

    The LINC (LInker of Nucleoskeleton and Cytoskeleton) complex, composed of proteins within the inner and the outer nuclear membranes, connects the nuclear lamina to the cytoskeleton. The importance of this complex has been highlighted by the discovery of mutations in genes encoding LINC complex proteins, which are causative for skeletal or cardiac myopathies. Herein, this review summarizes structure, function, and interactions of major components of the LINC complex, highlights how mutations in these proteins may lead to cardiac disease, and outlines future challenges in the field. PMID:24481844

  1. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination.

    PubMed

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-03-18

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  2. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    PubMed Central

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-01-01

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

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

    PubMed

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

    2016-02-01

    The assembly of proteins into high-order complexes is a general mechanism for these biomolecules to implement their versatile functions in cells. Natural evolution has developed various assembling pathways for specific protein complexes to maintain their stability and proper activities. Previous studies have provided numerous examples of the misassembly of protein complexes leading to severe biological consequences. Although the research focusing on protein complexes has started to move beyond the static representation of quaternary structures to the dynamic aspect of their assembly, the current understanding of the assembly mechanism of protein complexes is still largely limited. To tackle this problem, we developed a new multiscale modeling framework. This framework combines a lower-resolution rigid-body-based simulation with a higher-resolution Cα-based simulation method so that protein complexes can be assembled with both structural details and computational efficiency. We applied this model to a homotrimer and a heterotetramer as simple test systems. Consistent with experimental observations, our simulations indicated very different kinetics between protein oligomerization and dimerization. The formation of protein oligomers is a multistep process that is much slower than dimerization but thermodynamically more stable. Moreover, we showed that even the same protein quaternary structure can have very diverse assembly pathways under different binding constants between subunits, which is important for regulating the functions of protein complexes. Finally, we revealed that the binding between subunits in a complex can be synergistically strengthened during assembly without considering allosteric regulation or conformational changes. Therefore, our model provides a useful tool to understand the general principles of protein complex assembly. PMID:26738810

  4. Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint

    PubMed Central

    Braunstein, Ilana; Miniowitz, Shirly; Moshe, Yakir; Hershko, Avram

    2007-01-01

    The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. It affects the activity of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets inhibitors of anaphase initiation for degradation. The mechanisms by which this system regulates APC/C remain obscure. Some models propose that the system promotes sequestration of the APC/C activator Cdc20 by binding to the checkpoint proteins Mad2 and BubR1. A different model suggests that a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Cdc20, and Mad2 inhibits APC/C in mitotic checkpoint [Sudakin V, Chan GKT, Yen TJ (2001) J Cell Biol 154:925–936]. We examined this problem by using extracts from nocodazole-arrested cells that reproduce some downstream events of the mitotic checkpoint system, such as lag kinetics of the degradation of APC/C substrate. Incubation of extracts with adenosine-5′-(γ-thio)triphosphate (ATP[γS]) stabilized the checkpoint-arrested state, apparently by stable thiophosphorylation of some proteins. By immunoprecipitation of APC/C from stably checkpoint-arrested extracts, followed by elution with increased salt concentration, we isolated inhibitory factors associated with APC/C. A part of the inhibitory material consists of Cdc20 associated with BubR1 and Mad2, and is thus similar to MCC. Contrary to the original MCC hypothesis, we find that MCC disassembles upon exit from the mitotic checkpoint. Thus, the requirement of the mitotic checkpoint system for the binding of Mad2 and BubR1 to Cdc20 may be for the assembly of the inhibitory complex rather than for Cdc20 sequestration. PMID:17360335

  5. SnapShot: SMC Protein Complexes Part II.

    PubMed

    Haering, Christian H; Gruber, Stephan

    2016-02-11

    This second of two SnapShots on SMC proteins depicts their roles at different stages of the eukaryotic cell cycle. The composition and architecture of SMC protein complexes and their regulators appear in SMC Protein Complexes Part I (available at http://www.cell.com/cell/pdf/S0092-8674%2815%2901690-6.pdf). To view this SnapShot, open or download the PDF. PMID:26871638

  6. Role of Ingested Amino Acids and Protein in the Promotion of Resistance Exercise-Induced Muscle Protein Anabolism.

    PubMed

    Reidy, Paul T; Rasmussen, Blake B

    2016-02-01

    The goal of this critical review is to comprehensively assess the evidence for the molecular, physiologic, and phenotypic skeletal muscle responses to resistance exercise (RE) combined with the nutritional intervention of protein and/or amino acid (AA) ingestion in young adults. We gathered the literature regarding the translational response in human skeletal muscle to acute exposure to RE and protein/AA supplements and the literature describing the phenotypic skeletal muscle adaptation to RE and nutritional interventions. Supplementation of protein/AAs with RE exhibited clear protein dose-dependent effects on translational regulation (protein synthesis) through mammalian target of rapamycin complex 1 (mTORC1) signaling, which was most apparent through increases in p70 ribosomal protein S6 kinase 1 (S6K1) phosphorylation, compared with postexercise recovery in the fasted or carbohydrate-fed state. These acute findings were critically tested via long-term exposure to RE training (RET) and protein/AA supplementation, and it was determined that a diminishing protein/AA supplement effect occurs over a prolonged exposure stimulus after exercise training. Furthermore, we found that protein/AA supplements, combined with RET, produced a positive, albeit minor, effect on the promotion of lean mass growth (when assessed in >20 participants/treatment); a negligible effect on muscle mass; and a negligible to no additional effect on strength. A potential concern we discovered was that the majority of the exercise training studies were underpowered in their ability to discern effects of protein/AA supplementation. Regardless, even when using optimal methodology and large sample sizes, it is clear that the effect size for protein/AA supplementation is low and likely limited to a subset of individuals because the individual variability is high. With regard to nutritional intakes, total protein intake per day, rather than protein timing or quality, appears to be more of a factor on

  7. A novel DNA replication origin identified in the human heat shock protein 70 gene promoter.

    PubMed Central

    Taira, T; Iguchi-Ariga, S M; Ariga, H

    1994-01-01

    A general and sensitive method for the mapping of initiation sites of DNA replication in vivo, developed by Vassilev and Johnson, has revealed replication origins in the region of simian virus 40 ori, in the regions upstream from the human c-myc gene and downstream from the Chinese hamster dihydrofolate reductase gene, and in the enhancer region of the mouse immunoglobulin heavy-chain gene. Here we report that the region containing the promoter of the human heat shock protein 70 (hsp70) gene was identified as a DNA replication origin in HeLa cells by this method. Several segments of the region were cloned into pUC19 and examined for autonomously replicating sequence (ARS) activity. The plasmids carrying the segments replicated episomally and semiconservatively when transfected into HeLa cells. The segments of ARS activity contained the sequences previously identified as binding sequences for a c-myc protein complex (T. Taira, Y. Negishi, F. Kihara, S. M. M. Iguchi-Ariga, and H. Ariga, Biochem. Biophys. Acta 1130:166-174, 1992). Mutations introduced within the c-myc protein complex binding sequences abolished the ARS activity. Moreover, the ARS plasmids stably replicated at episomal state for a long time in established cell lines. The results suggest that the promoter region of the human hsp70 gene plays a role in DNA replication as well as in transcription. Images PMID:8065368

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

    PubMed

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

    2013-01-01

    The identification of protein complexes plays a key role in understanding major cellular processes and biological functions. Various computational algorithms have been proposed to identify protein complexes from protein-protein interaction (PPI) networks. In this paper, we first introduce a new seed-selection strategy for seed-growth style algorithms. Cliques rather than individual vertices are employed as initial seeds. After that, a result-modification approach is proposed based on this seed-selection strategy. Predictions generated by higher order clique seeds are employed to modify results that are generated by lower order ones. The performance of this seed-selection strategy and the result-modification approach are tested by using the entropy-based algorithm, which is currently the best seed-growth style algorithm to detect protein complexes from PPI networks. In addition, we investigate four pairs of strategies for this algorithm in order to improve its accuracy. The numerical experiments are conducted on a Saccharomyces cerevisiae PPI network. The group of best predictions consists of 1711 clusters, with the average f-score at 0.68 after removing all similar and redundant clusters. We conclude that higher order clique seeds can generate predictions with higher accuracy and that our improved entropy-based algorithm outputs more reasonable predictions than the original one. PMID:23112006

  9. Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

    PubMed

    Kalveram, Birte; Ikegami, Tetsuro

    2013-04-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells. PMID:23325696

  10. Toscana Virus NSs Protein Promotes Degradation of Double-Stranded RNA-Dependent Protein Kinase

    PubMed Central

    Kalveram, Birte

    2013-01-01

    Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses—i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus—has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells. PMID:23325696

  11. Lytic Promoters Express Protein during Herpes Simplex Virus Latency

    PubMed Central

    Russell, Tiffany A.; Tscharke, David C.

    2016-01-01

    Herpes simplex virus (HSV) has provided the prototype for viral latency with previously well-defined acute or lytic and latent phases. More recently, the deep quiescence of HSV latency has been questioned with evidence that lytic genes can be transcribed in this state. However, to date the only evidence that these transcripts might be translated has come from immunological studies that show activated T cells persist in the nervous system during latency. Here we use a highly sensitive Cre-marking model to show that lytic and latent phases are less clearly defined in two significant ways. First, around half of the HSV spread leading to latently infected sites occurred beyond the initial acute infection and second, we show direct evidence that lytic promoters can drive protein expression during latency. PMID:27348812

  12. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Voigtländer, J.; Niedermeier, D.; Wex, H.; Stratmann, F.

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

  13. The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression.

    PubMed

    Sadasivam, Subhashini; Duan, Shenghua; DeCaprio, James A

    2012-03-01

    Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex-comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4-serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like protein p130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from p130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis. PMID:22391450

  14. Protein factors in Blastocladiella emersonii cell extracts recognize similar sequence elements in the promoters of the genes encoding cAMP-dependent protein kinase subunits.

    PubMed

    de Oliveira, J C; Marques, M V; Gomes, S L

    1997-08-01

    Blastocladiella emersonii contains a single cAMP-dependent protein kinase (PKA), which is similar to the mammalian type II isoforms. Its activity is regulated during development by changes in the levels of the catalytic (C) and regulatory (R) subunits, which occur in parallel with changes in levels of the corresponding mRNAs, suggesting coordinate transcriptional control of the expression of both subunits. Both R and C mRNA levels are low in vegetative cells, rise sharply during sporulation and decrease to basal levels again after germination. To investigate sequence elements common to both Blastocladiella R and C gene promoters, which might be involved in the coordinate regulation of these genes, their 5'-flanking regions were analyzed by gel mobility shift and DNase I footprinting assays. We determined that different DNA-protein complexes are generated when fragments of the R and C gene promoters are incubated with extracts from cells expressing (sporulating cells) or not expressing (vegetative cells) both subunits, and competition experiments suggested that similar protein factors bind to both promoters. DNase I footprinting experiments have indicated that a sequence common to both R and C promoters, and similar to mammalian E-boxes, binds factors present in extracts from vegetative and sporulating cells, whereas sequences flanking the E-boxes in both promoters showed a change in the pattern of DNase I digestion only when the vegetative cell extract was used. This result suggests that the composition of the protein complexes binding to these regions changes during sporulation. PMID:9294034

  15. Independent RNA polymerase II preinitiation complex dynamics and nucleosome turnover at promoter sites in vivo

    PubMed Central

    Grimaldi, Yoselin; Ferrari, Paolo; Strubin, Michel

    2014-01-01

    Transcription by all three eukaryotic RNA polymerases involves the assembly of a large preinitiation complex (PIC) at gene promoters. The PIC comprises several general transcription factors (GTFs), including TBP, and the respective RNA polymerase. It has been suggested that some GTFs remain stably bound at active promoters to facilitate multiple transcription events. Here we used two complementary approaches to show that, in G1-arrested yeast cells, TBP exchanges very rapidly even at the most highly active RNA Pol II promoters. A similar situation is observed at RNA Pol III promoters. In contrast, TBP remains stably bound at RNA Pol I promoters. We also provide evidence that, unexpectedly, PIC dynamics are neither the cause nor the consequence of nucleosome exchange at most of the RNA Pol II promoters we analyzed. These results point to a stable reinitiation complex at RNA Pol I promoters and suggest independent PIC and nucleosome turnover at many RNA Pol II promoters. PMID:24298073

  16. Generation of an Artificial Double Promoter for Protein Expression in Bacillus subtilis through a Promoter Trap System

    PubMed Central

    Yang, Mingming; Zhang, Weiwei; Ji, Shengyue; Cao, Pinghua; Chen, Yulin; Zhao, Xin

    2013-01-01

    Bacillus subtilis is an attractive host for production of recombinant proteins. Promoters and expression plasmid backbones have direct impacts on the efficiency of gene expression. To screen and isolate strong promoters, a promoter trap vector pShuttleF was developed in this study. Using the vector, approximately 1000 colonies containing likely promoters from Bacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09 exhibited the highest β-Gal activities in both Escherichia coli and B. subtilis. The activity of pShuttle-09 in B. subtilis was eight times of that of the P43 promoter, a commonly used strong promoter for B. subtilis. A sequence analysis showed that pShuttle-09 contained PluxS and truncated luxS in-frame fused with the reporter gene as well as another fragment upstream of PluxS containing a putative promoter. This putative promoter was a hybrid promoter and its β-Gal activity was higher than PluxS. Reconstructing the hybrid promoter from pShuttle-09 to PlapS further improved the β-Gal production by 60%. The usefulness of our promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. Thus, our data provide additional evidence to support the concept of using a promoter trap system to create new promoters. PMID:23409173

  17. Nucleoprotein complex formation by the enhancer binding protein nifA.

    PubMed

    Wang, X Y; Kolb, A; Cannon, W; Buck, M

    1997-09-01

    The nitrogen fixation protein NifA is a member of the protein family activating transcription by the alternative eubacterial sigmaN (sigma54) RNA polymerase holoenzyme. Binding sites for NifA, upstream activator sequences (UASs), are remotely located. Interaction between holoenzyme bound in a closed promoter complex and NiFA is facilitated by bending of the intervening DNA by integration host factor (IHF). We have examined NifA contact with the Klebsiella pneumoniae nifH promoter UAS in the presence and absence of holoenzyme and IHF. Footprints with UV light were made on 5-BrdU-substituted DNA and DNase I and laser UV footprints on conventional DNA templates. Results establish that the consensus thymidine residues of the UAS motif 5'-TGT are in close proximity to NifA. Reactivity suggests that each UAS thymidine is not structurally equivalent. Titration of NifA binding to the UAS in the presence or absence of the closed promoter complex indicates that the interaction of NifA with the UAS is not strongly co-operative with holoenzyme or IHF, a result supportive of an activation mechanism not reliant upon simple recruitment of factors to the promoter. Laser footprints demonstrated that holoenzyme suppressed reactivity of promoter consensus -14, -15 and -16 T residues, indicating close contact. Binding of holoenzyme resulted in a specific increase in 5-BrdU reactivity at -9 within the holoenzyme binding site, likely reflecting DNA distortion. Enhanced -9 reactivity required sigmaNN-terminal sequences that are necessary for activation. Since T-9 is melted in open complexes the closed complex appears poised for melting. Open promoter complex formation was accompanied by a distinct change in laser footprint signal at -11, consistent with the view that nucleation of strand separation occurs within or close to the -12 promoter element. PMID:9254707

  18. DNA binding and regulatory effects of transcription factors SP1 and USF at the rat amyloid precursor protein gene promoter.

    PubMed Central

    Hoffman, P W; Chernak, J M

    1995-01-01

    Two DNA elements which we have termed SAA and GAG have been shown to control expression of the rat amyloid precursor protein (APP) gene, and the region containing the SAA element has been shown to interact with nuclear proteins [Hoffman and Chernak (1994) Biochem. Biophys. Res. Commun. 201, 610-617]. In this report we study DNA sequences and proteins which influence the activity of the SAA element. An oligonucleotide containing the SAA element is specifically bound by nuclear proteins derived from rat PC12 cells, consistently forming four complexes designated C25, C30, C35 and C40 in electrophoretic mobility shift assays (EMSAs). We demonstrate that the C25, C30 and C40 complexes involve the binding of nuclear proteins to an SP1 consensus sequence located within the SAA element and that the C25 complex contains a protein antigenically related to the human SP1 protein. We establish further that the C35 complex requires a USF recognition site located within the SAA element and contains a protein antigenically related to the human upstream stimulatory factor (USF) protein. Using APP promoter/luciferase reporter gene constructs, we demonstrate that both the SP1 and the USF sites can play a role in the transcriptional activity of the SAA element. Finally, we show that complexes similar to the C25, C30 and C35 complexes are formed by rat cortex nuclear extracts and the SAA element in EMSA experiments, suggesting the relevance of our in vitro observations to the in vivo functioning of the rat APP promoter. Images PMID:7610052

  19. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    SciTech Connect

    Yonezawa, Takayuki; Lee, Ji-Won; Hibino, Ayaka; Asai, Midori; Hojo, Hironori; Cha, Byung-Yoon; Teruya, Toshiaki; Nagai, Kazuo; Chung, Ung-Il; Yagasaki, Kazumi; and others

    2011-06-03

    Highlights: {yields} Harmine promotes the activity and mRNA expression of ALP. {yields} Harmine enhances the expressions of osteocalcin mRNA and protein. {yields} Harmine induces osteoblastic mineralization. {yields} Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. {yields} BMP signaling pathways are involved in the actions of harmine. -- Abstract: Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a {beta}-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related {beta}-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of

  20. Capillary electrophoresis methods for the determination of covalent polyphenol-protein complexes.

    PubMed

    Trombley, John D; Loegel, Thomas N; Danielson, Neil D; Hagerman, Ann E

    2011-09-01

    The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine. PMID:21400190

  1. Recording information on protein complexes in an information management system

    PubMed Central

    Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

    2011-01-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  2. Mass Spectrometry of Protein Complexes: From Origins to Applications

    NASA Astrophysics Data System (ADS)

    Mehmood, Shahid; Allison, Timothy M.; Robinson, Carol V.

    2015-04-01

    Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase.

  3. Rap1 promotes multiple pancreatic islet cell functions and signals through mammalian target of rapamycin complex 1 to enhance proliferation.

    PubMed

    Kelly, Patrick; Bailey, Candice L; Fueger, Patrick T; Newgard, Christopher B; Casey, Patrick J; Kimple, Michelle E

    2010-05-21

    Recent studies have implicated Epac2, a guanine-nucleotide exchange factor for the Rap subfamily of monomeric G proteins, as an important regulator of insulin secretion from pancreatic beta-cells. Although the Epac proteins were originally identified as cAMP-responsive activators of Rap1 GTPases, the role of Rap1 in beta-cell biology has not yet been defined. In this study, we examined the direct effects of Rap1 signaling on beta-cell biology. Using the Ins-1 rat insulinoma line, we demonstrate that activated Rap1A, but not related monomeric G proteins, promotes ribosomal protein S6 phosphorylation. Using isolated rat islets, we show that this signaling event is rapamycin-sensitive, indicating that it is mediated by the mammalian target of rapamycin complex 1-p70 S6 kinase pathway, a known growth regulatory pathway. This newly defined beta-cell signaling pathway acts downstream of cAMP, in parallel with the stimulation of cAMP-dependent protein kinase, to drive ribosomal protein S6 phosphorylation. Activated Rap1A promotes glucose-stimulated insulin secretion, islet cell hypertrophy, and islet cell proliferation, the latter exclusively through mammalian target of rapamycin complex 1, suggesting that Rap1 is an important regulator of beta-cell function. This newly defined signaling pathway may yield unique targets for the treatment of beta-cell dysfunction in diabetes. PMID:20339002

  4. Tumor metastatic promoter ABCE1 interacts with the cytoskeleton protein actin and increases cell motility.

    PubMed

    Han, Xu; Tian, Ye; Tian, Dali

    2016-06-01

    ABCE1, a member of the ATP-binding cassette (ABC) family, is a candidate tumor metastatic promoter in lung cancer. Overexpression of ABCE1 is correlated with aggressive growth and metastasis in lung cancer cells. However, the exact mechanism remains unclear. In the present study, GST pull-down assay provided evidence of the possible interaction between ABCE1 and β-actin using GST-ABCE1 as a bait protein. Co-immunoprecipitation manifested ABCE1 formed complexes with β-actin in vivo. ABCE1 overexpression significantly increased the migration of lung cancer cells which may be attributed to the promotion of F-actin rearrangements. Taken together, these data suggest that overexpression of ABCE1 produces an obvious effect on the motility of lung cancer cells through cytoskeleton rearrangement. PMID:27109616

  5. Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo.

    PubMed

    Grdzelishvili, V Z; Chapman, S N; Dawson, W O; Lewandowski, D J

    2000-09-15

    The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3'-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS Lett. 173, 247-250) and 4828 (K. Lehto, G. L. Grantham, and W. O. Dawson (1990), Virology 174, 145-157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between -35 and +10 and -95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between -69 and +12, whereas the boundaries of the fully active promoter were between -157 and +54. Computer analysis predicted two stem-loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem-loop structure. Deletion analysis suggested that the upper part of this stem-loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role. PMID:11017798

  6. Parathyroid Hormone–Related Protein Promotes Epithelial–Mesenchymal Transition

    PubMed Central

    Ardura, Juan Antonio; Rayego-Mateos, Sandra; Rámila, David; Ruiz-Ortega, Marta

    2010-01-01

    Epithelial–mesenchymal transition (EMT) is an important process that contributes to renal fibrogenesis. TGF-β1 and EGF stimulate EMT. Recent studies suggested that parathyroid hormone–related protein (PTHrP) promotes fibrogenesis in the damaged kidney, apparently dependent on its interaction with vascular endothelial growth factor (VEGF), but whether it also interacts with TGF-β and EGF to modulate EMT is unknown. Here, PTHrP(1-36) increased TGF-β1 in cultured tubuloepithelial cells and TGF-β blockade inhibited PTHrP-induced EMT-related changes, including upregulation of α-smooth muscle actin and integrin-linked kinase, nuclear translocation of Snail, and downregulation of E-cadherin and zonula occludens-1. PTHrP(1-36) also induced EGF receptor (EGFR) activation; inhibition of protein kinase C and metalloproteases abrogated this activation. Inhibition of EGFR activation abolished these EMT-related changes, the activation of ERK1/2, and upregulation of TGF-β1 and VEGF by PTHrP(1-36). Moreover, inhibition of ERK1/2 blocked EMT induced by either PTHrP(1-36), TGF-β1, EGF, or VEGF. In vivo, obstruction of mouse kidneys led to changes consistent with EMT and upregulation of TGF-β1 mRNA, p-EGFR protein, and PTHrP. Taken together, these data suggest that PTHrP, TGF-β, EGF, and VEGF might cooperate through activation of ERK1/2 to induce EMT in renal tubuloepithelial cells. PMID:19959711

  7. Proteomic comparison of etioplast and chloroplast protein complexes.

    PubMed

    Plöscher, Matthias; Reisinger, Veronika; Eichacker, Lutz A

    2011-08-12

    Angiosperms grown in darkness develop etioplasts during skotomorphogenesis. It is well known that etioplasts accumulate large quantities of protochlorophyllideoxidoreductase, are devoid of chlorophyll and are the site to assemble the photosynthetic machinery during photomorphogenesis. Proteomic investigation of the membrane protein complexes by Native PAGE, in combination with CyDye labelling and mass spectrometric analysis revealed that etioplasts and chloroplasts share a number of membrane protein complexes characteristic for electron transport, chlorophyll and protein synthesis as well as fatty acid biosynthesis. The complex regulatory function in both developmental states is discussed. PMID:21440687

  8. UpSET recruits HDAC complexes and restricts chromatin accessibility and histone acetylation at promoter regions

    PubMed Central

    Rincon-Arano, Hector; Halow, Jessica; Delrow, Jeffrey J.; Parkhurst, Susan M.; Groudine, Mark

    2012-01-01

    Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here we describe upSET, a transcriptional regulator encoding a SET domain-containing protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to up-regulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin. PMID:23177352

  9. PROSTAGLANDIN E2 MODIFIES SMAD2 AND PROMOTES SMAD2-SMAD4 COMPLEX FORMATION

    PubMed Central

    Yang, Chen; Chen, Chen; Sorokin, Andrey

    2014-01-01

    We report that PGE2 promotes Smad2-Smad4 complex formation and this phenomenon could be blocked by DIDS, an anion transporter inhibitor. Our data suggest that PGE2 had no effects on Smad2 phosphorylation, suggesting that PGE2-mediated Smad2-Smad4 complex formation is independent of TGF-β signaling and that PGE2 induced Smad2 modification which is different from TGF-β-mediated phosphorylation. We demonstrate that in primary human glomerular mesangial cells PGE2 caused modification of Smad2 as detected by Smad2N antibody, raised against a peptide near the N-terminus of Smad2. We hypothesize that Smad2 protein is post-translationaly modified by PGE2. Direct evidence of Smad2 modification by PGE2 was achieved by avidin pulldown assay which showed that endogenous Smad2 and recombinant Smad2 protein were attached by biotin-labeled PGE2. Taken together, our results provided evidence that post-translational modification of Smad2 could be a mechanism for the action of PGE2 in the pathogenesis of human pathologies. PMID:24613014

  10. Intermediates in the assembly of mitotic checkpoint complexes and their role in the regulation of the anaphase-promoting complex

    PubMed Central

    Kaisari, Sharon; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Hershko, Avram

    2016-01-01

    The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system. PMID:26755599

  11. The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression

    PubMed Central

    Sadasivam, Subhashini; Duan, Shenghua; DeCaprio, James A.

    2012-01-01

    Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex—comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4—serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like protein p130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from p130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis. PMID:22391450

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

    PubMed Central

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

    2009-01-01

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

  13. An endophilin–dynamin complex promotes budding of clathrin-coated vesicles during synaptic vesicle recycling

    PubMed Central

    Sundborger, Anna; Soderblom, Cynthia; Vorontsova, Olga; Evergren, Emma; Hinshaw, Jenny E.; Shupliakov, Oleg

    2011-01-01

    Clathrin-mediated vesicle recycling in synapses is maintained by a unique set of endocytic proteins and interactions. We show that endophilin localizes in the vesicle pool at rest and in spirals at the necks of clathrin-coated pits (CCPs) during activity in lamprey synapses. Endophilin and dynamin colocalize at the base of the clathrin coat. Protein spirals composed of these proteins on lipid tubes in vitro have a pitch similar to the one observed at necks of CCPs in living synapses, and lipid tubules are thinner than those formed by dynamin alone. Tubulation efficiency and the amount of dynamin recruited to lipid tubes are dramatically increased in the presence of endophilin. Blocking the interactions of the endophilin SH3 domain in situ reduces dynamin accumulation at the neck and prevents the formation of elongated necks observed in the presence of GTPγS. Therefore, endophilin recruits dynamin to a restricted part of the CCP neck, forming a complex, which promotes budding of new synaptic vesicles. PMID:21172823

  14. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    SciTech Connect

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

  15. Protein structures in SDS micelle-protein complexes.

    PubMed Central

    Parker, W; Song, P S

    1992-01-01

    Sodium dodecyl sulfate (SDS) is used more often than any other detergent as an excellent denaturing or "unfolding" detergent. However, formation of ordered structure (alpha-helix or beta-sheet) in certain peptides is known to be induced by interaction with SDS micelles. The SDS-induced structures formed by these peptides are amphiphilic, having both a hydrophobic and a hydrophilic face. Previous work in this area has revealed that SDS induces helical folding in a wide variety of non-helical proteins. Here, we describe the interaction of several structurally unrelated proteins with SDS micelles and the correlation of these structures to helical amphiphilic regions present in the primary sequence. It is likely that the ability of native nonordered protein structures to form induced amphiphilic ordered structures is rather common. PMID:1600087

  16. Embracing proteins: structural themes in aptamer-protein complexes.

    PubMed

    Gelinas, Amy D; Davies, Douglas R; Janjic, Nebojsa

    2016-02-01

    Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer-protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer-protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity. PMID:26919170

  17. Prion protein promotes kidney iron uptake via its ferrireductase activity.

    PubMed

    Haldar, Swati; Tripathi, Ajai; Qian, Juan; Beserra, Amber; Suda, Srinivas; McElwee, Matthew; Turner, Jerrold; Hopfer, Ulrich; Singh, Neena

    2015-02-27

    Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrP(C)) from its normal conformation to an aggregated, PrP-scrapie (PrP(Sc)) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrP(C) in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrP(C) is lacking. Kidney provides a relevant model for this evaluation because PrP(C) is expressed in the kidney, and ∼370 μg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrP(C) promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of (59)Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP(-/-)) mouse kidney relative to PrP(+/+) controls. Selective in vivo radiolabeling of plasma NTBI with (59)Fe revealed similar results. Expression of exogenous PrP(C) in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of (59)Fe-NTBI and to a smaller extent (59)Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrP(Δ51-89)) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrP(C) to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrP(C) promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism. PMID:25572394

  18. Prion Protein Promotes Kidney Iron Uptake via Its Ferrireductase Activity*

    PubMed Central

    Haldar, Swati; Tripathi, Ajai; Qian, Juan; Beserra, Amber; Suda, Srinivas; McElwee, Matthew; Turner, Jerrold; Hopfer, Ulrich; Singh, Neena

    2015-01-01

    Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrPC) from its normal conformation to an aggregated, PrP-scrapie (PrPSc) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrPC in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrPC is lacking. Kidney provides a relevant model for this evaluation because PrPC is expressed in the kidney, and ∼370 μg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrPC promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of 59Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP−/−) mouse kidney relative to PrP+/+ controls. Selective in vivo radiolabeling of plasma NTBI with 59Fe revealed similar results. Expression of exogenous PrPC in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of 59Fe-NTBI and to a smaller extent 59Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrPΔ51–89) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrPC to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrPC promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism. PMID:25572394

  19. Factorial combinations of protein interactions generate a multiplicity of florigen activation complexes in wheat and barley.

    PubMed

    Li, Chengxia; Lin, Huiqiong; Dubcovsky, Jorge

    2015-10-01

    The FLOWERING LOCUS T (FT) protein is a central component of a mobile flowering signal (florigen) that is transported from leaves to the shoot apical meristem (SAM). Two FT monomers and two DNA-binding bZIP transcription factors interact with a dimeric 14-3-3 protein bridge to form a hexameric protein complex. This complex, designated as the 'florigen activation complex' (FAC), plays a critical role in flowering. The wheat homologue of FT, designated FT1 (= VRN3), activates expression of VRN1 in the leaves and the SAM, promoting flowering under inductive long days. In this study, we show that FT1, other FT-like proteins, and different FD-like proteins, can interact with multiple wheat and barley 14-3-3 proteins. We also identify the critical amino acid residues in FT1 and FD-like proteins required for their interactions, and demonstrate that 14-3-3 proteins are necessary bridges to mediate the FT1-TaFDL2 interaction. Using in vivo bimolecular fluorescent complementation (BiFC) assays, we demonstrate that the interaction between FT1 and 14-3-3 occurs in the cytoplasm, and that this complex is then translocated to the nucleus, where it interacts with TaFDL2 to form a FAC. We also demonstrate that a FAC including FT1, TaFDL2 and Ta14-3-3C can bind to the VRN1 promoter in vitro. Finally, we show that relative transcript levels of FD-like and 14-3-3 genes vary among tissues and developmental stages. Since FD-like proteins determine the DNA specificity of the FACs, variation in FD-like gene expression can result in spatial and temporal modulation of the effects of mobile FT-like signals. PMID:26252567

  20. PEGylated Albumin-Based Polyion Complex Micelles for Protein Delivery.

    PubMed

    Jiang, Yanyan; Lu, Hongxu; Chen, Fan; Callari, Manuela; Pourgholami, Mohammad; Morris, David L; Stenzel, Martina H

    2016-03-14

    An increasing amount of therapeutic agents are based on proteins. However, proteins as drug have intrinsic problems such as their low hydrolytic stability. Delivery of proteins using nanoparticles has increasingly been the focus of interest with polyion complex micelles, prepared from charged block copolymer and the oppositely charged protein, as an example of an attractive carrier for proteins. Inspired by this approach, a more biocompatible pathway has been developed here, which replaces the charged synthetic polymer with an abundant protein, such as albumin. Although bovine serum albumin (BSA) was observed to form complexes with positively charged proteins directly, the resulting protein nanoparticle were not stable and aggregated to large precipitates over the course of a day. Therefore, maleimide functionalized poly(oligo (ethylene glycol) methyl ether methacrylate) (MI-POEGMEMA) (Mn = 26000 g/mol) was synthesized to generate a polymer-albumin conjugate, which was able to condense positively charged proteins, here lysozyme (Lyz) as a model. The PEGylated albumin polyion complex micelle with lysozyme led to nanoparticles between 15 and 25 nm in size depending on the BSA to Lyz ratio. The activity of the encapsulated protein was tested using Sprouty 1 (C-12; Spry1) proteins, which can act as an endogenous angiogenesis inhibitor. Condensation of Spry1 with the PEGylated albumin could improve the anticancer efficacy of Spry1 against the breast cancer cells lowering the IC50 value of the protein. Furthermore, the high anticancer efficacy of the POEGMEMA-BSA/Spry1 complex micelle was verified by effectively inhibiting the growth of three-dimensional MCF-7 multicellular tumor spheroids. The PEGylated albumin complex micelle has great potential as a drug delivery vehicle for a new generation of cancer pharmaceuticals. PMID:26809948

  1. Coevolution Drives the Emergence of Complex Traits and Promotes Evolvability

    PubMed Central

    Zaman, Luis; Meyer, Justin R.; Devangam, Suhas; Bryson, David M.; Lenski, Richard E.; Ofria, Charles

    2014-01-01

    The evolution of complex organismal traits is obvious as a historical fact, but the underlying causes—including the role of natural selection—are contested. Gould argued that a random walk from a necessarily simple beginning would produce the appearance of increasing complexity over time. Others contend that selection, including coevolutionary arms races, can systematically push organisms toward more complex traits. Methodological challenges have largely precluded experimental tests of these hypotheses. Using the Avida platform for digital evolution, we show that coevolution of hosts and parasites greatly increases organismal complexity relative to that otherwise achieved. As parasites evolve to counter the rise of resistant hosts, parasite populations retain a genetic record of past coevolutionary states. As a consequence, hosts differentially escape by performing progressively more complex functions. We show that coevolution's unique feedback between host and parasite frequencies is a key process in the evolution of complexity. Strikingly, the hosts evolve genomes that are also more phenotypically evolvable, similar to the phenomenon of contingency loci observed in bacterial pathogens. Because coevolution is ubiquitous in nature, our results support a general model whereby antagonistic interactions and natural selection together favor both increased complexity and evolvability. PMID:25514332

  2. Profiles of embryonic nuclear protein binding to the proximal promoter region of the soybean β-conglycinin α subunit gene.

    PubMed

    Yoshino, M; Tsutsumi, K; Kanazawa, A

    2015-01-01

    β-Conglycinin, a major component of seed storage protein in soybean, comprises three subunits: α, α' and β. The expression of genes for these subunits is strictly controlled during embryogenesis. The proximal promoter region up to 245 bp upstream of the transcription start site of the α subunit gene sufficiently confers spatial and temporal control of transcription in embryos. Here, the binding profile of nuclear proteins in the proximal promoter region of the α subunit gene was analysed. DNase I footprinting analysis indicated binding of proteins to the RY element and DNA regions including box I, a region conserved in cognate gene promoters. An electrophoretic mobility shift assay (EMSA) using different portions of box I as a probe revealed that multiple portions of box I bind to nuclear proteins. In addition, an EMSA using nuclear proteins extracted from embryos at different developmental stages indicated that the levels of major DNA-protein complexes on box I increased during embryo maturation. These results are consistent with the notion that box I is important for the transcriptional control of seed storage protein genes. Furthermore, the present data suggest that nuclear proteins bind to novel motifs in box I including 5'-TCAATT-3' rather than to predicted cis-regulatory elements. PMID:24943483

  3. Promoting Task-Based Pragmatics Instruction in EFL Classroom Contexts: The Role of Task Complexity

    ERIC Educational Resources Information Center

    Kim, Youjin; Taguchi, Naoko

    2015-01-01

    Robinson's (2001) Cognition Hypothesis claims that more complex tasks promote interaction and language development. This study examined the effect of task complexity in the learning of request-making expressions. Task complexity was operationalized as [+/- reasoning] following Robinson's framework. The study employed a pretest-posttest research…

  4. Identification and characterization of a prostate-specific androgen-independent protein-binding site in the probasin promoter.

    PubMed Central

    Yeung, Lillian H Y; Read, Jason T; Sorenson, Pernille; Nelson, Colleen C; Jia, William; Rennie, Paul S

    2003-01-01

    In this study we investigated the combination of transcription factors and proteins binding to the proximal part of the prostate-specific probasin (PB) promoter. Using DNaseI in vitro footprinting, several protected regions were identified on the proximal PB promoter (nucleotides -286 to +28 relative to the transcription start site) when nuclear extracts from LNCaP, a human prostate cancer cell line, were used. Four of the protected areas were observed only when LNCaP nuclear extracts treated with synthetic androgen (10 nM R1881) were used. Two other regions, referred to as FPI and FPII, showed protection regardless of the presence or absence of androgen. When DNaseI footprinting was done using other prostate and non-prostate nuclear extracts, protection of the FPII region was only seen in prostate cell lines. These androgen-independent regions were further tested for tissue and binding specificity using the electrophoretic mobility-shift assay. Eight complexes formed with the FPI probe while four complexes were observed with the FPII probe on incubation with the tested nuclear extracts. Methylation protection assays reveal that prostate cancer cell lines yield slightly different protection patterns for some of the protein complexes formed with non-prostate-derived cell lines, suggesting the presence of prostate-enriched or -exclusive proteins. Site-directed mutagenesis of the protected nucleotides within FPII resulted in a significant reduction in expression from the PB promoter. Identification of proteins binding to the FPII region revealed the participation of nuclear factor I (NF-I) or a closely related protein, although other unknown proteins are also involved. Defining the DNA and protein components that dictate prostate-specific expression of the PB promoter in an androgen-independent manner would provide a strong basis for the design and development of a gene therapy for systemic treatment of androgen-independent prostate cancer. PMID:12540291

  5. Identification of Post-translational Modifications of Plant Protein Complexes

    PubMed Central

    Piquerez, Sophie J. M.; Balmuth, Alexi L.; Sklenář, Jan; Jones, Alexandra M.E.; Rathjen, John P.; Ntoukakis, Vardis

    2014-01-01

    Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein. PMID:24637539

  6. Protein Connectivity in Chemotaxis Receptor Complexes.

    PubMed

    Eismann, Stephan; Endres, Robert G

    2015-12-01

    The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET) measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures. PMID:26646441

  7. Protein Connectivity in Chemotaxis Receptor Complexes

    PubMed Central

    Eismann, Stephan; Endres, Robert G.

    2015-01-01

    The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET) measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures. PMID:26646441

  8. Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes.

    PubMed

    Chymkowitch, Pierre; Nguéa, Aurélie P; Aanes, Håvard; Koehler, Christian J; Thiede, Bernd; Lorenz, Susanne; Meza-Zepeda, Leonardo A; Klungland, Arne; Enserink, Jorrit M

    2015-06-01

    Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs. PMID:25800674

  9. Coupling protein complex analysis to peptide based proteomics.

    PubMed

    Gao, Qiang; Madian, Ashraf G; Liu, Xiuping; Adamec, Jiri; Regnier, Fred E

    2010-12-01

    Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes. PMID:21036361

  10. Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

    PubMed

    Yin, Baoru; Zhang, Rujing; Yao, Ping

    2015-01-01

    The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry. PMID:25803397

  11. Building a Hierarchical Organization of Protein Complexes Out of Protein Association Data

    PubMed Central

    Stojmirović, Aleksandar; Yu, Yi-Kuo

    2014-01-01

    Organizing experimentally determined protein associations as a hierarchy can be a good approach to elucidating the content of protein complexes and the modularity of subcomplexes. Several challenges exist. First, intrinsically sticky proteins, such as chaperones, are often falsely assigned to many functionally unrelated complexes. Second, the reported collections of proteins may not be true “complexes” in the sense that they bind together and perform a joint cellular function. Third, due to imperfect sensitivity of protein detection methods, both false positive and false negative assignments of a protein to complexes may occur. We mitigate the first issue by down-weighting sticky proteins by their occurrence frequencies. We approach the other two problems by merging nearly identical complexes and by constructing a directed acyclic graph (DAG) based on the relationship of partial inclusion. The constructed DAG, within which smaller complexes form parts of the larger, can reveal how different complexes are joined. By merging almost identical complexes one can deemphasize the influence of false positives, while allowing false negatives to be rescued by other nearly identical association data. We investigate several protein weighting schemes and compare their corresponding DAGs using yeast and human complexes. We find that the scheme incorporating weights based on information flow in the network of direct protein–protein interactions produces biologically most meaningful DAGs. In either yeast or human, isolated nodes form a large proportion of the final hierarchy. While most connected components encompass very few nodes, the largest one for each species contains a sizable portion of all nodes. By considering examples of subgraphs composed of nodes containing a specified protein, we illustrate that the graphs' topological features can correctly suggest the biological roles of protein complexes. The input data, final results and the source code are available at ftp

  12. Cross-linking Measurements of In Vivo Protein Complex Topologies*

    PubMed Central

    Zheng, Chunxiang; Yang, Li; Hoopmann, Michael R.; Eng, Jimmy K.; Tang, Xiaoting; Weisbrod, Chad R.; Bruce, James E.

    2011-01-01

    Identification and measurement of in vivo protein interactions pose critical challenges in the goal to understand biological systems. The measurement of structures and topologies of proteins and protein complexes as they exist in cells is particularly challenging, yet critically important to improve understanding of biological function because proteins exert their intended function only through the structures and interactions they exhibit in vivo. In the present study, protein interactions in E. coli cells were identified in our unbiased cross-linking approach, yielding the first in vivo topological data on many interactions and the largest set of identified in vivo cross-linked peptides produced to date. These data show excellent agreement with protein and complex crystal structures where available. Furthermore, our unbiased data provide novel in vivo topological information that can impact understanding of biological function, even for cases where high resolution structures are not yet available. PMID:21697552

  13. Graph theory and stability analysis of protein complex interaction networks.

    PubMed

    Huang, Chien-Hung; Chen, Teng-Hung; Ng, Ka-Lok

    2016-04-01

    Protein complexes play an essential role in many biological processes. Complexes can interact with other complexes to form protein complex interaction network (PCIN) that involves in important cellular processes. There are relatively few studies on examining the interaction topology among protein complexes; and little is known about the stability of PCIN under perturbations. We employed graph theoretical approach to reveal hidden properties and features of four species PCINs. Two main issues are addressed, (i) the global and local network topological properties, and (ii) the stability of the networks under 12 types of perturbations. According to the topological parameter classification, we identified some critical protein complexes and validated that the topological analysis approach could provide meaningful biological interpretations of the protein complex systems. Through the Kolmogorov-Smimov test, we showed that local topological parameters are good indicators to characterise the structure of PCINs. We further demonstrated the effectiveness of the current approach by performing the scalability and data normalization tests. To measure the robustness of PCINs, we proposed to consider eight topological-based perturbations, which are specifically applicable in scenarios of targeted, sustained attacks. We found that the degree-based, betweenness-based and brokering-coefficient-based perturbations have the largest effect on network stability. PMID:26997661

  14. Biochemical isolation of Argonaute protein complexes by Ago-APP

    PubMed Central

    Hauptmann, Judith; Schraivogel, Daniel; Bruckmann, Astrid; Manickavel, Sudhir; Jakob, Leonhard; Eichner, Norbert; Pfaff, Janina; Urban, Marc; Sprunck, Stefanie; Hafner, Markus; Tuschl, Thomas; Deutzmann, Rainer; Meister, Gunter

    2015-01-01

    During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells. PMID:26351695

  15. SnapShot: SMC Protein Complexes Part I.

    PubMed

    Haering, Christian H; Gruber, Stephan

    2016-01-14

    This first of two SnapShots on SMC proteins depicts the composition and architecture of SMC protein complexes and their regulators. Their roles at different stages of the cell cycle will appear in Part II. To view this SnapShot, open or download the PDF. PMID:26771499

  16. Linking structural features of protein complexes and biological function.

    PubMed

    Sowmya, Gopichandran; Breen, Edmond J; Ranganathan, Shoba

    2015-09-01

    Protein-protein interaction (PPI) establishes the central basis for complex cellular networks in a biological cell. Association of proteins with other proteins occurs at varying affinities, yet with a high degree of specificity. PPIs lead to diverse functionality such as catalysis, regulation, signaling, immunity, and inhibition, playing a crucial role in functional genomics. The molecular principle of such interactions is often elusive in nature. Therefore, a comprehensive analysis of known protein complexes from the Protein Data Bank (PDB) is essential for the characterization of structural interface features to determine structure-function relationship. Thus, we analyzed a nonredundant dataset of 278 heterodimer protein complexes, categorized into major functional classes, for distinguishing features. Interestingly, our analysis has identified five key features (interface area, interface polar residue abundance, hydrogen bonds, solvation free energy gain from interface formation, and binding energy) that are discriminatory among the functional classes using Kruskal-Wallis rank sum test. Significant correlations between these PPI interface features amongst functional categories are also documented. Salt bridges correlate with interface area in regulator-inhibitors (r = 0.75). These representative features have implications for the prediction of potential function of novel protein complexes. The results provide molecular insights for better understanding of PPIs and their relation to biological functions. PMID:26131659

  17. Phase transitions of multivalent proteins can promote clustering of membrane receptors

    PubMed Central

    Banjade, Sudeep; Rosen, Michael K

    2014-01-01

    Clustering of proteins into micrometer-sized structures at membranes is observed in many signaling pathways. Most models of clustering are specific to particular systems, and relationships between physical properties of the clusters and their molecular components are not well understood. We report biochemical reconstitution on supported lipid bilayers of protein clusters containing the adhesion receptor Nephrin and its cytoplasmic partners, Nck and N-WASP. With Nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Dynamics and thermodynamics of the clusters are modulated by the valencies and affinities of the interacting species. In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes. Interactions between multivalent proteins could be a general mechanism for cytoplasmic adaptor proteins to organize membrane receptors into micrometer-scale signaling zones. DOI: http://dx.doi.org/10.7554/eLife.04123.001 PMID:25321392

  18. Heat shock protein 27 promotes cell proliferation through activator protein-1 in lung cancer

    PubMed Central

    ZHANG, SAI; HU, YANGMIN; HUANG, YUWEN; XU, HUIMIN; WU, GONGXIONG; DAI, HAIBIN

    2015-01-01

    Heat shock protein 27 (HSP27) is an important regulator involved in the development of lung cancer. However, limited evidence exists concerning the underlying molecular mechanisms of its action. The results of the present study revealed that HSP27 was highly expressed in the lung cancer tissues of mice. In an in vitro model, the overexpression of HSP27 promoted cell proliferation, while HSP27 knockdown inhibited cell proliferation. HSP27 promoted cell proliferation in vitro by directly upregulating the expression of HSP27 target genes, which required the activation of the activator protein-1 (AP-1) signaling pathway. This was evaluated by the phosphorylation status of an important pathway component, c-Jun in lung cancer tissue and cells. These results suggested that HSP27 has a promotional role in lung cancer, and therefore indicated a novel mechanism involving lung cancer cell proliferation, which may underlie poor responses to therapy. Therefore, HSP27 may be a suitable therapeutic target for the treatment of lung cancer. PMID:26137108

  19. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  20. Optimized Affinity Capture of Yeast Protein Complexes.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented. PMID:27371596

  1. [Complex management of partnerships during a health promotion campaign].

    PubMed

    Renaud, Lise; Caron-Bouchard, Monique; Martel, Guillaume; Gagnon, Louis; Pelletier, Marie-Claude

    2009-01-01

    This article discusses an analysis of partnerships in the context of health promotion. The 5/30 Health Challenge, or "Défi Santé 5/30", is a campaign to promote healthy eating habits in Quebec. The authors employ this as a case study in order to 1) describe the actors and the nature of their involvement during the campaign's development, design and dissemination; 2) illustrate the interaction of these actors during the conceptualization and rollout of the campaign; 3) propose a paradigm that supports the identification of factors that contribute to or impede partner relationships. The "Défi Santé 5/30" example demonstrates that the creation and maintenance of a partnership network depends on the following key factors: dialogue between partners and the organization responsible for the campaign; the participation of partners at every stage of the campaign (no matter how many there are); allocation of sufficient time for the conceptualization of campaign materials. Dialogue between partners and the central organizer must be guaranteed through the establishment and use of a managerial contract that clearly outlines the role of each actor in the campaign. Further, the partners' activities during the campaign should be regulated through both a formal agreement and a code of ethics. Any campaign's efficiency is directly linked to these factors, among others. The study of partnerships between public, public-private, and private organizations within the framework of health promotion campaigns, thus, merits further study. In addition, to maintain alliances with partners, it is important to demonstrate the benefits of such arrangements to each partner and to equally ensure the contributions of each, be they public, private, media, or community-based organizations. PMID:19863021

  2. Bringing single-molecule spectroscopy to macromolecular protein complexes

    PubMed Central

    Joo, Chirlmin; Fareh, Mohamed; Kim, V. Narry

    2013-01-01

    Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. PMID:23200186

  3. Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.

    PubMed

    Su, C; Wang, F; Ciolek, D; Pan, Y C

    1994-11-15

    Electrophoresis of proteins and protein-protein complexes in polyacrylamide gels under native conditions using a horizontal gel apparatus is described. The advantage of this system is that it permits the detection of both negatively and positively charged proteins as well as protein-protein complexes in the same gel. During electrophoresis, a continuous gel sandwiched between two glass plates is placed horizontally on the platform and submerged in a reservoir buffer. The sample wells are made along the center of the gel, allowing positively and negatively charged proteins to migrate toward the cathode and anode, respectively. Several proteins with varying molecular weights and isoelectric point (pI) values and pairs of proteins capable of forming protein-protein complexes were chosen as model systems to illustrate the methodology. The effects of several parameters on the performance of the gel system including protein molecular weight, pI, and gel concentration were also examined and the results obtained by this method are comparable to those obtained by the vertical system. Following electrophoresis, both negatively and positively charged proteins as well as protein-protein complexes can be transferred by electroblotting onto polyvinylidene difluoride membranes for further analyses. PMID:7695108

  4. The Structure of the BfrB-Bfd Complex Reveals Protein-Protein Interactions Enabling Iron Release from Bacterioferritin

    SciTech Connect

    Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M; Battaile, Kevin P; Vakser, Ilya A; Rivera, Mario

    2012-09-11

    Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis.

  5. The structure of the BfrB-Bfd complex reveals protein-protein interactions enabling iron release from bacterioferritin

    PubMed Central

    Yao, Huili; Wang, Yan; Lovell, Scott; Kumar, Ritesh; Ruvinsky, Anatoly M.; Battaile, Kevin P.; Vakser, Ilya A.; Rivera, Mario

    2012-01-01

    Ferritin-like molecules are unique to cellular iron homeostasis because they can store iron at concentrations much higher than those dictated by the solubility of Fe3+. Very little is known about the protein interactions that deliver iron for storage, or promote the mobilization of stored iron from ferritin-like molecules. Here, we report the X-ray crystal structure of Pseudomonas aeruginosa bacterioferritin (Pa-BfrB) in complex with bacterioferritin-associated ferredoxin (Pa-Bfd) at 2.0 Å resolution. As the first example of a ferritin-like molecule in complex with a cognate partner, the structure provides unprecedented insight into the complementary interface that enables the [2Fe-2S] cluster of Pa-Bfd to promote heme-mediated electron transfer through the BfrB protein dielectric (~18 Å), a process that is necessary to reduce the core ferric mineral and facilitate mobilization of Fe2+. The Pa-BfrB-Bfd complex also revealed the first structure of a Bfd, thus providing a first view to what appears to be a versatile metal binding domain ubiquitous to the large Fer2_BFD family of proteins and enzymes with diverse functions. Residues at the Pa-BfrB-Bfd interface are highly conserved in Bfr and Bfd sequences from a number of pathogenic bacteria, suggesting that the specific recognition between Pa-BfrB and Pa-Bfd is of widespread significance to the understanding of bacterial iron homeostasis. PMID:22812654

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

    PubMed Central

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

    1996-01-01

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

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

    SciTech Connect

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

    2004-10-01

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

  8. Negative Ions Enhance Survival of Membrane Protein Complexes.

    PubMed

    Liko, Idlir; Hopper, Jonathan T S; Allison, Timothy M; Benesch, Justin L P; Robinson, Carol V

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein. Graphical Abstract ᅟ. PMID:27106602

  9. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  10. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-04-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.