Science.gov

Sample records for global regulatory protein

  1. Global Regulatory Pathways in the Alphaproteobacteria

    SciTech Connect

    2007-04-27

    A major goal for microbiologists in the twenty-first century is to develop an understanding of the microbial cell in all its complexity. In addition to understanding the function of individual gene products we need to focus on how the cell regulates gene expression at a global level to respond to different environmental parameters. Development of genomic technologies such as complete genome sequencing, proteomics, and global comparisons of mRNA expression patterns allows us to begin to address this issue. This proposal focuses on a number of phylogenetically related bacteria that are involved in environmentally important processes such as carbon sequestration and bioremediation. Genome sequencing projects of a number of these bacteria have revealed the presence of a small family of regulatory genes found thus far only in the alpha-proteobacteria. These genes encode proteins that are related to the global regulatory protein RosR in Rhizobium etli, which is involved in determining nodulation competitiveness in this bacterium. Our goal is to examine the function of the proteins encoded by this gene family in several of the bacteria containing homologs to RosR. We will construct gene disruption mutations in a number of these bacteria and characterize the resulting mutant strains using two-dimensional gel electrophoresis and genetic and biochemical techniques. We will thus determine if the other proteins also function as global regulators of gene expression. Using proteomics methods we will identify the specific proteins whose expression varies depending on the presence or absence of the RosR homolog. Over fifty loci regulated by RosR have been identified in R. etli using transposon mutagenesis; this will serve as out benchmark to which we will compare the other regulons. We expect to identify genes regulated by RosR homologs in several bacterial species, including, but not limited to Rhodopseudomonas palustris and Sphingomonas aromaticivorans. In this way we will

  2. Global Summit on Regulatory Science 2013.

    PubMed

    Howard, Paul C; Tong, Weida; Weichold, Frank; Healy, Marion; Slikker, William

    2014-12-01

    Regulatory science has been defined as the science that is used to develop regulatory decisions by government bodies. Regulatory science encompasses many scientific disciplines that oversee many studies producing a wide array of data. These may include fundamental research into the cellular interaction or response to a particular chemical or substance, hazard-assessment and dose-response studies in animal species, neurophysiological or neurobehavioral studies, best practices for the generation and analysis of genomics data, bioinformatics approaches, and mathematical modeling of risk. The Global Summit on Regulatory Science is an international conference with a mission to explore emerging and innovative technologies, and provide a platform to enhance translation of basic science into regulatory applications. The Third Global Summit on Regulatory Science which focused on nanotechnology is discussed. PMID:25128336

  3. FliZ is a global regulatory protein affecting the expression of flagellar and virulence genes in individual Xenorhabdus nematophila bacterial cells.

    PubMed

    Jubelin, Grégory; Lanois, Anne; Severac, Dany; Rialle, Stéphanie; Longin, Cyrille; Gaudriault, Sophie; Givaudan, Alain

    2013-10-01

    Heterogeneity in the expression of various bacterial genes has been shown to result in the presence of individuals with different phenotypes within clonal bacterial populations. The genes specifying motility and flagellar functions are coordinately regulated and form a complex regulon, the flagellar regulon. Complex interplay has recently been demonstrated in the regulation of flagellar and virulence gene expression in many bacterial pathogens. We show here that FliZ, a DNA-binding protein, plays a key role in the insect pathogen, Xenorhabdus nematophila, affecting not only hemolysin production and virulence in insects, but efficient swimming motility. RNA-Seq analysis identified FliZ as a global regulatory protein controlling the expression of 278 Xenorhabdus genes either directly or indirectly. FliZ is required for the efficient expression of all flagellar genes, probably through its positive feedback loop, which controls expression of the flhDC operon, the master regulator of the flagellar circuit. FliZ also up- or downregulates the expression of numerous genes encoding non-flagellar proteins potentially involved in key steps of the Xenorhabdus lifecycle. Single-cell analysis revealed the bimodal expression of six identified markers of the FliZ regulon during exponential growth of the bacterial population. In addition, a combination of fluorescence-activated cell sorting and RT-qPCR quantification showed that this bimodality generated a mixed population of cells either expressing ("ON state") or not expressing ("OFF state") FliZ-dependent genes. Moreover, studies of a bacterial population exposed to a graded series of FliZ concentrations showed that FliZ functioned as a rheostat, controlling the rate of transition between the "OFF" and "ON" states in individuals. FliZ thus plays a key role in cell fate decisions, by transiently creating individuals with different potentials for motility and host interactions. PMID:24204316

  4. Global Analysis of Photosynthesis Transcriptional Regulatory Networks

    PubMed Central

    Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.

    2014-01-01

    Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

  5. Global analysis of photosynthesis transcriptional regulatory networks.

    PubMed

    Imam, Saheed; Noguera, Daniel R; Donohue, Timothy J

    2014-12-01

    Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

  6. Fur-Mediated Global Regulatory Circuits in Pathogenic Neisseria Species

    PubMed Central

    Yu, Chunxiao

    2012-01-01

    The ferric uptake regulator (Fur) protein has been shown to function as a repressor of transcription in a number of diverse microorganisms. However, recent studies have established that Fur can function at a global level as both an activator and a repressor of transcription through both direct and indirect mechanisms. Fur-mediated indirect activation occurs via the repression of additional repressor proteins, or small regulatory RNAs, thereby activating transcription of a previously silent gene. Fur mediates direct activation through binding of Fur to the promoter regions of genes. Whereas the repressive mechanism of Fur has been thoroughly investigated, emerging studies on direct and indirect Fur-mediated activation mechanisms have revealed novel global regulatory circuits. PMID:22885296

  7. Phosphorylation of the AfsR product, a global regulatory protein for secondary-metabolite formation in Streptomyces coelicolor A3(2).

    PubMed Central

    Hong, S K; Kito, M; Beppu, T; Horinouchi, S

    1991-01-01

    The AfsR protein is essential for the biosynthesis at the wild-type level of A-factor, actinorhodin, and undecylprodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans. Because overexpression of the afsR gene caused some deleterious effect on these strains, a multicopy plasmid carrying the whole afsR gene was introduced into Streptomyces griseus, from which a crude cell lysate was prepared as a protein source. The AfsR protein was purified to homogeneity from the cytoplasmic fraction through several steps of chromatography, including affinity column chromatography with ATP-agarose and use of anti-AfsR antibody for its detection. The molecular weight of AfsR was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration to be 105,300, which is in good agreement with that deduced from the nucleotide sequence of afsR. The purified AfsR protein was found to be phosphorylated through the transfer of the gamma-phosphate group of ATP in the presence of the cell extracts of S. coelicolor A3(2) and S. lividans. This phosphorylation proceeded very rapidly, and no competition was observed with CTP, GTP, UTP, or cyclic AMP. In the cell extract of S. griseus, no activity phosphorylating the AfsR protein was detected, suggesting that this activity is not generally present in Streptomyces spp. but is specific to certain species. It is conceivable that the extent of phosphorylation of the AfsR protein modulates its regulatory activity which, in turn, regulates expression of some target gene(s) involved in the secondary-metabolite formation in S. coelicolor A3(2). Images PMID:2007554

  8. The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins

    PubMed Central

    Rising, Lisa; Mak, Raymond; Webb, Kristofor; Kaiser, Stephen E.; Zuzow, Nathan; Riviere, Paul; Yang, Bing; Fenech, Emma; Tang, Xin; Lindsay, Scott A.; Christianson, John C.; Hampton, Randolph Y.; Wasserman, Steven A.; Bennett, Eric J.

    2015-01-01

    Summary Insults to endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as a previously uncharacterized and important facet of eukaryotic translational control. PMID:26051182

  9. Global market for dairy proteins.

    PubMed

    Lagrange, Veronique; Whitsett, Dacia; Burris, Cameron

    2015-03-01

    This review examines the global market for dairy ingredients by assessing the global demand for dairy products in relation to major dairy ingredient categories. Each broad category of dairy ingredients is reviewed including its definition, production and trade status, key applications, and future trends. Ingredient categories examined include whole and skim milk powders (WMPs, SMPs), whey protein concentrates (WPCs) and whey protein isolates (WPIs), milk protein concentrates (MPCs) and milk protein isolates (MPIs), caseins, and caseinates. Increases in world population and improvements in socioeconomic conditions will continue to drive the demand for dairy products and ingredients in the future. Dairy proteins are increasingly recognized to have nutritional and functional advantages compared to many protein sources, and the variety of ingredients with different protein concentrations, functionality, and flavor can meet the needs of the increasingly global dairy consumption. A thorough understanding of the variety of ingredients, how the ingredients are derived from milk, and how the demand from particular markets affects the supply situation are critical elements in understanding the current ingredient marketplace. PMID:25757893

  10. Topological origin of global attractors in gene regulatory networks

    NASA Astrophysics Data System (ADS)

    Zhang, YunJun; Ouyang, Qi; Geng, Zhi

    2015-02-01

    Fixed-point attractors with global stability manifest themselves in a number of gene regulatory networks. This property indicates the stability of regulatory networks against small state perturbations and is closely related to other complex dynamics. In this paper, we aim to reveal the core modules in regulatory networks that determine their global attractors and the relationship between these core modules and other motifs. This work has been done via three steps. Firstly, inspired by the signal transmission in the regulation process, we extract the model of chain-like network from regulation networks. We propose a module of "ideal transmission chain (ITC)", which is proved sufficient and necessary (under certain condition) to form a global fixed-point in the context of chain-like network. Secondly, by examining two well-studied regulatory networks (i.e., the cell-cycle regulatory networks of Budding yeast and Fission yeast), we identify the ideal modules in true regulation networks and demonstrate that the modules have a superior contribution to network stability (quantified by the relative size of the biggest attraction basin). Thirdly, in these two regulation networks, we find that the double negative feedback loops, which are the key motifs of forming bistability in regulation, are connected to these core modules with high network stability. These results have shed new light on the connection between the topological feature and the dynamic property of regulatory networks.

  11. [Regulatory proteins of vertebrate eye tissues].

    PubMed

    Krasnov, M S; Grigorian, E N; Iamskova, V P; Boguslavskiĭ, D V; Iamskov, I A

    2003-01-01

    In our work the new proteins likely belonged to the microenvironment of pigmented epithelium cells and retinal neurons in mammalian eye were studied. We attempted to understand the role of these proteins in the maintenance of normal morphological and functional state of these eye tissues. Earlier for the first time we identified the adhesion molecules with physico-chemical and biological properties much different from other known cell adhesion molecules of bovine eye. Probably, they represent one family of low molecular weigh, highly glicosylated proteins, that express biological activity in extremely low doses--10(-10) mg/ml. The homogeneity of studying proteins is confirmed by HPLC and SDS-electrophoresis in PAAG. It is shown also that these proteins are N-glycosylated, because they contain mannose and N-acetilglucosamine residues. They demonstrate as well a high calcium-binding activity, with Kd corresponded to 10(-4)-10(-6) mg/ml. For a study of the biological effect of these glycoproteins in extremely low doses, a new experimental model was proposed and developed. It was the cultivation in vitro of the posterior part of the eye obtained from the newt Pleurodeles waltl. In short-time culture system it was demonstrated that the studied glycoproteins could stabilize pigment epithelium cell differentiation and cellular interactions in the neural retina in vitro. In addition, glycoproteins, obtained from the pigmented epithelium of bovine eye could decrease the rate of bipolar cell apoptosis in the neural retina. Therefore, the novel adhesion glycoproteins, expressing their biological activity in extremely low doses, pretend to be the regulatory molecules with vivid gomeostatic effects necessary for the delicate adjustment of cell behavior action and function in sensory tissues. PMID:12881976

  12. Global regulatory standards for the approval of biosimilars.

    PubMed

    Mounho, Barbara; Phillips, Audrey; Holcombe, Kay; Grampp, Gustavo; Lubiniecki, Tony; Mollerup, Inger; Jones, Carolyn

    2010-01-01

    On March 23, 2010, President Barack Obama signed into law the Patient Protection and Affordable Care Act, which contains the Biologics Price Competition and Innovation Act. Biosimilars have an important role in the United States health care system, and this new law creates an abbreviated approval pathway for biosimilar products in the U.S. A biosimilar is a biologic product demonstrated to be highly similar to an approved innovator biologic product ("reference product"). While the law provides general information on the standards to demonstrate biosimilarity, Congress has authorized the FDA to define the scientific standards and content of biosimilar applications. There is an increasing global interest in the development of biosimilar products, and several regulatory authorities around the world, as well as the World Health Organization (WHO), have established regulatory guidelines for the approval of biosimilars. The scientific standards and requirements in the biosimilar guidelines of the WHO and other health authorities, including the European Union, Canada, Japan, and South Africa, are reviewed in this paper. The similarities as well as the differences among the policies adopted by these regulatory authorities may provide the FDA valuable information as the agency develops its standards and approaches for the approval of biosimilars in the U.S. At the same time, while establishing such approaches, the FDA has the opportunity to demonstrate leadership in addressing significant safety and other issues related to multi-source biologics and biosimilars that remain a global challenge. PMID:24479248

  13. [Role of Academia in Regulatory Science for Global Drug Development].

    PubMed

    Tsukamoto, Katsura; Takenaka, Toichi

    2016-01-01

    As diseases know no national boundaries, drug development must be designed at a global level. Drugs are highly regulated to maximize the benefits to public health, which is assessed on a regional basis. The complexity and diversity of stakeholders increase dramatically once multiple international regions are involved. Each stakeholder in drug development depends on customized criteria to make decisions for its own benefit. Thus, a huge gap exists among drug discovery researchers, developers, clinicians, patients, and regulatory bodies. With reasonable scientific evidence gathered and analyzed, mutual agreement can be reached. We believe that this important role of regulatory science and academic involvement will create harmony. By practicing diverse, innovative regulatory scientific research, academia has the potential to become the core of communication among various stakeholder groups. Furthermore, another important responsibility of academia, i.e., knowledge, provides additional aspects to the field of drug development. Those who understand regulatory science can contribute to the efficient achievement of innovative, effective, safe drugs. Thus, research and education are essential roles of academia to allow a better understanding of the balance between benefits and risks. Communication and knowledge will promote the prompt delivery of better medical products to patients in need. PMID:27040336

  14. Energy-coupled outer membrane transport proteins and regulatory proteins.

    PubMed

    Braun, Volkmar; Endriss, Franziska

    2007-06-01

    FhuA and FecA are two examples of energy-coupled outer membrane import proteins of gram-negative bacteria. FhuA transports iron complexed by the siderophore ferrichrome and serves as a receptor for phages, a toxic bacterial peptide, and a toxic protein. FecA transports diferric dicitrate and regulates transcription of an operon encoding five ferric citrate (Fec) transport genes. Properties of FhuA mutants selected according to the FhuA crystal structure are described. FhuA mutants in the TonB box, the hatch, and the beta-barrel are rather robust. TonB box mutants in FhuA FecA, FepA, Cir, and BtuB are compared; some mutations are suppressed by mutations in TonB. Mutant studies have not revealed a ferrichrome diffusion pathway, and tolerance to mutations in the region linking the TonB box to the hatch does not disclose a mechanism for how energy transfer from the cytoplasmic membrane to FhuA changes the conformation of FhuA such that bound substrates are released, the pore is opened, and substrates enter the periplasm, or how surface loops change their conformation such that TonB-dependent phages bind irreversibly and release their DNA into the cells. The FhuA and FecA crystal structures do not disclose the mechanism of these proteins, but they provide important information for specific functional studies. FecA is also a regulatory protein that transduces a signal from the cell surface into the cytoplasm. The interacting subdomains of the proteins in the FecA --> FecR --> FecI --> RNA polymerase signal transduction pathway resulting in fecABCDE transcription have been determined. Energy-coupled transporters transport not only iron and vitamin B12, but also other substrates of very low abundance such as sugars across the outer membrane; transcription regulation of the transport genes may occur similarly to that of the Fec transport genes. PMID:17370038

  15. Nanosilver and global public health: international regulatory issues.

    PubMed

    Faunce, Thomas; Watal, Aparna

    2010-06-01

    Silver in nanoparticle form is used extensively worldwide in hospital and general practice settings, in dressings as a treatment for external wounds, burns and ulcers. Nanosilver is also an increasingly important coating over embedded medical devices, inhibiting the development of biofilm. Nanosilver disinfectant sprays and polymer coatings are being widely promoted as protective against viral infections. In addition, nanosilver is widely used for its antibacterial properties in food processing and packaging, as well as in consumer products used for domestic cleaning and clothing. This article argues that medical devices, therapeutic products, and domestic food and goods containing nanosilver, although offering therapeutic benefits, must be subject to precautionary regulation owing to associated public health and environmental risks, particularly from large volumes of nanosilver in waste water. The article first examines the use of nanosilver in a variety of contemporary medical and domestic products, the utilization of which may assist in resolving global public health problems, such as restricted access to safe food, water and medical care. It then discusses the mechanisms of toxicity for nanosilver, whether it should be classified as a new chemical entity for regulatory purposes and whether its increased usage poses significant environmental and public health risks. The article next critically analyses representative international regulatory regimes (the USA, EU, UK and Australia) for medical and domestic use of nanosilver. The conclusion includes a set of recommendations for improving international regulation of nanosilver. PMID:20528456

  16. Functional Classification of Immune Regulatory Proteins

    SciTech Connect

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

    2013-05-01

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

  17. Global Analysis of Posttranslational Protein Arginylation

    PubMed Central

    Rai, Reena; Bailey, Aaron O; Yates, John R; Wolf, Yuri I; Zebroski, Henry; Kashina, Anna

    2007-01-01

    Posttranslational arginylation is critical for embryogenesis, cardiovascular development, and angiogenesis, but its molecular effects and the identity of proteins arginylated in vivo are largely unknown. Here we report a global analysis of this modification on the protein level and identification of 43 proteins arginylated in vivo on highly specific sites. Our data demonstrate that unlike previously believed, arginylation can occur on any N-terminally exposed residue likely defined by a structural recognition motif on the protein surface, and that it preferentially affects a number of physiological systems, including cytoskeleton and primary metabolic pathways. The results of our study suggest that protein arginylation is a general mechanism for regulation of protein structure and function and outline the potential role of protein arginylation in cell metabolism and embryonic development. PMID:17896865

  18. CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS

    EPA Science Inventory

    IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic...

  19. Global Hopf bifurcation in the ZIP regulatory system.

    PubMed

    Claus, Juliane; Ptashnyk, Mariya; Bohmann, Ansgar; Chavarría-Krauser, Andrés

    2015-10-01

    Regulation of zinc uptake in roots of Arabidopsis thaliana has recently been modeled by a system of ordinary differential equations based on the uptake of zinc, expression of a transporter protein and the interaction between an activator and inhibitor. For certain parameter choices the steady state of this model becomes unstable upon variation in the external zinc concentration. Numerical results show periodic orbits emerging between two critical values of the external zinc concentration. Here we show the existence of a global Hopf bifurcation with a continuous family of stable periodic orbits between two Hopf bifurcation points. The stability of the orbits in a neighborhood of the bifurcation points is analyzed by deriving the normal form, while the stability of the orbits in the global continuation is shown by calculation of the Floquet multipliers. From a biological point of view, stable periodic orbits lead to potentially toxic zinc peaks in plant cells. Buffering is believed to be an efficient way to deal with strong transient variations in zinc supply. We extend the model by a buffer reaction and analyze the stability of the steady state in dependence of the properties of this reaction. We find that a large enough equilibrium constant of the buffering reaction stabilizes the steady state and prevents the development of oscillations. Hence, our results suggest that buffering has a key role in the dynamics of zinc homeostasis in plant cells. PMID:25312412

  20. Circuitry Linking the Csr and Stringent Response Global Regulatory Systems

    PubMed Central

    Edwards, Adrianne N.; Patterson-Fortin, Laura M.; Vakulskas, Christopher A.; Mercante, Jeffrey W.; Potrykus, Katarzyna; Vinella, Daniel; Camacho, Martha I.; Fields, Joshua A.; Thompson, Stuart A.; Georgellis, Dimitris; Cashel, Michael; Babitzke, Paul; Romeo, Tony

    2011-01-01

    Summary CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR, and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry. PMID:21488981

  1. Quaternion maps of global protein structure.

    PubMed

    Hanson, Andrew J; Thakur, Sidharth

    2012-09-01

    The geometric structures of proteins are vital to the understanding of biochemical interactions. However, there is much yet to be understood about the spatial arrangements of the chains of amino acids making up any given protein. In particular, while conventional analysis tools like the Ramachandran plot supply some insight into the local relative orientation of pairs of amino acid residues, they provide little information about the global relative orientations of large groups of residues. We apply quaternion maps to families of coordinate frames defined naturally by amino acid residue structures as a way to expose global spatial relationships among residues within proteins. The resulting visualizations enable comparisons of absolute orientations as well as relative orientations, and thus generalize the framework of the Ramachandran plot. There are a variety of possible quaternion frames and visual representation strategies that can be chosen, and very complex quaternion maps can result. Just as Ramachandran plots are useful for addressing particular questions and not others, quaternion tools have characteristic domains of relevance. In particular, quaternion maps show great potential for answering specific questions about global residue alignment in crystallographic data and statistical orientation properties in Nuclear Magnetic Resonance (NMR) data that are very difficult to treat by other methods. PMID:23099777

  2. Regulatory challenges for in vitro diagnostics in a global environment.

    PubMed

    Longwell, A

    1994-06-01

    U.S. medical products are marketed globally and are designed to meet needs of medical practitioners and their patients throughout the world. However, differences in how these products are regulated in different countries can pose challenges for the global marketer. This paper explores some of the differences between proposed and extant U.S. and European regulations for in vitro diagnostic products in terms of documentation, records, and labelling. It will describe some of the practical implications of these differences. PMID:7804632

  3. Redox control of iron regulatory protein 2 stability.

    PubMed

    Hausmann, Anja; Lee, Julie; Pantopoulos, Kostas

    2011-02-18

    Iron regulatory protein 2 (IRP2) is a critical switch for cellular and systemic iron homeostasis. In iron-deficient or hypoxic cells, IRP2 binds to mRNAs containing iron responsive elements (IREs) and regulates their expression. Iron promotes proteasomal degradation of IRP2 via the F-box protein FBXL5. Here, we explored the effects of oxygen and cellular redox status on IRP2 stability. We show that iron-dependent decay of tetracycline-inducible IRP2 proceeds efficiently under mild hypoxic conditions (3% oxygen) but is compromised in severe hypoxia (0.1% oxygen). A treatment of cells with exogenous H(2)O(2) protects IRP2 against iron and increases its IRE-binding activity. IRP2 is also stabilized during menadione-induced oxidative stress. These data demonstrate that the degradation of IRP2 in iron-replete cells is not only oxygen-dependent but also sensitive to redox perturbations. PMID:21281640

  4. Global view of the protein universe

    PubMed Central

    Nepomnyachiy, Sergey; Ben-Tal, Nir; Kolodny, Rachel

    2014-01-01

    To explore protein space from a global perspective, we consider 9,710 SCOP (Structural Classification of Proteins) domains with up to 70% sequence identity and present all similarities among them as networks: In the “domain network,” nodes represent domains, and edges connect domains that share “motifs,” i.e., significantly sized segments of similar sequence and structure. We explore the dependence of the network on the thresholds that define the evolutionary relatedness of the domains. At excessively strict thresholds the network falls apart completely; for very lax thresholds, there are network paths between virtually all domains. Interestingly, at intermediate thresholds the network constitutes two regions that can be described as “continuous” versus “discrete.” The continuous region comprises a large connected component, dominated by domains with alternating alpha and beta elements, and the discrete region includes the rest of the domains in isolated islands, each generally corresponding to a fold. We also construct the “motif network,” in which nodes represent recurring motifs, and edges connect motifs that appear in the same domain. This network also features a large and highly connected component of motifs that originate from domains with alternating alpha/beta elements (and some all-alpha domains), and smaller isolated islands. Indeed, the motif network suggests that nature reuses such motifs extensively. The networks suggest evolutionary paths between domains and give hints about protein evolution and the underlying biophysics. They provide natural means of organizing protein space, and could be useful for the development of strategies for protein search and design. PMID:25071170

  5. Reviewing the regulatory barriers for nanomedicine: global questions and challenges.

    PubMed

    Bowman, Diana M; Gatof, Jake

    2015-01-01

    Nanomedicine will play an increasing role in prevention and treatment across the entire healthcare spectrum. However, their precise market size, economic value and areas of application remain unclear. This opacity, including the question of what constitutes nanomedicine matters, especially when considered alongside the key regulatory questions and concerns. This article begins by placing these key questions into context in relation to the current scientific state of the art, focusing particular attention on the human health and safety context. In exploring these central questions surrounding the regulation of nanomedicine, this perspective also explores existing and suggested frameworks that aim to deal with emerging technologies more generally. It then outlines priority areas for action and general conclusions specific to nanomedicine. PMID:26470990

  6. Regulatory Elements of the Staphylococcus aureus Protein A (Spa) Promoter†

    PubMed Central

    Gao, Jinxin; Stewart, George C.

    2004-01-01

    Staphylococcal protein A (Spa) is an important virulence factor of Staphylococcus aureus. Transcription of the spa determinant occurs during the exponential growth phase and is repressed when the cells enter the postexponential growth phase. Regulation of spa expression has been found to be complicated, with regulation involving multiple factors, including Agr, SarA, SarS, SarT, Rot, and MgrA. Our understanding of how these factors work on the spa promoter to regulate spa expression is incomplete. To identify regulatory sites within the spa promoter, analysis of deletion derivatives of the promoter in host strains deficient in one or more of the regulatory factors was undertaken, and several critical features of spa regulation were revealed. The transcriptional start sites of spa were determined by primer extension. The spa promoter sequences were subcloned in front of a promoterless chloramphenicol acetyltransferase reporter gene. Various lengths of spa truncations with the same 3′ end were constructed, and the resultant plasmids were transduced into strains with different regulatory genetic backgrounds. Our results identified upstream promoter sequences necessary for Agr system regulation of spa expression. The cis elements for SarS activity, an activator of spa expression, and for SarA activity, a repressor of spa expression, were identified. The well-characterized SarA consensus sequence on the spa promoter was found to be insufficient for SarA repression of the spa promoter. Full repression required the presence of a second consensus site adjacent to the SarS binding site. Sequences directly upstream of the core promoter sequence were found to stimulate transcription. PMID:15175287

  7. Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling.

    PubMed

    Londino, James D; Gulick, Dexter; Isenberg, Jeffrey S; Mallampalli, Rama K

    2015-12-25

    Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling. PMID:26534964

  8. Laboratory tests for disorders of complement and complement regulatory proteins.

    PubMed

    Shih, Angela R; Murali, Mandakolathur R

    2015-12-01

    The complement pathway is a cascade of proteases that is involved in immune surveillance and innate immunity, as well as adaptive immunity. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, complement regulatory proteins, or complement deficiencies, and has been linked to a number of hematologic disorders, including paroxysmal noctural hemoglobinuria (PNH), hereditary angioedema (HAE), and atypical hemolytic-uremic syndrome (aHUS). Here, current laboratory tests for disorders of the complement pathway are reviewed, and their utility and limitations in hematologic disorders and systemic diseases are discussed. Current therapeutic advances targeting the complement pathway in treatment of complement-mediated hematologic disorders are also reviewed. PMID:26437749

  9. Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa

    PubMed Central

    2014-01-01

    Background Saccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins. Results Two-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis. Conclusions This study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance

  10. Structural Analysis of the Global Multimedia Scenario: Technological, Market, Environmental, and Regulatory Issues.

    ERIC Educational Resources Information Center

    Nicolo, Enrico; Sapio, Bartolomeo

    1996-01-01

    Presents a strategic evaluation of the global multimedia scenario, considering both stand-alone workstations and distributed multimedia in the worldwide interactive network, including educational databases on the Internet. Discusses 50 technological, market, environmental, and regulatory factors and estimates their impacts on each other using WISE…

  11. [The intracellular localization of the regulatory proteins of the densovirus of German cockroach, Blattella germanica].

    PubMed

    Martynova, E U; Kapelinskaia, T V; Schal, C; Mukha, D V

    2014-01-01

    The intracellular localization of the regulatory proteins encoded by the genome of the densovirus of German cockroach was analyzed using western-blotting of nuclear and cytoplasmic extracts of the densovirus-infected passaging cells tissue culture BGE-2. Two of the three regulatory proteins, NS1 and NS3, were shown to possess mainly nuclear localization, while NS2 protein was distributed between the nucleus and cytoplasm. Data obtained provide new information necessary for prediction of the functions of densovirus regulatory proteins. Intracellular localization of NS3 protein was described for the densoviruses for the first time. PMID:25850305

  12. Re-engineering cellular physiology by rewiring high-level global regulatory genes

    PubMed Central

    Fitzgerald, Stephen; Dillon, Shane C.; Chao, Tzu-Chiao; Wiencko, Heather L.; Hokamp, Karsten; Cameron, Andrew D. S.; Dorman, Charles J.

    2015-01-01

    Knowledge of global regulatory networks has been exploited to rewire the gene control programmes of the model bacterium Salmonella enterica serovar Typhimurium. The product is an organism with competitive fitness that is superior to that of the wild type but tuneable under specific growth conditions. The paralogous hns and stpA global regulatory genes are located in distinct regions of the chromosome and control hundreds of target genes, many of which contribute to stress resistance. The locations of the hns and stpA open reading frames were exchanged reciprocally, each acquiring the transcription control signals of the other. The new strain had none of the compensatory mutations normally associated with alterations to hns expression in Salmonella; instead it displayed rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon. Thus the expression patterns of global regulators can be adjusted artificially to manipulate microbial physiology, creating a new and resilient organism. PMID:26631971

  13. Re-engineering cellular physiology by rewiring high-level global regulatory genes.

    PubMed

    Fitzgerald, Stephen; Dillon, Shane C; Chao, Tzu-Chiao; Wiencko, Heather L; Hokamp, Karsten; Cameron, Andrew D S; Dorman, Charles J

    2015-01-01

    Knowledge of global regulatory networks has been exploited to rewire the gene control programmes of the model bacterium Salmonella enterica serovar Typhimurium. The product is an organism with competitive fitness that is superior to that of the wild type but tuneable under specific growth conditions. The paralogous hns and stpA global regulatory genes are located in distinct regions of the chromosome and control hundreds of target genes, many of which contribute to stress resistance. The locations of the hns and stpA open reading frames were exchanged reciprocally, each acquiring the transcription control signals of the other. The new strain had none of the compensatory mutations normally associated with alterations to hns expression in Salmonella; instead it displayed rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon. Thus the expression patterns of global regulators can be adjusted artificially to manipulate microbial physiology, creating a new and resilient organism. PMID:26631971

  14. Regulatory Underpinnings of Global Health Security: FDA's Roles in Preventing, Detecting, and Responding to Global Health Threats

    PubMed Central

    Bond, Katherine C.; Maher, Carmen

    2014-01-01

    In February 2014, health officials from around the world announced the Global Health Security Agenda, a critical effort to strengthen national and global systems to prevent, detect, and respond to infectious disease threats and to foster stronger collaboration across borders. With its increasing global roles and broad range of regulatory responsibilities in ensuring the availability, safety, and security of medical and food products, the US Food and Drug Administration (FDA) is engaged in a range of efforts in support of global health security. This article provides an overview of FDA's global health security roles, focusing on its responsibilities related to the development and use of medical countermeasures (MCMs) for preventing, detecting, and responding to global infectious disease and other public health emergency threats. The article also discusses several areas—antimicrobial resistance, food safety, and supply chain integrity—in which FDA's global health security roles continue to evolve and extend beyond MCMs and, in some cases, beyond traditional infectious disease threats. PMID:25254912

  15. Steroidogenic Acute Regulatory Protein Overexpression Correlates with Protein Kinase A Activation in Adrenocortical Adenoma.

    PubMed

    Zhou, Weiwei; Wu, Luming; Xie, Jing; Su, Tingwei; Jiang, Lei; Jiang, Yiran; Cao, Yanan; Liu, Jianmin; Ning, Guang; Wang, Weiqing

    2016-01-01

    The association of pathological features of cortisol-producing adrenocortical adenomas (ACAs) with somatic driver mutations and their molecular classification remain unclear. In this study, we explored the association between steroidogenic acute regulatory protein (StAR) expression and the driver mutations activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling to identify the pathological markers of ACAs. Immunohistochemical staining for StAR and mutations in the protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) and guanine nucleotide binding protein, alpha stimulating (GNAS) genes were examined in 97 ACAs. The association of StAR expression with the clinical and mutational features of the ACAs was analyzed. ACAs with mutations in PRKACA, GNAS, and PRKAR1A showed strong immunopositive staining for StAR. The concordance between high StAR expression and mutations activating cAMP/PKA signaling in the ACAs was 99.0%. ACAs with high expression of StAR had significantly smaller tumor volume (P < 0.001) and higher urinary cortisol per tumor volume (P = 0.032) than those with low expression of StAR. Our findings suggest that immunohistochemical staining for StAR is a reliable pathological approach for the diagnosis and classification of ACAs with cAMP/PKA signaling-activating mutations. PMID:27606678

  16. The Evolution of the Secreted Regulatory Protein Progranulin.

    PubMed

    Palfree, Roger G E; Bennett, Hugh P J; Bateman, Andrew

    2015-01-01

    Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

  17. The Evolution of the Secreted Regulatory Protein Progranulin

    PubMed Central

    Palfree, Roger G. E.; Bennett, Hugh P. J.; Bateman, Andrew

    2015-01-01

    Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

  18. An experimentally supported model of the Bacillus subtilis global transcriptional regulatory network.

    PubMed

    Arrieta-Ortiz, Mario L; Hafemeister, Christoph; Bate, Ashley Rose; Chu, Timothy; Greenfield, Alex; Shuster, Bentley; Barry, Samantha N; Gallitto, Matthew; Liu, Brian; Kacmarczyk, Thadeous; Santoriello, Francis; Chen, Jie; Rodrigues, Christopher D A; Sato, Tsutomu; Rudner, David Z; Driks, Adam; Bonneau, Richard; Eichenberger, Patrick

    2015-11-01

    Organisms from all domains of life use gene regulation networks to control cell growth, identity, function, and responses to environmental challenges. Although accurate global regulatory models would provide critical evolutionary and functional insights, they remain incomplete, even for the best studied organisms. Efforts to build comprehensive networks are confounded by challenges including network scale, degree of connectivity, complexity of organism-environment interactions, and difficulty of estimating the activity of regulatory factors. Taking advantage of the large number of known regulatory interactions in Bacillus subtilis and two transcriptomics datasets (including one with 38 separate experiments collected specifically for this study), we use a new combination of network component analysis and model selection to simultaneously estimate transcription factor activities and learn a substantially expanded transcriptional regulatory network for this bacterium. In total, we predict 2,258 novel regulatory interactions and recall 74% of the previously known interactions. We obtained experimental support for 391 (out of 635 evaluated) novel regulatory edges (62% accuracy), thus significantly increasing our understanding of various cell processes, such as spore formation. PMID:26577401

  19. Transcriptional and translational regulatory responses to iron limitation in the globally distributed marine bacterium Candidatus Pelagibacter ubique

    SciTech Connect

    Smith, Daniel P.; Kitner, J. B.; Norbeck, Angela D.; Clauss, Therese RW; Lipton, Mary S.; Schwalbach, M. S.; Steindler, L.; Nicora, Carrie D.; Smith, Richard D.; Giovannoni, Stephen J.

    2010-05-05

    Abstract Background: Iron is recognized as an important micronutrient that limits microbial plankton productivity over vast regions of the oceans. We investigated the gene expression responses of Candidatus Pelagibacter ubique cultures to iron limitation in natural seawater media supplemented with a siderophore to chelate iron. Methodology/Principal Findings: Microarray data indicated transcription of the periplasmic iron binding protein sfuC increased by 16-fold, and iron transporter subunits, iron-sulfur center assembly genes, and the putative ferroxidase rubrerythrin transcripts increased to a lesser extent. Quantitative peptide mass spectrometry revealed that sfuC protein abundance increased 27-fold, despite an average decrease of 59% across the global proteome. Two RNA-binding proteins, CspE and CspL, correlated well with iron availability, suggesting that they may contribute to the observed differences between the transcriptome and proteome. Conclusions/Significance: We propose sfuC as a marker gene for indicating iron limitation in marine metatranscriptomic and metaproteomic ecological surveys. The marked proteome reduction was not directly correlated to changes in the transcriptome, implicating post-transcriptional regulatory mechanisms as modulators of protein expression. We propose a model in which the RNA-binding activity of cspE and cspL selectively enables protein synthesis of the iron acquisition protein sfuC during transient growth-limiting episodes of iron scarcity.

  20. Iron misregulation and neurodegenerative disease in mouse models that lack iron regulatory proteins.

    PubMed

    Ghosh, Manik C; Zhang, De-Liang; Rouault, Tracey A

    2015-09-01

    Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are two cytosolic proteins that maintain cellular iron homeostasis by binding to RNA stem loops known as iron responsive elements (IREs) that are found in the untranslated regions of target mRNAs that encode proteins involved in iron metabolism. IRPs modify the expression of iron metabolism genes, and global and tissue-specific knockout mice have been made to evaluate the physiological significance of these iron regulatory proteins (Irps). Here, we will discuss the results of the studies that have been performed with mice engineered to lack the expression of one or both Irps and made in different strains using different methodologies. Both Irp1 and Irp2 knockout mice are viable, but the double knockout (Irp1(-/-)Irp2(-/-)) mice die before birth, indicating that these Irps play a crucial role in maintaining iron homeostasis. Irp1(-/-) mice develop polycythemia and pulmonary hypertension, and when these mice are challenged with a low iron diet, they die early of abdominal hemorrhages, suggesting that Irp1 plays an essential role in erythropoiesis and in the pulmonary and cardiovascular systems. Irp2(-/-) mice develop microcytic anemia, erythropoietic protoporphyria and a progressive neurological disorder, indicating that Irp2 has important functions in the nervous system and erythropoietic homeostasis. Several excellent review articles have recently been published on Irp knockout mice that mainly focus on Irp1(-/-) mice (referenced in the introduction). In this review, we will briefly describe the phenotypes and physiological implications of Irp1(-/-) mice and discuss the phenotypes observed for Irp2(-/-) mice in detail with a particular emphasis on the neurological problems of these mice. PMID:25771171

  1. A strategy to quantitate global phosphorylation of bone matrix proteins.

    PubMed

    Sroga, Grażyna E; Vashishth, Deepak

    2016-04-15

    Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials. PMID:26851341

  2. Local conformational fluctuations can modulate the coupling between proton binding and global structural transitions in proteins

    PubMed Central

    Whitten, Steven T.; García-Moreno E., Bertrand; Hilser, Vincent J.

    2005-01-01

    Local conformational fluctuations in proteins can affect the coupling between ligand binding and global structural transitions. This finding was established by monitoring quantitatively how the population distribution in the ensemble of microstates of staphylococcal nuclease was affected by proton binding. Analysis of acid unfolding and proton-binding data with an ensemble-based model suggests that local fluctuations: (i) can be effective modulators of ligand-binding affinities, (ii) are important determinants of the cooperativity of ligand-driven global structural transitions, and (iii) are well represented thermodynamically as local unfolding processes. These studies illustrate how an ensemble-based description of proteins can be used to describe quantitatively the interdependence of local conformational fluctuations, ligand-binding processes, and global structural transitions. This level of understanding of the relationship between conformation, energy, and dynamics is required for a detailed mechanistic understanding of allostery, cooperativity, and other complex functional and regulatory properties of macromolecules. PMID:15767576

  3. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

    NASA Astrophysics Data System (ADS)

    Lloyd, David J.; St Jean, David J.; Kurzeja, Robert J. M.; Wahl, Robert C.; Michelsen, Klaus; Cupples, Rod; Chen, Michelle; Wu, John; Sivits, Glenn; Helmering, Joan; Komorowski, Renée; Ashton, Kate S.; Pennington, Lewis D.; Fotsch, Christopher; Vazir, Mukta; Chen, Kui; Chmait, Samer; Zhang, Jiandong; Liu, Longbin; Norman, Mark H.; Andrews, Kristin L.; Bartberger, Michael D.; van, Gwyneth; Galbreath, Elizabeth J.; Vonderfecht, Steven L.; Wang, Minghan; Jordan, Steven R.; Véniant, Murielle M.; Hale, Clarence

    2013-12-01

    Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

  4. Modular architecture of protein structures and allosteric communications: potential implications for signaling proteins and regulatory linkages

    PubMed Central

    del Sol, Antonio; Araúzo-Bravo, Marcos J; Amoros, Dolors; Nussinov, Ruth

    2007-01-01

    Background Allosteric communications are vital for cellular signaling. Here we explore a relationship between protein architectural organization and shortcuts in signaling pathways. Results We show that protein domains consist of modules interconnected by residues that mediate signaling through the shortest pathways. These mediating residues tend to be located at the inter-modular boundaries, which are more rigid and display a larger number of long-range interactions than intra-modular regions. The inter-modular boundaries contain most of the residues centrally conserved in the protein fold, which may be crucial for information transfer between amino acids. Our approach to modular decomposition relies on a representation of protein structures as residue-interacting networks, and removal of the most central residue contacts, which are assumed to be crucial for allosteric communications. The modular decomposition of 100 multi-domain protein structures indicates that modules constitute the building blocks of domains. The analysis of 13 allosteric proteins revealed that modules characterize experimentally identified functional regions. Based on the study of an additional functionally annotated dataset of 115 proteins, we propose that high-modularity modules include functional sites and are the basic functional units. We provide examples (the Gαs subunit and P450 cytochromes) to illustrate that the modular architecture of active sites is linked to their functional specialization. Conclusion Our method decomposes protein structures into modules, allowing the study of signal transmission between functional sites. A modular configuration might be advantageous: it allows signaling proteins to expand their regulatory linkages and may elicit a broader range of control mechanisms either via modular combinations or through modulation of inter-modular linkages. PMID:17531094

  5. Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP.

    PubMed

    Bader, G; Schiffmann, S; Herrmann, A; Fischer, M; Gütlich, M; Auerbach, G; Ploom, T; Bacher, A; Huber, R; Lemm, T

    2001-10-01

    Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory protein (GFRP). The 2.6 A resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the interaction of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins. PMID:11580249

  6. Role of basic leucine zipper proteins in transcriptional regulation of the steroidogenic acute regulatory protein gene

    PubMed Central

    Manna, Pulak R.; Dyson, Matthew T.; Stocco, Douglas M.

    2016-01-01

    The regulation of steroidogenic acute regulatory protein (StAR) gene transcription by cAMP-dependent mechanisms occurs in the absence of a consensus cAMP response element (CRE, TGACGTGA). This regulation is coordinated by multiple transcription factors that bind to sequence-specific elements located approximately 150 bp upstream of the transcription start site. Among the proteins that bind within this region, the basic leucine zipper (bZIP) family of transcription factors, i.e. CRE binding protein (CREB)/CRE modulator (CREM)/activating transcription factor (ATF), activator protein 1 (AP-1; Fos/Jun), and CCAAT enhancer binding protein β (C/EBPβ), interact with an overlapping region (−81/−72 bp) in the StAR promoter, mediate stimulus-transcription coupling of cAMP signaling and play integral roles in regulating StAR gene expression. These bZIP proteins are structurally similar and bind to DNA sequences as dimers; however, they exhibit discrete transcriptional activities, interact with several transcription factors and other properties that contribute in their regulatory functions. The 5′-flanking −81/−72 bp region of the StAR gene appears to function as a key element within a complex cAMP response unit by binding to different bZIP members, and the StAR promoter displays variable states of cAMP responsivity contingent upon the occupancy of these cis-elements with these transcription factors. The expression and activities of CREB/CREM/ATF, Fos/Jun and C/EBPβ have been demonstrated to be mediated by a plethora of extracellular signals, and the phosphorylation of these proteins at several Ser and Thr residues allows recruitment of the transcriptional coactivator CREB binding protein (CBP) or its functional homolog p300 to the StAR promoter. This review will focus on the current level of understanding of the roles of selective bZIP family proteins within the complex series of processes involved in regulating StAR gene transcription. PMID:19150388

  7. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  8. Regulatory protein BBD18 of the lyme disease spirochete: essential role during tick acquisition?

    PubMed

    Hayes, Beth M; Dulebohn, Daniel P; Sarkar, Amit; Tilly, Kit; Bestor, Aaron; Ambroggio, Xavier; Rosa, Patricia A

    2014-01-01

    The Lyme disease spirochete Borrelia burgdorferi senses and responds to environmental cues as it transits between the tick vector and vertebrate host. Failure to properly adapt can block transmission of the spirochete and persistence in either vector or host. We previously identified BBD18, a novel plasmid-encoded protein of B. burgdorferi, as a putative repressor of the host-essential factor OspC. In this study, we investigate the in vivo role of BBD18 as a regulatory protein, using an experimental mouse-tick model system that closely resembles the natural infectious cycle of B. burgdorferi. We show that spirochetes that have been engineered to constitutively produce BBD18 can colonize and persist in ticks but do not infect mice when introduced by either tick bite or needle inoculation. Conversely, spirochetes lacking BBD18 can persistently infect mice but are not acquired by feeding ticks. Through site-directed mutagenesis, we have demonstrated that abrogation of spirochete infection in mice by overexpression of BBD18 occurs only with bbd18 alleles that can suppress OspC synthesis. Finally, we demonstrate that BBD18-mediated regulation does not utilize a previously described ospC operator sequence required by B. burgdorferi for persistence in immunocompetent mice. These data lead us to conclude that BBD18 does not represent the putative repressor utilized by B. burgdorferi for the specific downregulation of OspC in the mammalian host. Rather, we suggest that BBD18 exhibits features more consistent with those of a global regulatory protein whose critical role occurs during spirochete acquisition by feeding ticks. IMPORTANCE Lyme disease, caused by Borrelia burgdorferi, is the most common arthropod-borne disease in North America. B. burgdorferi is transmitted to humans and other vertebrate hosts by ticks as they take a blood meal. Transmission between vectors and hosts requires the bacterium to sense changes in the environment and adapt. However, the mechanisms

  9. Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

    PubMed Central

    Zhao, Yang; Xie, Shaojun; Batelli, Giorgia; Wang, Bangshing; Duan, Cheng-Guo; Wang, Xingang; Xing, Lu; Lei, Mingguang; Yan, Jun; Zhu, Xiaohong; Zhu, Jian-Kang

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. PMID:26943172

  10. Bovine viral diarrhea virus structural protein E2 as a complement regulatory protein.

    PubMed

    Ostachuk, Agustín

    2016-07-01

    Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, family Flaviviridae, and is one of the most widely distributed viruses in cattle worldwide. Approximately 60 % of cattle in endemic areas without control measures are infected with BVDV during their lifetime. This wide prevalence of BVDV in cattle populations results in significant economic losses. BVDV is capable of establishing persistent infections in its host due to its ability to infect fetuses, causing immune tolerance. However, this cannot explain how the virus evades the innate immune system. The objective of the present work was to test the potential activity of E2 as a complement regulatory protein. E2 glycoprotein, produced both in soluble and transmembrane forms in stable CHO-K1 cell lines, was able to reduce complement-mediated cell lysis up to 40 % and complement-mediated DNA fragmentation by 50 %, in comparison with cell lines not expressing the glycoprotein. This work provides the first evidence of E2 as a complement regulatory protein and, thus, the finding of a mechanism of immune evasion by BVDV. Furthermore, it is postulated that E2 acts as a self-associated molecular pattern (SAMP), enabling the virus to avoid being targeted by the immune system and to be recognized as self. PMID:27038454

  11. De Novo Evolution of Complex, Global and Hierarchical Gene Regulatory Mechanisms

    PubMed Central

    Jenkins, Dafyd J.

    2010-01-01

    Gene regulatory networks exhibit complex, hierarchical features such as global regulation and network motifs. There is much debate about whether the evolutionary origins of such features are the results of adaptation, or the by-products of non-adaptive processes of DNA replication. The lack of availability of gene regulatory networks of ancestor species on evolutionary timescales makes this a particularly difficult problem to resolve. Digital organisms, however, can be used to provide a complete evolutionary record of lineages. We use a biologically realistic evolutionary model that includes gene expression, regulation, metabolism and biosynthesis, to investigate the evolution of complex function in gene regulatory networks. We discover that: (i) network architecture and complexity evolve in response to environmental complexity, (ii) global gene regulation is selected for in complex environments, (iii) complex, inter-connected, hierarchical structures evolve in stages, with energy regulation preceding stress responses, and stress responses preceding growth rate adaptations and (iv) robustness of evolved models to mutations depends on hierarchical level: energy regulation and stress responses tend not to be robust to mutations, whereas growth rate adaptations are more robust and non-lethal when mutated. These results highlight the adaptive and incremental evolution of complex biological networks, and the value and potential of studying realistic in silico evolutionary systems as a way of understanding living systems. Electronic supplementary material The online version of this article (doi:10.1007/s00239-010-9369-4) contains supplementary material, which is available to authorized users. PMID:20680619

  12. Global De Novo Protein-Protein Interactome Elucidates Interactions of Drought-Responsive Proteins in Horse Gram (Macrotyloma uniflorum).

    PubMed

    Bhardwaj, Jyoti; Gangwar, Indu; Panzade, Ganesh; Shankar, Ravi; Yadav, Sudesh Kumar

    2016-06-01

    Inspired by the availability of de novo transcriptome of horse gram (Macrotyloma uniflorum) and recent developments in systems biology studies, the first ever global protein-protein interactome (PPI) map was constructed for this highly drought-tolerant legume. Large-scale studies of PPIs and the constructed database would provide rationale behind the interplay at cascading translational levels for drought stress-adaptive mechanisms in horse gram. Using a bidirectional approach (interolog and domain-based), a high-confidence interactome map and database for horse gram was constructed. Available transcriptomic information for shoot and root tissues of a sensitive (M-191; genotype 1) and a drought-tolerant (M-249; genotype 2) genotype of horse gram was utilized to draw comparative PPI subnetworks under drought stress. High-confidence 6804 interactions were predicted among 1812 proteins covering about one-fourth of the horse gram proteome. The highest number of interactions (33.86%) in horse gram interactome matched with Arabidopsis PPI data. The top five hub nodes mostly included ubiquitin and heat-shock-related proteins. Higher numbers of PPIs were found to be responsive in shoot tissue (416) and root tissue (2228) of genotype 2 compared with shoot tissue (136) and root tissue (579) of genotype 1. Characterization of PPIs using gene ontology analysis revealed that kinase and transferase activities involved in signal transduction, cellular processes, nucleocytoplasmic transport, protein ubiquitination, and localization of molecules were most responsive to drought stress. Hence, these could be framed in stress adaptive mechanisms of horse gram. Being the first legume global PPI map, it would provide new insights into gene and protein regulatory networks for drought stress tolerance mechanisms in horse gram. Information compiled in the form of database (MauPIR) will provide the much needed high-confidence systems biology information for horse gram genes, proteins, and

  13. 76 FR 32364 - Collaboration in Regulatory Science and Capacity To Advance Global Access to Safe Vaccines and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-06

    ...The Food and Drug Administration (FDA) announces its intention to accept and consider a single source application for award of a cooperative agreement to the World Health Organization (WHO) in support of collaboration in regulatory science and capacity of National Regulatory Authorities (NRAs) to advance global access to safe and effective vaccines and other biologicals that meet international......

  14. Proteomics analysis of global regulatory cascades involved in clavulanic acid production and morphological development in Streptomyces clavuligerus.

    PubMed

    Ferguson, Nicole L; Peña-Castillo, Lourdes; Moore, Marcus A; Bignell, Dawn R D; Tahlan, Kapil

    2016-04-01

    The genus Streptomyces comprises bacteria that undergo a complex developmental life cycle and produce many metabolites of importance to industry and medicine. Streptomyces clavuligerus produces the β-lactamase inhibitor clavulanic acid, which is used in combination with β-lactam antibiotics to treat certain β-lactam resistant bacterial infections. Many aspects of how clavulanic acid production is globally regulated in S. clavuligerus still remains unknown. We conducted comparative proteomics analysis using the wild type strain of S. clavuligerus and two mutants (ΔbldA and ΔbldG), which are defective in global regulators and vary in their ability to produce clavulanic acid. Approximately 33.5 % of the predicted S. clavuligerus proteome was detected and 192 known or putative regulatory proteins showed statistically differential expression levels in pairwise comparisons. Interestingly, the expression of many proteins whose corresponding genes contain TTA codons (predicted to require the bldA tRNA for translation) was unaffected in the bldA mutant. PMID:26790415

  15. Analysis of Protein Phosphatase-1 and Aurora Protein Kinase Suppressors Reveals New Aspects of Regulatory Protein Function in Saccharomyces cerevisiae

    PubMed Central

    Ghosh, Anuprita; Cannon, John F.

    2013-01-01

    Protein phosphatase-1 (PP1) controls many processes in eukaryotic cells. Modulation of mitosis by reversing phosphorylation of proteins phosphorylated by aurora protein kinase is a critical function for PP1. Overexpression of the sole PP1, Glc7, in budding yeast, Saccharomyces cerevisiae, is lethal. This work shows that lethality requires the function of Glc7 regulatory proteins Sds22, Reg2, and phosphorylated Glc8. This finding shows that Glc7 overexpression induced cell death requires a specific subset of the many Glc7-interacting proteins and therefore is likely caused by promiscuous dephosphorylation of a variety of substrates. Additionally, suppression can occur by reducing Glc7 protein levels by high-copy Fpr3 without use of its proline isomerase domain. This divulges a novel function of Fpr3. Most suppressors of GLC7 overexpression also suppress aurora protein kinase, ipl1, temperature-sensitive mutations. However, high-copy mutant SDS22 genes show reciprocal suppression of GLC7 overexpression induced cell death or ipl1 temperature sensitivity. Sds22 binds to many proteins besides Glc7. The N-terminal 25 residues of Sds22 are sufficient to bind, directly or indirectly, to seven proteins studied here including the spindle assembly checkpoint protein, Bub3. These data demonstrate that Sds22 organizes several proteins in addition to Glc7 to perform functions that counteract Ipl1 activity or lead to hyper Glc7 induced cell death. These data also emphasize that Sds22 targets Glc7 to nuclear locations distinct from Ipl1 substrates. PMID:23894419

  16. Future Regulatory Science through a Global Product Development Strategy to Overcome the Device Lag.

    PubMed

    Tsuchii, Isao

    2016-01-01

    Environment that created "medical device lag (MDL)" has changed dramatically, and currently that term is not heard often. This was mainly achieved through the leadership of three groups: government, which determined to overcome MDL and took steps to do so; medical societies, which exhibited accountability in trial participation; and MD companies, which underwent a change in mindset that allowed comprehensive tripartite cooperation to reach the current stage. In particular, the global product development strategy (GPDS) of companies in a changing social environment has taken a new-turn with international harmonization trends, like Global Harmonization Task Force and International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. As a result, this evolution has created opportunities for treatment with cutting-edge MDs in Japanese society. Simultaneously, it has had a major impact on the planning process of GPDS of companies. At the same time, the interest of global companies has shifted to emerging economies for future potential profit since Japan no longer faces MDL issue. This economic trend makes MDLs a greater problem for manufacturers. From the regulatory science viewpoint, this new environment has not made it easy to plan a global strategy that will be adaptable to local societies. Without taking hasty action, flexible thinking from the global point of view is necessary to enable the adjustment of local strategies to fit the situation on the ground so that the innovative Japanese medical technology can be exported to a broad range of societies. PMID:27040334

  17. Regulation of Airway Inflammation by G-protein Regulatory Motif Peptides of AGS3 protein

    PubMed Central

    Choi, IL-Whan; Ahn, Do Whan; Choi, Jang-Kyu; Cha, Hee-Jae; Ock, Mee Sun; You, EunAe; Rhee, SangMyung; Kim, Kwang Chul; Choi, Yung Hyun; Song, Kyoung Seob

    2016-01-01

    Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFβ but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment. PMID:27270970

  18. Improving global access to new vaccines: intellectual property, technology transfer, and regulatory pathways.

    PubMed

    Crager, Sara Eve

    2014-11-01

    The 2012 World Health Assembly Global Vaccine Action Plan called for global access to new vaccines within 5 years of licensure. Current approaches have proven insufficient to achieve sustainable vaccine pricing within such a timeline. Paralleling the successful strategy of generic competition to bring down drug prices, a clear consensus is emerging that market entry of multiple suppliers is a critical factor in expeditiously bringing down prices of new vaccines. In this context, key target objectives for improving access to new vaccines include overcoming intellectual property obstacles, streamlining regulatory pathways for biosimilar vaccines, and reducing market entry timelines for developing-country vaccine manufacturers by transfer of technology and know-how. I propose an intellectual property, technology, and know-how bank as a new approach to facilitate widespread access to new vaccines in low- and middle-income countries by efficient transfer of patented vaccine technologies to multiple developing-country vaccine manufacturers. PMID:25211753

  19. [Improving global access to new vaccines: intellectual property, technology transfer, and regulatory pathways].

    PubMed

    Crager, Sara Eve

    2015-01-01

    The 2012 World Health Assembly Global Vaccine Action Plan called for global access to new vaccines within 5 years of licensure. Current approaches have proven insufficient to achieve sustainable vaccine pricing within such a timeline. Paralleling the successful strategy of generic competition to bring down drug prices, a clear consensus is emerging that market entry of multiple suppliers is a critical factor in expeditiously bringing down prices of new vaccines. In this context, key target objectives for improving access to new vaccines include overcoming intellectual property obstacles, streamlining regulatory pathways for biosimilar vaccines, and reducing market entry timelines for developing-country vaccine manufacturers by transfer of technology and know-how. I propose an intellectual property, technology, and know-how bank as a new approach to facilitate widespread access to new vaccines in low- and middle-income countries by efficient transfer of patented vaccine technologies to multiple developing-country vaccine manufacturers. PMID:25791189

  20. The global regulatory landscape regarding micronutrient fortification of condiments and seasonings.

    PubMed

    Mejia, Luis A; Bower, Allyson M

    2015-11-01

    Fortification of staple foods has been a successful strategy for combatting micronutrient deficiency. Recently, fortification of condiments and seasonings has been considered as a new approach to mitigate micronutrient deficiencies worldwide. The regulatory environment of already existing programs must be examined to assess their safety, efficacy, and sustainability as this strategy expands globally. The objective of this review is to summarize the global regulatory landscape for the fortification of condiments and seasonings. Presently, legislation regarding the fortification of condiments and seasonings is primarily voluntary and limited to a few nations in Asia. The only dietary vehicles addressed are salt, soy sauce, and fish sauce, and the micronutrients addressed are iron and iodine. A marketing-driven introduction of fortified seasoning powders with iron, and indirectly with iodine, is also gaining popularity in Africa, Central America, and Caribbean countries. It is recommended that legislation regarding food fortification be mandatory in nature and follow established CODEX and World Trade Organization principles as well as World Health Organization/Food and Agriculture Organization of the United Nations fortification guidelines to ensure that these programs are safe, effective, and sustainable. PMID:26251126

  1. Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms

    PubMed Central

    Allen, Mary Ann; Andrysik, Zdenek; Dengler, Veronica L; Mellert, Hestia S; Guarnieri, Anna; Freeman, Justin A; Sullivan, Kelly D; Galbraith, Matthew D; Luo, Xin; Kraus, W Lee; Dowell, Robin D; Espinosa, Joaquin M

    2014-01-01

    The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms. DOI: http://dx.doi.org/10.7554/eLife.02200.001 PMID:24867637

  2. Capacity for a global vaccine safety system: the perspective of national regulatory authorities.

    PubMed

    Graham, Janice E; Borda-Rodriguez, Alexander; Huzair, Farah; Zinck, Emily

    2012-07-13

    Confidence in vaccine safety is critical to national immunization strategies and to global public health. To meet the Millenium Development Goals, and buoyed by the success of new vaccines produced in developing countries, the World Health Organization has been developing a strategy to establish a global system for effective vaccine pharmacovigilance in all countries. This paper reports the findings of a qualitative survey, conducted for the WHO Global Vaccine Safety Blueprint project, on the perspectives of national regulatory authorities responsible for vaccine safety in manufacturing and procuring countries. Capacity and capabilities of detecting, reporting and responding to adverse events following immunization (AEFI), and expectations of minimum capacity necessary for vaccine pharmacovigilance were explored. Key barriers to establishing a functional national vaccine safety system in developing countries were identified. The lack of infrastructure, information technology for stable communications and data exchange, and human resources affect vaccine safety monitoring in developing countries. A persistent "fear of reporting" in several low and middle income countries due to insufficient training and insecure employment underlies a perceived lack of political will in many governments for vaccine pharmacovigilance. Regulators recommended standardized and internationally harmonized safety reporting forms, improved surveillance mechanisms, and a global network for access and exchange of safety data independent of industry. PMID:22658930

  3. From local to global changes in proteins: a network view.

    PubMed

    Vuillon, Laurent; Lesieur, Claire

    2015-04-01

    To fulfill the biological activities in living organisms, proteins are endowed with dynamics, robustness and adaptability. The three properties co-exist because they allow global changes in structure to arise from local perturbations (dynamics). Robustness refers to the ability of the protein to incur such changes without suffering loss of function; adaptability is the emergence of a new biological activity. Since loss of function may jeopardize the survival of the organism and lead to disease, adaptability may occur through the combination of two local perturbations that together rescue the initial function. The review highlights the relevancy of computational network analysis to understand how a local change produces global changes. PMID:25791607

  4. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

    SciTech Connect

    Mao, Grace; Brody, James P.

    2007-11-09

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s{sup -1}. We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.

  5. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  6. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  7. Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics*

    PubMed Central

    Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J.; Ly, Tony; Lamond, Angus I.

    2013-01-01

    Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ∼5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution. PMID:23242552

  8. A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus

    PubMed Central

    Beccari, Leonardo; Yakushiji-Kaminatsui, Nayuta; Woltering, Joost M.; Necsulea, Anamaria; Lonfat, Nicolas; Rodríguez-Carballo, Eddie; Mascrez, Benedicte; Yamamoto, Shiori; Kuroiwa, Atsushi

    2016-01-01

    During vertebrate limb development, Hoxd genes are regulated following a bimodal strategy involving two topologically associating domains (TADs) located on either side of the gene cluster. These regulatory landscapes alternatively control different subsets of Hoxd targets, first into the arm and subsequently into the digits. We studied the transition between these two global regulations, a switch that correlates with the positioning of the wrist, which articulates these two main limb segments. We show that the HOX13 proteins themselves help switch off the telomeric TAD, likely through a global repressive mechanism. At the same time, they directly interact with distal enhancers to sustain the activity of the centromeric TAD, thus explaining both the sequential and exclusive operating processes of these two regulatory domains. We propose a model in which the activation of Hox13 gene expression in distal limb cells both interrupts the proximal Hox gene regulation and re-enforces the distal regulation. In the absence of HOX13 proteins, a proximal limb structure grows without any sign of wrist articulation, likely related to an ancestral fish-like condition. PMID:27198226

  9. A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus.

    PubMed

    Beccari, Leonardo; Yakushiji-Kaminatsui, Nayuta; Woltering, Joost M; Necsulea, Anamaria; Lonfat, Nicolas; Rodríguez-Carballo, Eddie; Mascrez, Benedicte; Yamamoto, Shiori; Kuroiwa, Atsushi; Duboule, Denis

    2016-05-15

    During vertebrate limb development, Hoxd genes are regulated following a bimodal strategy involving two topologically associating domains (TADs) located on either side of the gene cluster. These regulatory landscapes alternatively control different subsets of Hoxd targets, first into the arm and subsequently into the digits. We studied the transition between these two global regulations, a switch that correlates with the positioning of the wrist, which articulates these two main limb segments. We show that the HOX13 proteins themselves help switch off the telomeric TAD, likely through a global repressive mechanism. At the same time, they directly interact with distal enhancers to sustain the activity of the centromeric TAD, thus explaining both the sequential and exclusive operating processes of these two regulatory domains. We propose a model in which the activation of Hox13 gene expression in distal limb cells both interrupts the proximal Hox gene regulation and re-enforces the distal regulation. In the absence of HOX13 proteins, a proximal limb structure grows without any sign of wrist articulation, likely related to an ancestral fish-like condition. PMID:27198226

  10. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    PubMed Central

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  11. 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

  12. The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle

    PubMed Central

    Zhou, Bo; Schrader, Jared M.; Kalogeraki, Virginia S.; Abeliuk, Eduardo; Dinh, Cong B.; Pham, James Q.; Cui, Zhongying Z.; Dill, David L.; McAdams, Harley H.; Shapiro, Lucy

    2015-01-01

    Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle. PMID:25569173

  13. Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein.

    PubMed

    Huemer, H P; Wang, Y; Garred, P; Koistinen, V; Oppermann, S

    1993-08-01

    Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the beta-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR

  14. The regulatory PII protein controls arginine biosynthesis in Arabidopsis.

    PubMed

    Ferrario-Méry, Sylvie; Besin, Evelyne; Pichon, Olivier; Meyer, Christian; Hodges, Michael

    2006-04-01

    In higher plants, PII is a nuclear-encoded plastid protein which is homologous to bacterial PII signalling proteins known to be involved in the regulation of nitrogen metabolism. A reduced ornithine, citrulline and arginine accumulation was observed in two Arabidopsis PII knock-out mutants in response to NH4+ resupply after N starvation. This difference could be explained by the regulation of a key enzyme of the arginine biosynthesis pathway, N-acetyl glutamate kinase (NAGK) by PII. In vitro assays using purified recombinant proteins showed the catalytic activation of Arabidopsis NAGK by PII giving the first evidence of a physiological role of the PII protein in higher plants. Using Arabidopsis transcriptome microarray (CATMA) and RT-PCR analyses, it was found that none of the genes involved in the arginine biosynthetic or catabolic pathways were differentially expressed in a PII knock-out mutant background. In conclusion, the observed changes in metabolite levels can be explained by the reduced activation of NAGK by PII. PMID:16545809

  15. Membrane orientation of the Na,K-ATPase regulatory membrane protein CHIF determined by solid-state NMR

    PubMed Central

    Franzin, Carla M.; Teriete, Peter; Marassi, Francesca M.

    2010-01-01

    Corticosteroid hormone-induced factor (CHIF) is a major regulatory subunit of the Na,K-ATPase, and a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here we present the structure of CHIF oriented in the membrane, determined by solid-state NMR orientation-dependent restraints. Because CHIF adopts a similar structure in lipid micelles and bilayers, it is possible to assign the solid-state NMR spectrum measured for 15N-labeled CHIF in oriented bilayers from the structure determined in micelles, to obtain the global orientation of the protein in the membrane. PMID:18098352

  16. Integrated biclustering of heterogeneous genome-wide datasets for the inference of global regulatory networks

    PubMed Central

    Reiss, David J; Baliga, Nitin S; Bonneau, Richard

    2006-01-01

    Background The learning of global genetic regulatory networks from expression data is a severely under-constrained problem that is aided by reducing the dimensionality of the search space by means of clustering genes into putatively co-regulated groups, as opposed to those that are simply co-expressed. Be cause genes may be co-regulated only across a subset of all observed experimental conditions, biclustering (clustering of genes and conditions) is more appropriate than standard clustering. Co-regulated genes are also often functionally (physically, spatially, genetically, and/or evolutionarily) associated, and such a priori known or pre-computed associations can provide support for appropriately grouping genes. One important association is the presence of one or more common cis-regulatory motifs. In organisms where these motifs are not known, their de novo detection, integrated into the clustering algorithm, can help to guide the process towards more biologically parsimonious solutions. Results We have developed an algorithm, cMonkey, that detects putative co-regulated gene groupings by integrating the biclustering of gene expression data and various functional associations with the de novo detection of sequence motifs. Conclusion We have applied this procedure to the archaeon Halobacterium NRC-1, as part of our efforts to decipher its regulatory network. In addition, we used cMonkey on public data for three organisms in the other two domains of life: Helicobacter pylori, Saccharomyces cerevisiae, and Escherichia coli. The biclusters detected by cMonkey both recapitulated known biology and enabled novel predictions (some for Halobacterium were subsequently confirmed in the laboratory). For example, it identified the bacteriorhodopsin regulon, assigned additional genes to this regulon with apparently unrelated function, and detected its known promoter motif. We have performed a thorough comparison of cMonkey results against other clustering methods, and find that

  17. Autoimmune Hepatitis: Progress from Global Immunosuppression to Personalised Regulatory T Cell Therapy

    PubMed Central

    Than, Nwe Ni; Jeffery, Hannah C.; Oo, Ye H.

    2016-01-01

    Autoimmune hepatitis (AIH) is an immune mediated liver injury. The precise aetiology of AIH is still unknown but current evidence suggests both genetic and environmental factors are involved. Breakdown in peripheral self-tolerance, and impaired functions of FOXP3+ regulatory T cell along with effector cell resistance to suppression at the tissue level seem to play an important role in AIH immunopathogenesis. AIH is predominantly a T lymphocytes driven disease but B lymphocytes are also involved in the immunopathology. Innate immune cells are crucial in the initial onset of disease and their response is followed by adaptive T (Th1, Th17, and cytotoxic T cells) and B cell responses evidenced by liver histology and peripheral blood serology. Standard treatment regimens involving steroid and immunosuppressive medications lead to global immune suppression requiring life-long therapy with many side effects. Biologic therapies have been attempted but duration of remission is short-lived. Future direction of diagnosis and treatment for AIH should be guided by “omics” and the immunology profile of the individual patient and clinicians should aim to deliver personalised medicine for their patients. Cell therapy such as infusion of autologous, antigen-specific, and liver-homing regulatory T cells to restore hepatic immune tolerance may soon be a potential future treatment for AIH patients. PMID:27446862

  18. The Arabidopsis pyruvate,orthophosphate dikinase regulatory proteins encode a novel, unprecedented Ser/Thr protein kinase primary structure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pyruvate,orthophosphate dikinase (PPDK) is a ubiquitous, low abundance metabolic enzyme of undetermined function in C3 plants. Its activity in C3 chloroplasts is light-regulated via reversible phosphorylation of an active-site Thr residue by the PPDK regulatory protein (RP), a most unusual, bifuncti...

  19. Global organization of protein complexome in the yeast Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

    Background Proteins in organisms, rather than act alone, usually form protein complexes to perform cellular functions. We analyze the topological network structure of protein complexes and their component proteins in the budding yeast in terms of the bipartite network and its projections, where the complexes and proteins are its two distinct components. Compared to conventional protein-protein interaction networks, the networks from the protein complexes show more homogeneous structures than those of the binary protein interactions, implying the formation of complexes that cause a relatively more uniform number of interaction partners. In addition, we suggest a new optimization method to determine the abundance and function of protein complexes, based on the information of their global organization. Estimating abundance and biological functions is of great importance for many researches, by providing a quantitative description of cell behaviors, instead of just a "catalogues" of the lists of protein interactions. Results With our new optimization method, we present genome-wide assignments of abundance and biological functions for complexes, as well as previously unknown abundance and functions of proteins, which can provide significant information for further investigations in proteomics. It is strongly supported by a number of biologically relevant examples, such as the relationship between the cytoskeleton proteins and signal transduction and the metabolic enzyme Eno2's involvement in the cell division process. Conclusions We believe that our methods and findings are applicable not only to the specific area of proteomics, but also to much broader areas of systems biology with the concept of optimization principle. PMID:21843333

  20. Regulatory roles of Oct proteins in the mammary gland.

    PubMed

    Qian, Xi; Zhao, Feng-Qi

    2016-06-01

    The expression of Oct-1 and -2 and their binding to the octamer motif in the mammary gland are developmentally and hormonally regulated, consistent with the expression of milk proteins. Both of these transcription factors constitutively bind to the proximal promoter of the milk protein gene β-casein and might be involved in the inhibition or activation of promoter activity via interactions with other transcription factors or cofactors at different developmental stages. In particular, the lactogenic hormone prolactin and glucocorticoids induce Oct-1 and Oct-2 binding and interaction with both the signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid receptor on the β-casein promoter to activate β-casein expression. In addition, increasing evidence has shown the involvement of another Oct factor, Oct-3/4, in mammary tumorigenesis, making Oct-3/4 an emerging prognostic marker of breast cancer and a molecular target for the gene-directed therapeutic intervention, prevention and treatment of breast cancer. This article is part of a Special Issue entitled: The Oct Transcription Factor Family, edited by Dr. Dean Tantin. PMID:27044595

  1. Regulatory effects of matrix protein variations on influenza virus growth.

    PubMed

    Yasuda, J; Toyoda, T; Nakayama, M; Ishihama, A

    1993-01-01

    Influenza virus A/WSN/33 forms large plaques (> 3 mm diameter) on MDCK cells whereas A/Aichi/2/68 forms only small plaques (< 1 mm diameter). Fast growing reassortants (AWM), isolated by mixed infection of MDCK cells with these two virus strains in the presence of anti-WSN antibodies, all carried the M gene from WSN. On MDCK cells, these reassortants produced progeny viruses as rapidly as did WSN, and the virus yield was as high as Aichi. The fast-growing reassortants overcame the growth inhibitory effect of lignins. Pulse-labeling experiments at various times after virus infection showed that the reassortant AWM started to synthesize viral proteins earlier than Aichi. Taken together, we conclude that upon infecting MDCK cells, the reassortant viruses advance rapidly into the growth cycle, thereby leading to an elevated level of progeny viruses in the early period of infection. Possible mechanisms of the M gene involvement in the determination of virus growth rate are discussed, in connection with multiple functions of the M proteins. PMID:8257290

  2. Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization

    PubMed Central

    Vinogradova, Maia V.; Malanina, Galina G.; Waitzman, Joshua S.; Rice, Sarah E.; Fletterick, Robert J.

    2013-01-01

    Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca2+-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca2+ signaling since Ca2+- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface. PMID:23805258

  3. Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization.

    PubMed

    Vinogradova, Maia V; Malanina, Galina G; Waitzman, Joshua S; Rice, Sarah E; Fletterick, Robert J

    2013-01-01

    Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca(2+)-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca(2+) signaling since Ca(2+)- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface. PMID:23805258

  4. The impact of RGS and other G-protein regulatory proteins on Gαi-mediated signaling in immunity.

    PubMed

    Kehrl, John H

    2016-08-15

    Leukocyte chemoattractant receptors are members of the G-protein coupled receptor (GPCR) family. Signaling downstream of these receptors directs the localization, positioning and homeostatic trafficking of leukocytes; as well as their recruitment to, and their retention at, inflammatory sites. Ligand induced changes in the molecular conformation of chemoattractant receptors results in the engagement of heterotrimeric G-proteins, which promotes α subunits to undergo GTP/GDP exchange. This results in the functional release of βγ subunits from the heterotrimers, thereby activating downstream effector molecules, which initiate leukocyte polarization, gradient sensing, and directional migration. Pertussis toxin ADP ribosylates Gαi subunits and prevents chemoattractant receptors from triggering Gαi nucleotide exchange. The use of pertussis toxin revealed the essential importance of Gαi subunit nucleotide exchange for chemoattractant receptor signaling. More recent studies have identified a range of regulatory mechanisms that target these receptors and their associated heterotrimeric G-proteins, thereby helping to control the magnitude, kinetics, and duration of signaling. A failure in these regulatory pathways can lead to impaired receptor signaling and immunopathology. The analysis of mice with targeted deletions of Gαi isoforms as well as some of these G-protein regulatory proteins is providing insights into their roles in chemoattractant receptor signaling. PMID:27071343

  5. A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

    PubMed Central

    Neupert, B; Menotti, E; Kühn, L C

    1995-01-01

    We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151. Images PMID:7544459

  6. Inhibition of GDP/GTP exchange on G alpha subunits by proteins containing G-protein regulatory motifs.

    PubMed

    Natochin, M; Gasimov, K G; Artemyev, N O

    2001-05-01

    A novel Galpha binding consensus sequence, termed G-protein regulatory (GPR) or GoLoco motif, has been identified in a growing number of proteins, which are thought to modulate G-protein signaling. Alternative roles of GPR proteins as nucleotide exchange factors or as GDP dissociation inhibitors for Galpha have been proposed. We investigated the modulation of the GDP/GTP exchange of Gialpha(1), Goalpha, and Gsalpha by three proteins containing GPR motifs (GPR proteins), LGN-585-642, Pcp2, and RapIGAPII-23-131, to elucidate the mechanisms of GPR protein function. The GPR proteins displayed similar patterns of interaction with Gialpha(1) with the following order of affinities: Gialpha(1)GDP > Gialpha(1)GDPAlF(4)(-) > or = Gialpha(1)GTPgammaS. No detectable binding of the GPR proteins to Gsalpha was observed. LGN-585-642, Pcp2, and RapIGAPII-23-131 inhibited the rates of spontaneous GTPgammaS binding and blocked GDP release from Gialpha(1) and Goalpha. The inhibitory effects of the GPR proteins on Gialpha(1) were significantly more potent, indicating that Gi might be a preferred target for these modulators. Our results suggest that GPR proteins are potent GDP dissociation inhibitors for Gialpha-like Galpha subunits in vitro, and in this capacity they may inhibit GPCR/Gi protein signaling in vivo. PMID:11318657

  7. Protein Surface Matching by Combining Local and Global Geometric Information

    PubMed Central

    Ellingson, Leif; Zhang, Jinfeng

    2012-01-01

    Comparison of the binding sites of proteins is an effective means for predicting protein functions based on their structure information. Despite the importance of this problem and much research in the past, it is still very challenging to predict the binding ligands from the atomic structures of protein binding sites. Here, we designed a new algorithm, TIPSA (Triangulation-based Iterative-closest-point for Protein Surface Alignment), based on the iterative closest point (ICP) algorithm. TIPSA aims to find the maximum number of atoms that can be superposed between two protein binding sites, where any pair of superposed atoms has a distance smaller than a given threshold. The search starts from similar tetrahedra between two binding sites obtained from 3D Delaunay triangulation and uses the Hungarian algorithm to find additional matched atoms. We found that, due to the plasticity of protein binding sites, matching the rigid body of point clouds of protein binding sites is not adequate for satisfactory binding ligand prediction. We further incorporated global geometric information, the radius of gyration of binding site atoms, and used nearest neighbor classification for binding site prediction. Tested on benchmark data, our method achieved a performance comparable to the best methods in the literature, while simultaneously providing the common atom set and atom correspondences. PMID:22815760

  8. Protein surface matching by combining local and global geometric information.

    PubMed

    Ellingson, Leif; Zhang, Jinfeng

    2012-01-01

    Comparison of the binding sites of proteins is an effective means for predicting protein functions based on their structure information. Despite the importance of this problem and much research in the past, it is still very challenging to predict the binding ligands from the atomic structures of protein binding sites. Here, we designed a new algorithm, TIPSA (Triangulation-based Iterative-closest-point for Protein Surface Alignment), based on the iterative closest point (ICP) algorithm. TIPSA aims to find the maximum number of atoms that can be superposed between two protein binding sites, where any pair of superposed atoms has a distance smaller than a given threshold. The search starts from similar tetrahedra between two binding sites obtained from 3D Delaunay triangulation and uses the Hungarian algorithm to find additional matched atoms. We found that, due to the plasticity of protein binding sites, matching the rigid body of point clouds of protein binding sites is not adequate for satisfactory binding ligand prediction. We further incorporated global geometric information, the radius of gyration of binding site atoms, and used nearest neighbor classification for binding site prediction. Tested on benchmark data, our method achieved a performance comparable to the best methods in the literature, while simultaneously providing the common atom set and atom correspondences. PMID:22815760

  9. Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry.

    PubMed

    Xiao, Yongsheng; Wang, Yinsheng

    2016-09-01

    Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein-nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:601-619, 2016. PMID:25376990

  10. Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences.

    PubMed

    De Jaeger, Geert; Scheffer, Stanley; Jacobs, Anni; Zambre, Mukund; Zobell, Oliver; Goossens, Alain; Depicker, Ann; Angenon, Geert

    2002-12-01

    Over the past decade, several high value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds. Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops. In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large scale production. PMID:12415287

  11. BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1α

    PubMed Central

    Rodriguez, Diego A; Zamorano, Sebastian; Lisbona, Fernanda; Rojas-Rivera, Diego; Urra, Hery; Cubillos-Ruiz, Juan R; Armisen, Ricardo; Henriquez, Daniel R; H Cheng, Emily; Letek, Michal; Vaisar, Tomas; Irrazabal, Thergiory; Gonzalez-Billault, Christian; Letai, Anthony; Pimentel-Muiños, Felipe X; Kroemer, Guido; Hetz, Claudio

    2012-01-01

    Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions. PMID:22510886

  12. Optimizing a global alignment of protein interaction networks

    PubMed Central

    Chindelevitch, Leonid; Ma, Cheng-Yu; Liao, Chung-Shou; Berger, Bonnie

    2013-01-01

    Motivation: The global alignment of protein interaction networks is a widely studied problem. It is an important first step in understanding the relationship between the proteins in different species and identifying functional orthologs. Furthermore, it can provide useful insights into the species’ evolution. Results: We propose a novel algorithm, PISwap, for optimizing global pairwise alignments of protein interaction networks, based on a local optimization heuristic that has previously demonstrated its effectiveness for a variety of other intractable problems. PISwap can begin with different types of network alignment approaches and then iteratively adjust the initial alignments by incorporating network topology information, trading it off for sequence information. In practice, our algorithm efficiently refines other well-studied alignment techniques with almost no additional time cost. We also show the robustness of the algorithm to noise in protein interaction data. In addition, the flexible nature of this algorithm makes it suitable for different applications of network alignment. This algorithm can yield interesting insights into the evolutionary dynamics of related species. Availability: Our software is freely available for non-commercial purposes from our Web site, http://piswap.csail.mit.edu/. Contact: bab@csail.mit.edu or csliao@ie.nthu.edu.tw Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24048352

  13. The global regulatory system Csr senses glucose through the phosphoenolpyruvate: carbohydrate phosphotransferase system.

    PubMed

    Pérez-Morales, Deyanira; Bustamante, Víctor H

    2016-02-01

    A novel connection between two regulatory systems controlling crucial biological processes in bacteria, the carbon storage regulator (Csr) system and the glucose-specific phosphotransferase system (PTS), is reported by Leng et al. in this issue. This involves the interaction of unphosphorylated EIIA(Glc), a component of the glucose-specific PTS, with the CsrD protein, which accelerates the decay of the CsrB and CsrC small RNAs via RNase E in Escherichia coli. As unphosphorylated EIIA(G) (lc) is generated in the presence of glucose, the PTS thus acts as a sensor of glucose for the Csr system. Interestingly, another pathway can operate for communication between the Csr system and the glucose-specific PTS. The absence of glucose generates phosphorylated EIIA(Glc) , which activates the enzyme adenylate cyclase to produce cyclic adenosine monophosphate (cAMP) that, in turn, binds to the regulator cAMP receptor protein (CRP). Leng et al. show that the complex cAMP-CRP modestly reduces CsrB decay independently of CsrD. On the other hand, a previous study indicates that the complex cAMP-CRP positively regulates the transcription of CsrB and CsrC in Salmonella enterica. Therefore, EIIA(G) (lc) could work as a molecular switch that regulates the activity of the Csr system, in response to its phosphorylation state determined by the presence or absence of glucose, in order to control gene expression. PMID:26593223

  14. Global, quantitative and dynamic mapping of protein subcellular localization

    PubMed Central

    Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

    2016-01-01

    Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

  15. Global Conservation of Protein Status between Cell Lines and Xenografts.

    PubMed

    Biau, Julian; Chautard, Emmanuel; Court, Frank; Pereira, Bruno; Verrelle, Pierre; Devun, Flavien; De Koning, Leanne; Dutreix, Marie

    2016-08-01

    Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery. PMID:27567954

  16. Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells

    PubMed Central

    Bourseau-Guilmain, E.; Menard, J. A.; Lindqvist, E.; Indira Chandran, V.; Christianson, H. C.; Cerezo Magaña, M.; Lidfeldt, J.; Marko-Varga, G.; Welinder, C.; Belting, M.

    2016-01-01

    Hypoxia promotes tumour aggressiveness and resistance of cancers to oncological treatment. The identification of cancer cell internalizing antigens for drug targeting to the hypoxic tumour niche remains a challenge of high clinical relevance. Here we show that hypoxia down-regulates the surface proteome at the global level and, more specifically, membrane proteome internalization. We find that hypoxic down-regulation of constitutive endocytosis is HIF-independent, and involves caveolin-1-mediated inhibition of dynamin-dependent, membrane raft endocytosis. Caveolin-1 overexpression inhibits protein internalization, suggesting a general negative regulatory role of caveolin-1 in endocytosis. In contrast to this global inhibitory effect, we identify several proteins that can override caveolin-1 negative regulation, exhibiting increased internalization at hypoxia. We demonstrate antibody-mediated cytotoxin delivery and killing specifically of hypoxic cells through one of these proteins, carbonic anhydrase IX. Our data reveal that caveolin-1 modulates cell-surface proteome turnover at hypoxia with potential implications for specific targeting of the hypoxic tumour microenvironment. PMID:27094744

  17. Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells.

    PubMed

    Bourseau-Guilmain, E; Menard, J A; Lindqvist, E; Indira Chandran, V; Christianson, H C; Cerezo Magaña, M; Lidfeldt, J; Marko-Varga, G; Welinder, C; Belting, M

    2016-01-01

    Hypoxia promotes tumour aggressiveness and resistance of cancers to oncological treatment. The identification of cancer cell internalizing antigens for drug targeting to the hypoxic tumour niche remains a challenge of high clinical relevance. Here we show that hypoxia down-regulates the surface proteome at the global level and, more specifically, membrane proteome internalization. We find that hypoxic down-regulation of constitutive endocytosis is HIF-independent, and involves caveolin-1-mediated inhibition of dynamin-dependent, membrane raft endocytosis. Caveolin-1 overexpression inhibits protein internalization, suggesting a general negative regulatory role of caveolin-1 in endocytosis. In contrast to this global inhibitory effect, we identify several proteins that can override caveolin-1 negative regulation, exhibiting increased internalization at hypoxia. We demonstrate antibody-mediated cytotoxin delivery and killing specifically of hypoxic cells through one of these proteins, carbonic anhydrase IX. Our data reveal that caveolin-1 modulates cell-surface proteome turnover at hypoxia with potential implications for specific targeting of the hypoxic tumour microenvironment. PMID:27094744

  18. Interaction and localization diversities of global and local hubs in human protein-protein interaction networks.

    PubMed

    Kiran, M; Nagarajaram, H A

    2016-08-16

    Hubs, the highly connected nodes in protein-protein interaction networks (PPINs), are associated with several characteristic properties and are known to perform vital roles in cells. We defined two classes of hubs, global (housekeeping) and local (tissue-specific) hubs. These two categories of hubs are distinct from each other with respect to their abundance, structure and function. However, how distinct are the spatial expression pattern and other characteristics of their interacting partners is still not known. Our investigations revealed that the partners of the local hubs compared with those of global hubs are conserved across the tissues in which they are expressed. Partners of local hubs show diverse subcellular localizations as compared with the partners of global hubs. We examined the nature of interacting domains in both categories of hubs and found that they are promiscuous in global hubs but not so in local hubs. Deletion of some of the local and global hubs has an impact on the characteristic path length of the network indicating that those hubs are inter-modular in nature. Our present study has, therefore, shed further light on the characteristic features of the local and global hubs in human PPIN. This knowledge of different topological aspects of hubs with regard to their types and subtypes is essential as it helps in better understanding of roles of hub proteins in various cellular processes under various conditions including those caused by host-pathogen interactions and therefore useful in prioritizing targets for drug design and repositioning. PMID:27400769

  19. Interaction between Major Nitrogen Regulatory Protein NIT2 and Pathway-Specific Regulatory Factor NIT4 Is Required for Their Synergistic Activation of Gene Expression in Neurospora crassa

    PubMed Central

    Feng, Bo; Marzluf, George A.

    1998-01-01

    In Neurospora crassa, the major nitrogen regulatory protein, NIT2, a member of the GATA family of transcription factors, controls positively the expression of numerous genes which specify nitrogen catabolic enzymes. Expression of the highly regulated structural gene nit-3, which encodes nitrate reductase, is dependent upon a synergistic interaction of NIT2 with a pathway-specific control protein, NIT4, a member of the GAL4 family of fungal regulatory factors. The NIT2 and NIT4 proteins both bind at specific recognition elements in the nit-3 promoter, but, in addition, we show that a direct protein-protein interaction between NIT2 and NIT4 is essential for optimal expression of the nit-3 structural gene. Neurospora possesses at least five different GATA factors which control different areas of cellular function, but which have a similar DNA binding specificity. Significantly, only NIT2, of the several Neurospora GATA factors examined, interacts with NIT4. We propose that protein-protein interactions of the individual GATA factors with additional pathway-specific regulatory factors determine each of their specific regulatory functions. PMID:9632783

  20. Governing effect of regulatory proteins for Cl(-)/HCO3(-) exchanger 2 activity.

    PubMed

    Jeong, Yon Soo; Hong, Jeong Hee

    2016-05-01

    Anion exchanger 2 (AE2) has a critical role in epithelial cells and is involved in the ionic homeostasis such as Cl(-) uptake and HCO3(-) secretion. However, little is known about the regulatory mechanism of AE2. The main goal of the present study was to investigate potential regulators, such as spinophilin (SPL), inositol-1,4,5-trisphosphate [IP3] receptors binding protein released with IP3 (IRBIT), STE20/SPS1-related proline/alanine-rich kinase (SPAK) kinase, and carbonic anhydrase XII (CA XII). We found that SPL binds to AE2 and markedly increased the Cl(-)/HCO3(-) exchange activity of AE2. Especially SPL 1-480 domain is required for enhancing AE2 activity. For other regulatory components that affect the fidelity of fluid and HCO3(-) secretion, IRBIT and SPAK had no effect on the activity of AE2 and no protein-protein interaction with AE2. It has been proposed that CA activity is closely associated with AE activity. In this study, we provide evidence that the basolateral membrane-associated CA isoform CA XII significantly increased the activity of AE2 and co-localized with AE2 to the plasma membrane. Collectively, SPL and CA XII enhanced the Cl(-)/HCO3(-) exchange activity of AE2. The modulating action of these regulatory proteins could serve as potential therapeutic targets for secretory diseases mediated by AE2. PMID:26716707

  1. Global Regulatory T-Cell Research from 2000 to 2015: A Bibliometric Analysis.

    PubMed

    Zongyi, Yin; Dongying, Chen; Baifeng, Li

    2016-01-01

    We aimed to analyze the global scientific output of regulatory T-cell (Treg) research and built a model to qualitatively and quantitatively evaluate publications from 2000 to 2015. Data were obtained from the Web of Science Core Collection (WoSCC) of Thomson Reuters on January 1, 2016. The bibliometric method and Citespace III were used to analyze authors, journals, publication outputs, institutions, countries, research areas, research hotspots, and trends. In total, we identified 35,741 publications on Treg research from 2000 to 2015, and observed that the annual publication rate increased with time. The Journal of Immunology published the highest number of articles, the leading country was the USA, and the leading institute was Harvard University. Sakaguchi, Hori, Fontenot, and Wang were the top authors in Treg research. Immunology accounted for the highest number of publications, followed by oncology, experimental medicine, cell biology, and hematology. Keyword analysis indicated that autoimmunity, inflammation, cytokine, gene expression, foxp3, and immunotherapy were the research hotspots, whereas autoimmune inflammation, gene therapy, granzyme B, RORγt, and th17 were the frontiers of Treg research. This bibliometric analysis revealed that Treg-related studies are still research hotspots, and that Treg-related clinical therapies are the research frontiers; however, further study and collaborations are needed worldwide. Overall, our findings provide valuable information for the editors of immunology journals to identify new perspectives and shape future research directions. PMID:27611317

  2. Drug policy and global regulatory capitalism: the case of new psychoactive substances (NPS).

    PubMed

    Seddon, Toby

    2014-09-01

    The recent emergence of vibrant markets in 'new psychoactive substances' or 'legal highs' has posed significant new challenges for drug policy. These partly concern what to do about them but the speed and complexity of change has also raised difficulties for how policy responses should be developed. Existing drug policy systems appear too slow and cumbersome to keep up with the pace of change, remaining locked in large part within 'old' ways of thinking that centre almost exclusively around the deployment (or not) of the criminal law and its related enforcement apparatus. In this paper, it is argued that we need to rethink the problem through the lens of regulation, in order to learn lessons from other sectors where more agile responses to changing markets and business innovation have often proved possible. By examining examples drawn from these other areas, an alternative policy-making framework can be developed, involving a more flexible mix of state regulation, civil society action and private law mechanisms. This new approach is founded on a recognition of the networked and polycentric character of effective market governance in an era of global regulatory capitalism. PMID:24768473

  3. Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation

    SciTech Connect

    Ansong, Charles; Yoon, Hyunjin; Porwollik, Steffen; Mottaz-Brewer, Heather; Petritis, Brianne O.; Jaitly, Navdeep; Adkins, Joshua N.; Mcclelland, Michael; Heffron, Fred; Smith, Richard D.

    2009-03-11

    In recent years the profound importance of sRNA-mediated translational/post-transcriptional regulation has been increasingly appreciated. However, the global role played by translational regulation in control of gene expression has never been elucidated in any organism for the simple reason that global proteomics methods required to accurately characterize post-transcriptional processes and the knowledge of translational control mechanisms have only become available within the last few years. The proteins Hfq and SmpB are essential for the biological activity of a range of regulatory sRNAs and thus provide a means to identify potential targets of sRNA regulation. We performed a sample-matched global proteomics and transcriptional analysis to examine the role of Hfq and SmpB in global protein translation and virulence using the Salmonella typhimurium model system. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all Salmonella proteins, respectively, with limited correlation between transcription and protein expression. This is the first report suggesting that SmpB could be a general translational regulator. The broad spectrum of proteins modulated by Hfq was also surprising including central metabolism, LPS biosynthesis, two-component regulatory systems, quorum sensing, SP1-TTSS, oxidative stress, fatty acid metabolism, nucleoside and nucleotide metabolism, envelope stress, aminoacyl-tRNA synthetases, amino acid biosynthesis, peptide transport, and motility.. The extent of global regulation of translation by Hfq is unexpected, with profound effects in all stages of Salmonella’s life cycle. Our results represent the first global systems-level analysis of translational regulation; the elucidated potential targets of sRNA regulation from our analysis will

  4. Proteomics technologies for the global identification and quantification of proteins.

    PubMed

    Brewis, Ian A; Brennan, P

    2010-01-01

    This review provides an introduction for the nonspecialist to proteomics and in particular the major approaches available for global protein identification and quantification. Proteomics technologies offer considerable opportunities for improved biological understanding and biomarker discovery. The central platform for proteomics is tandem mass spectrometry (MS) but a number of other technologies, resources, and expertise are absolutely required to perform meaningful experiments. These include protein separation science (and protein biochemistry in general), genomics, and bioinformatics. There are a range of workflows available for protein (or peptide) separation prior to tandem MS and subsequent bioinformatics analysis to achieve protein identifications. The predominant approaches are 2D electrophoresis (2DE) and subsequent MS, liquid chromatography-MS (LC-MS), and GeLC-MS. Beyond protein identification, there are a number of well-established options available for protein quantification. Difference gel electrophoresis (DIGE) following 2DE is one option but MS-based methods (most commonly iTRAQ-Isobaric Tags for Relative and Absolute Quantification or SILAC-Stable Isotope Labeling by Amino Acids) are now the preferred options. Sample preparation is critical to performing good experiments and subcellular fractionation can additionally provide protein localization information compared with whole cell lysates. Differential detergent solubilization is another valid option. With biological fluids, it is possible to remove the most abundant proteins by immunodepletion. Sample enrichment is also used extensively in certain analyses and most commonly in phosphoproteomics with the initial purification of phosphopeptides. Proteomics produces considerable datasets and resources to facilitate the necessary extended analysis of this data are improving all the time. Beyond the opportunities afforded by proteomics there are definite challenges to achieving full proteomic coverage

  5. Binding of regulatory subunits of cyclic AMP-dependent protein kinase to cyclic CMP agarose.

    PubMed

    Hammerschmidt, Andreas; Chatterji, Bijon; Zeiser, Johannes; Schröder, Anke; Genieser, Hans-Gottfried; Pich, Andreas; Kaever, Volkhard; Schwede, Frank; Wolter, Sabine; Seifert, Roland

    2012-01-01

    The bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis as well as soluble guanylyl cyclase α(1)β(1) synthesize the cyclic pyrimidine nucleotide cCMP. These data raise the question to which effector proteins cCMP binds. Recently, we reported that cCMP activates the regulatory subunits RIα and RIIα of cAMP-dependent protein kinase. In this study, we used two cCMP agarose matrices as novel tools in combination with immunoblotting and mass spectrometry to identify cCMP-binding proteins. In agreement with our functional data, RIα and RIIα were identified as cCMP-binding proteins. These data corroborate the notion that cAMP-dependent protein kinase may serve as a cCMP target. PMID:22808067

  6. Influence of Crystal Packing on Global Protein Conformation

    NASA Astrophysics Data System (ADS)

    Ahlstrom, Logan; Miyashita, Osamu

    2011-10-01

    X-ray crystallography is the most robust method for solving protein structure. However, packing forces in the crystal lattice select just a snapshot of a protein's conformational ensemble, whereas proteins are flexible and can adopt different conformations. Here we compare molecular dynamics (MD) simulations in solution and the crystal lattice to add dynamical insight to the static X-ray images of proteins. As a model system, we consider the λ Cro dimer, whose solved X-ray structures range from a ``closed'' to an ``open'' global conformation. Free energy profiles depicting the conformational space sampled by the dimer in solution show some reported structures correspond to stable states. Yet other conformations, while accessible, lie higher in energy, indicating the effect of crystal packing. Subsequent crystal MD simulations estimated the strength of packing interfaces in the lattice, showing the influence of crystal form and mutation in stabilizing different dimer conformations. Our quantitative results will aid analysis of X-ray data in establishing protein structure-function relationships.

  7. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    SciTech Connect

    Volkman, B.F.

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a {open_quote}receiver domain{close_quote} in the family of {open_quote}two-component{close_quote} regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  8. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

    PubMed Central

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-01-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma. PMID:27573585

  9. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma.

    PubMed

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-09-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma. PMID:27573585

  10. Proteomic Shifts in Embryonic Stem Cells with Gene Dose Modifications Suggest the Presence of Balancer Proteins in Protein Regulatory Networks

    PubMed Central

    Mao, Lei; Zabel, Claus; Herrmann, Marion; Nolden, Tobias; Mertes, Florian; Magnol, Laetitia; Chabert, Caroline; Hartl, Daniela; Herault, Yann; Delabar, Jean Maurice; Manke, Thomas; Himmelbauer, Heinz; Klose, Joachim

    2007-01-01

    Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of “balancer” proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the “elasticity” of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions. PMID:18043732

  11. [The effect of extremely low doses of the novel regulatory plant proteins ].

    PubMed

    Krasnov, M S; Margasiuk, D V; Iamskov, I A; Iamskova, V P

    2003-01-01

    Searching and study on regulatory proteins, which can keep under control the scope of important processes as like as cell adhesion, proliferation, differentiation and morphogenesis, is an actual aim of the current biochemistry. Recently we have identified S-100 proteins in plants of following species: plantain (Plantago major L.), aloe (Aloe arborescens L.), and bilberry (Vaccinum myrtillus L.). Extraction and purification of S-100 proteins gotten from these plants were performed by the method we developed earlier for adhesion proteins of animal tissues. Homogeneity of the studied plant proteins was evaluated and confirmed by HPLC and SDS-electrophoresis in PAAG. Both, plant and animal proteins have appeared to be biologically active at extremely low doses. The tests were performed by adhesiometrical method in short-term tissue culture of mouse's liver in vitro. As a result it was established that the plant proteins insert a membranotropic effect being added in extremely low doses, corresponding to 10(-10)-10(-13) mg/ml. Keeping in mind that the plantain is well known remedy for wound protection and healing, in several experiments we studied the biological effect of plant S-100 proteins on animal cells. It was found that S-100 proteins obtained from plantain influences proliferation of human fibroblasts in vitro. It was found that after the treatment with this protein in low doses the cell growth rate increases essentially. PMID:12881977

  12. Neurons Lacking Iron Regulatory Protein-2 Are Highly Resistant to the Toxicity of Hemoglobin

    PubMed Central

    Regan, Raymond F.; Chen, Mai; Li, Zhi; Zhang, Xuefeng; Benvenisti-Zarom, Luna; Chen-Roetling, Jing

    2008-01-01

    The effect of iron regulatory protein-2 (IRP2) on ferritin expression and neuronal vulnerability to hemoglobin was assessed in primary cortical cell cultures prepared from wild-type and IRP2 knockout mice. Baseline levels of H and L-ferritin subunits were significantly increased in IRP2 knockout neurons and astrocytes. Hemoglobin was toxic to wild-type neurons in mixed neuron-astrocyte cultures, with an LC50 near 3 µM for a 24 hour exposure. Neuronal death was reduced by 85–95% in knockout cultures, and also in cultures containing knockout neurons plated on wild-type astrocytes. Protein carbonylation, reactive oxygen species formation, and heme oxygenase-1 expression after hemoglobin treatment were also attenuated by IRP2 gene deletion. These results suggest that IRP2 binding activity increases the vulnerability of neurons to hemoglobin, possibly by reducing ferritin expression. Therapeutic strategies that target this regulatory mechanism may be beneficial after hemorrhagic CNS injuries. PMID:18571425

  13. Collapsin Response Mediator Protein-2 (Crmp2) Regulates Trafficking by Linking Endocytic Regulatory Proteins to Dynein Motors*

    PubMed Central

    Rahajeng, Juliati; Giridharan, Sai S. P.; Naslavsky, Naava; Caplan, Steve

    2010-01-01

    Endocytosis is a conserved cellular process in which nutrients, lipids, and receptors are internalized and transported to early endosomes, where they are sorted and either channeled to degradative pathways or recycled to the plasma membrane. MICAL-L1 and EHD1 are important regulatory proteins that control key endocytic transport steps. However, the precise mechanisms by which they mediate transport, and particularly the mode by which they connect to motor proteins, have remained enigmatic. Here we have identified the collapsin response mediator protein-2 (Crmp2) as an interaction partner of MICAL-L1 in non-neuronal cells. Crmp2 interacts with tubulin dimers and kinesin and negatively regulates dynein-based transport in neuronal cells, but its expression and function in non-neuronal cells have remained poorly characterized. Upon Crmp2 depletion, we observed dramatic relocalization of internalized transferrin (Tf) from peripheral vesicles to the endocytic recycling compartment (ERC), similar to the effect of depleting either MICAL-L1 or EHD1. Moreover, Tf relocalization to the ERC could be inhibited by interfering with microtubule polymerization, consistent with a role for uncoupled motor protein-based transport upon depletion of Crmp2, MICAL-L1, or EHD1. Finally, transfection of dynamitin, a component of the dynactin complex whose overexpression inhibits dynein activity, prevented the relocalization of internalized Tf to the ERC upon depletion of Crmp2, MICAL-L1, or EHD1. These data provide the first trafficking regulatory role for Crmp2 in non-neuronal cells and support a model in which Crmp2 is an important endocytic regulatory protein that links MICAL-L1·EHD1-based vesicular transport to dynein motors. PMID:20801876

  14. Global Low Frequency Protein Motions in Long-Range Allosteric Signaling

    NASA Astrophysics Data System (ADS)

    McLeish, Tom; Rogers, Thomas; Townsend, Philip; Burnell, David; Pohl, Ehmke; Wilson, Mark; Cann, Martin; Richards, Shane; Jones, Matthew

    2015-03-01

    We present a foundational theory for how allostery can occur as a function of low frequency dynamics without a change in protein structure. Elastic inhomogeneities allow entropic ``signalling at a distance.'' Remarkably, many globular proteins display just this class of elastic structure, in particular those that support allosteric binding of substrates (long-range co-operative effects between the binding sites of small molecules). Through multi-scale modelling of global normal modes we demonstrate negative co-operativity between the two cAMP ligands without change to the mean structure. Crucially, the value of the co-operativity is itself controlled by the interactions around a set of third allosteric ``control sites.'' The theory makes key experimental predictions, validated by analysis of variant proteins by a combination of structural biology and isothermal calorimetry. A quantitative description of allostery as a free energy landscape revealed a protein ``design space'' that identified the key inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, by analyzing naturally occurring CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. The methodology establishes the means to engineer allosteric mechanisms that are driven by low frequency dynamics.

  15. Regulatory Protein-Protein Interactions in Primary Metabolism: The Case of the Cysteine Synthase Complex

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sulfur is an essential nutrient for plant growth and development. In plant sulfur assimilation, cysteine biosynthesis plays a central role in fixing inorganic sulfur from the environment into the metabolic precursor for cellular thiol-containing compounds. A key regulatory feature of this process ...

  16. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques.

    PubMed

    Dhore, C R; Cleutjens, J P; Lutgens, E; Cleutjens, K B; Geusens, P P; Kitslaar, P J; Tordoir, J H; Spronk, H M; Vermeer, C; Daemen, M J

    2001-12-01

    In the present study, we examined the expression of regulators of bone formation and osteoclastogenesis in human atherosclerosis because accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. The most striking finding of this study was the constitutive immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein in nondiseased aortas and the absence of bone morphogenetic protein (BMP)-2, BMP-4, osteopontin, and osteonectin in nondiseased aortas and early atherosclerotic lesions. When atherosclerotic plaques demonstrated calcification or bone formation, BMP-2, BMP-4, osteopontin, and osteonectin were upregulated. Interestingly, this upregulation was associated with a sustained immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein. The 2 modulators of osteoclastogenesis (osteoprotegerin [OPG] and its ligand, OPGL) were present in the nondiseased vessel wall and in early atherosclerotic lesions. In advanced calcified lesions, OPG was present in bone structures, whereas OPGL was only present in the extracellular matrix surrounding calcium deposits. The observed expression patterns suggest a tight regulation of the expression of bone matrix regulatory proteins during human atherogenesis. The expression pattern of both OPG and OPGL during atherogenesis might suggest a regulatory role of these proteins not only in osteoclastogenesis but also in atherosclerotic calcification. PMID:11742876

  17. The cAMP-binding proteins of Leishmania are not the regulatory subunits of cAMP-dependent protein kinase.

    PubMed

    Banerjee, C; Sarkar, D

    2001-09-01

    The most commonly used method to determine the cAMP binding activity in cytosolic extracts of promastigotes of Leishmania spp. underestimated by approximately 11.5-fold the total amount of [(3)H]cAMP bound, when compared with results obtained by the modified Millipore filter technique. Three cAMP-binding proteins (BPI, BPII and BPIII) were partially purified and characterized. The native molecular masses of BPI, BPII and BPIII were estimated to be 105, 155 and 145 kDa, respectively. The binding of [(3)H]cAMP to these proteins was affected to different extents by several cAMP analogues. Antibodies directed against the types I and II regulatory subunits of PKA did not cross-react with the leishmanial extract. Photoaffinity labeling of the cytosolic extracts with 8-N(3)-[(32)P]cAMP specifically labeled a band of M(r) 116000 and a band of M(r) 80000 partially saturable by cAMP. From these results, it is concluded that the leishmanial cAMP-binding proteins appear to belong to a different class distinct from the regulatory subunits of cAMP-dependent protein kinases. PMID:11544092

  18. The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis

    PubMed Central

    Jones, Danielle M.; Murray, Christian M.; Ketelaar, KassaDee J.; Thomas, Joseph J.; Villalobos, Jose A.; Wallace, Ian S.

    2016-01-01

    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites. PMID:27252710

  19. The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis.

    PubMed

    Jones, Danielle M; Murray, Christian M; Ketelaar, KassaDee J; Thomas, Joseph J; Villalobos, Jose A; Wallace, Ian S

    2016-01-01

    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites. PMID:27252710

  20. SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium-triggered fusion

    PubMed Central

    Bharat, Tanmay A M; Malsam, Jörg; Hagen, Wim J H; Scheutzow, Andrea; Söllner, Thomas H; Briggs, John A G

    2014-01-01

    Synaptic vesicles fuse with the plasma membrane in response to Ca2+ influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large-scale, automated cryo-electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca2+-triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high-energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp. PMID:24493260

  1. RNA regulatory networks diversified through curvature of the PUF protein scaffold

    PubMed Central

    Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P.; Nevil, Markus; Campbell, Zachary T.; Tanaka Hall, Traci M.; Wickens, Marvin

    2015-01-01

    Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks. PMID:26364903

  2. RNA regulatory networks diversified through curvature of the PUF protein scaffold

    SciTech Connect

    Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P.; Nevil, Markus; Campbell, Zachary T.; Tanaka Hall, Traci M.; Wickens, Marvin

    2015-09-14

    Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.

  3. RNA regulatory networks diversified through curvature of the PUF protein scaffold

    DOE PAGESBeta

    Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P.; Nevil, Markus; Campbell, Zachary T.; Tanaka Hall, Traci M.; Wickens, Marvin

    2015-09-14

    Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extendedmore » interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.« less

  4. RNA regulatory networks diversified through curvature of the PUF protein scaffold.

    PubMed

    Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P; Nevil, Markus; Campbell, Zachary T; Tanaka Hall, Traci M; Wickens, Marvin

    2015-01-01

    Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p-RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks. PMID:26364903

  5. Disruption of a global regulatory gene to enhance central carbon flux into phenylalanine biosynthesis in Escherichia coli.

    PubMed

    Tatarko, M; Romeo, T

    2001-07-01

    Genetic engineering of microbes for commercial metabolite production traditionally has sought to alter the levels and/or intrinsic activities of key enzymes in relevant biosynthetic pathway(s). Microorganisms exploit similar strategies for flux control, but also coordinate flux through sets of related pathways by using global regulatory circuits. We have engineered a global regulatory system of Escherichia coli, Csr (carbon storage regulator), to increase precursor for aromatic amino acid biosynthesis. Disruption of csrA increases gluconeogenesis, decreases glycolysis, and thus elevates phosphoenolpyruvate, a limiting precursor of aromatics. A strain in which the aromatic (shikimate) pathway had been optimized produced twofold more phenylalanine when csrA was disrupted. Overexpression of tktA (transketolase) to increase the other precursor, erythrose-4-phosphate, yielded approximately 1.4-fold enhancement, while both changes were additive. These effects of csrA were not mediated by increasing the regulatory enzymes of phenylalanine biosynthesis. This study introduces the concept of "global metabolic engineering" for second-generation strain improvement. PMID:11375660

  6. Bacterial Iron–Sulfur Regulatory Proteins As Biological Sensor-Switches

    PubMed Central

    Crack, Jason C.; Green, Jeffrey; Hutchings, Matthew I.; Thomson, Andrew J.

    2012-01-01

    Abstract Significance: In recent years, bacterial iron–sulfur cluster proteins that function as regulators of gene transcription have emerged as a major new group. In all cases, the cluster acts as a sensor of the environment and enables the organism to adapt to the prevailing conditions. This can range from mounting a response to oxidative or nitrosative stress to switching between anaerobic and aerobic respiratory pathways. The sensitivity of these ancient cofactors to small molecule reactive oxygen and nitrogen species, in particular, makes them ideally suited to function as sensors. Recent Advances: An important challenge is to obtain mechanistic and structural information about how these regulators function and, in particular, how the chemistry occurring at the cluster drives the subsequent regulatory response. For several regulators, including FNR, SoxR, NsrR, IscR, and Wbl proteins, major advances in understanding have been gained recently and these are reviewed here. Critical Issues: A common theme emerging from these studies is that the sensitivity and specificity of the cluster of each regulatory protein must be exquisitely controlled by the protein environment of the cluster. Future Directions: A major future challenge is to determine, for a range of regulators, the key factors for achieving control of sensitivity/specificity. Such information will lead, eventually, to a system understanding of stress response, which often involves more than one regulator. Antioxid. Redox Signal. 17, 1215–1231. PMID:22239203

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

    PubMed Central

    Jaarsma, Dick; Hoogenraad, Casper C.

    2015-01-01

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

  8. Promoter analysis reveals globally differential regulation of human long non-coding RNA and protein-coding genes

    SciTech Connect

    Alam, Tanvir; Medvedeva, Yulia A.; Jia, Hui; Brown, James B.; Lipovich, Leonard; Bajic, Vladimir B.; Mantovani, Roberto

    2014-10-02

    Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future.

  9. Promoter analysis reveals globally differential regulation of human long non-coding RNA and protein-coding genes

    DOE PAGESBeta

    Alam, Tanvir; Medvedeva, Yulia A.; Jia, Hui; Brown, James B.; Lipovich, Leonard; Bajic, Vladimir B.; Mantovani, Roberto

    2014-10-02

    Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptionalmore » regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future.« less

  10. Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes

    PubMed Central

    Jia, Hui; Brown, James B.; Lipovich, Leonard; Bajic, Vladimir B.

    2014-01-01

    Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future. PMID:25275320

  11. Mys protein regulates protein kinase A activity by interacting with regulatory type Ialpha subunit during vertebrate development.

    PubMed

    Kotani, Tomoya; Iemura, Shun-ichiro; Natsume, Tohru; Kawakami, Koichi; Yamashita, Masakane

    2010-02-12

    During embryonic development, protein kinase A (PKA) plays a key role in cell fate specification by antagonizing the Hedgehog (Hh) signaling pathway. However, the mechanism by which PKA activity is regulated remains unknown. Here we show that the Misty somites (Mys) protein regulates the level of PKA activity during embryonic development in zebrafish. We isolate PKA regulatory type Ialpha subunit (Prkar1a) as a protein interacting with Mys by pulldown assay in HEK293 cells followed by mass spectrometry analysis. We show an interaction between endogenous Mys and Prkar1a in the zebrafish embryo. Mys binds to Prkar1a in its C terminus region, termed PRB domain, and activates PKA in vitro. Conversely, knockdown of Mys in zebrafish embryos results in reduction in PKA activity. We also show that knockdown of Mys induces ectopic activation of Hh target genes in the eyes, neural tube, and somites downstream of Smoothened, a protein essential for transduction of Hh signaling activity. The altered patterning of gene expression is rescued by activation of PKA. Together, our results reveal a molecular mechanism of regulation of PKA activity that is dependent on a protein-protein interaction and demonstrate that PKA activity regulated by Mys is indispensable for negative regulation of the Hh signaling pathway in Hh-responsive cells. PMID:20018846

  12. Structure of the Na,K-ATPase regulatory protein FXYD1 in micelles†

    PubMed Central

    Teriete, Peter; Franzin, Carla M.; Choi, Jungyuen; Marassi, Francesca M.

    2008-01-01

    FXYD1 is a major regulatory subunit of the Na,K-ATPase, and the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinases A and C in heart and skeletal muscle sarcolemma. It is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here we present the three-dimensional structure of FXYD1 determined in micelles by NMR spectroscopy. Structure determination was made possible by measuring residual dipolar couplings in weakly oriented micelle samples of the protein. This allowed us to obtain the relative orientations of the helical segments of the protein, and also provided information about the protein dynamics. The structural analysis was further facilitated by the inclusion of distance restraints, obtained from paramagnetic spin label relaxation enhancements, and by refinement with a micelle depth restraint, derived from paramagnetic Mn line broadening effects. The structure of FXYD1 provides the foundation for understanding its intra-membrane association with the Na,K-ATPase α subunit, and suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein, to modulate its interaction with the α subunit. PMID:17511473

  13. Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens.

    PubMed

    Smith, L Cody; Clark, Jessica C; Bisesi, Joseph H; Ferguson, P Lee; Sabo-Attwood, Tara

    2016-09-01

    The diverse biological effects of xenoestrogens may be explained by their ability to differentially recruit co-regulatory proteins to the estrogen receptor (ER). We employed high-throughput receptor affinity binding and co-regulatory protein recruitment screening assays based on fluorescence polarization and time resolved florescence resonance energy transfer (TR-FRET), respectively, to assess xenoestrogen-specific binding and co-regulatory protein recruitment to the ER. Then we used a functional proteomic assay based on co-immunoprecipitation of ER-bound proteins to isolate and identify intact co-regulatory proteins recruited to a ligand-activated ER. Through these approaches, we revealed differential binding affinity of bisphenol-A (BPA) and genistein (GEN) to the human ERα (ESR1) and ligand-dependent recruitment of SRC-1 and SRC-3 peptides. Recruitment profiles were variable for each ligand and in some cases were distinct compared to 17β-estradiol (E2). For example, E2 and GEN recruited both SRC-1 and -3 peptides whereas BPA recruited only SRC-1 peptides. Results of the functional proteomic assay showed differential recruitment between ligands where E2 recruited the greatest number of proteins followed by BPA then GEN. A number of proteins share previously identified relationships with ESR1 as determined by STRING analysis. Although there was limited overlap in proteins identified between treatments, all ligands recruited proteins involved in cell growth as determined by subnetwork enrichment analysis (p<0.05). A comparative, in silico analysis revealed that fewer interactions exist between zebrafish (Danio rerio) esr1 and zebrafish orthologs of proteins identified in our functional proteomic analysis. Taken together these results identify recruitment of known and previously unknown co-regulatory proteins to ESR1 and highlight new methods to assay recruitment of low abundant and intact, endogenous co-regulatory proteins to ESR1 or other nuclear receptors, in

  14. Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

    NASA Technical Reports Server (NTRS)

    Mednieks, M. I.; Popova, I.; Grindeland, R. E.

    1992-01-01

    Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

  15. Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA

    SciTech Connect

    Walden, William E.; Selezneva, Anna I.; Dupuy, Jérôme; Volbeda, Anne; Fontecilla-Camps, Juan C.; Theil, Elizabeth C.; Volz1, Karl

    2011-07-27

    Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 angstrom resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by {approx}30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator or enzyme.

  16. Rat beta 1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins.

    PubMed

    Kirigiti, P; Yang, Y F; Li, X; Li, B; Midson, C N; Machida, C A

    2000-03-01

    beta 1-Adrenergic receptors (beta1-ARs) serve as important regulators of central nervous system (CNS)-mediated behavior and several neural functions, including mood, memory, neuroendocrine control, and stimulation of autonomic function. Using beta 1-AR-luciferase reporter recombinants, we have previously determined that important beta 1-AR genetic elements controlling expression within the C6 glioma cell line are contained within the region -396 to -299, relative to the translational start site. By conducting progressive internal deletions of the rat beta 1-AR 5' flanking region and with the use of beta 1-AR-luciferase recombinants, we have verified that this region contains the primary beta 1-AR promoter and/or major regulatory elements. To begin the identification of protein factors involved in beta 1-AR transcriptional activity conferred by this beta 1-AR region and flanking sequences, we conducted electrophoretic mobility shift assays using defined beta 1-AR DNA subregion probes. One probe (GS-1), encompassing the region -396 to -367, was found to produce two major and two minor mobility shift complexes when bound to nuclear extracts from the beta 1-AR expresser C6 cell line. UV-crosslinking of DNA-protein complexes, coupled with DNase I digestion, indicated that this beta 1-AR region interacts with one major protein of approximately 117 kDa molecular weight and additional minor proteins. GS-1 DNA-protein complexes were observed using beta 1-AR expresser tissues in the CNS, including cortex, hippocampus, and olfactory bulb. No DNA-protein complexes were observed when using nuclear extracts from beta 1-AR nonexpresser tissues; in some cases, using L6 cells, previously characterized to express little or no beta1-ARs, a reduction in intensities of the DNA-protein complexes was observed. Competition experiments indicate that nuclear protein binds to one of two subregions within the GS-1 sequence that contain AP-2-like consensus elements. Recombinant AP-2 protein

  17. Exceptionally high heterologous protein levels in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences.

    PubMed

    De Jaeger, Geert; Angenon, Geert; Depicker, Ann

    2003-01-01

    Seeds are concentrated sources of protein and thus may be ideal 'bioreactors' for the production of heterologous proteins. For this application, strong seed-specific expression signals are required. A set of expression cassettes were designed using 5' and 3' regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) from Phaseolus vulgaris, and evaluated for the production of heterologous proteins in dicotyledonous plant species. A murine single-chain variable fragment (scFv) was chosen as model protein because of the current industrial interest to produce antibodies and derived fragments in crops. Because the highest scFv accumulation in seed had previously been achieved in the endoplasmic reticulum (ER), the scFv-encoding sequence was provided with signal sequences for accumulation in the ER. Transgenic Arabidopsis seed stocks, expressing the scFv under control of the 35S promoter, contained scFv accumulation levels in the range of 1% of total soluble protein (TSP). However, the seed storage promoter constructs boosted the scFv to exceptionally high levels. Maximum scFv levels were obtained in homozygous seed stocks, being 12.5% of TSP under control of the arc5-I regulatory sequences and even up to 36.5% of TSP upon replacing the arc5-I promoter by the beta-phaseolin promoter of Phaseolus vulgaris. Even at such very high levels, the scFv proteins retain their full antigen-binding activity. Moreover, the presence of very high scFv levels has only minory effects on seed germination and no effect on seed production. These results demonstrate that the expression levels of arcelin 5-I and beta-phaseolin seed storage protein genes can be transferred to heterologous proteins, giving exceptionally high levels of heterologous proteins, which can be of great value for the molecular farming industry by raising production yield and lowering bio-mass production and purification costs. Finally, the feasibility of heterologous protein production using the

  18. Protein phosphatase 2A regulatory subunits perform distinct functional roles in the maize pathogen Fusarium verticillioides.

    PubMed

    Shin, Joon-Hee; Kim, Jung-Eun; Malapi-Wight, Martha; Choi, Yoon-E; Shaw, Brian D; Shim, Won-Bo

    2013-06-01

    Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is still incomplete. We have demonstrated previously that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. In general, three PP2A subunits, structural A, regulatory B and catalytic C, make up a heterotrimer complex to perform regulatory functions. Significantly, we identified two PP2A regulatory subunits in the F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. In this study, we hypothesized that Ppr1 and Ppr2 are involved in the regulation of fumonisin biosynthesis and/or cell development in F. verticillioides, and generated a series of mutants to determine the functional role of Ppr1 and Ppr2. The PPR1 deletion strain (Δppr1) resulted in drastic growth defects, but increased microconidia production. The PPR2 deletion mutant strain (Δppr2) showed elevated fumonisin production, similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cells with a central septation site, whereas Δppr2 showed early hyphal branching during conidia germination. A kernel rot assay showed that the mutants were slow to colonize kernels, but this is probably a result of growth defects rather than a virulence defect. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out distinct roles in the regulation of fumonisin biosynthesis and fungal development. PMID:23452277

  19. Chronic Low Level Complement Activation within the Eye Is Controlled by Intraocular Complement Regulatory Proteins

    PubMed Central

    Sohn, Jeong-Hyeon; Kaplan, Henry J.; Suk, Hye-Jung; Bora, Puran S.; Bora, Nalini S.

    2007-01-01

    Purpose To explore the role of the complement system and complement regulatory proteins in an immune-privileged organ, the eye. Methods Eyes of normal Lewis rats were analyzed for the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay-acceleration factor (DAF), membrane inhibitor of reactive lysis (MIRL, CD59), and cell surface regulator of complement (Crry), using immunohistochemistry, Western blot analysis, and reverse transcription–polymerase chain reaction (RT-PCR). Zymosan, a known activator of the alternative pathway of complement system was injected into the anterior chamber of the eye of Lewis rats. Animals were also injected intracamerally with 5 μl (25 μg) of neutralizing monoclonal antibody (mAb) against rat Crry (5I2) or CD59 (6D1) in an attempt to develop antibody induced anterior uveitis; control animals received 5 μl of sterile phosphate-buffered saline (PBS), OX-18 (25 μg), G-16-510E3 (25 μg), or MOPC-21 (25 μg). The role of complement system in antibody-induced uveitis was explored by intraperitoneal injection of 35 U cobra venom factor (CVF), 24 hours before antibody injection. Immunohistochemical staining and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with Western blot analysis were used to detect the presence of membrane attack complex (MAC) and C3 activation products, respectively, in normal and antibody-injected rat eyes. Results Complement activation product MAC was present in the normal rat eye, and intraocular injection of zymosan induced severe anterior uveitis. The complement regulatory proteins, MCP, DAF, CD59, and Crry, were identified in the normal rat eye. Soluble forms of Crry and CD59 were also detected in normal rat aqueous humor. Severe anterior uveitis developed in Lewis rats injected with a neutralizing mAb against Crry, with increased formation of C3 split products. Systemic complement depletion by CVF prevented the induction of anterior uveitis by anti

  20. Cytosolic Na+ Controls an Epithelial Na+ Channel Via the Go Guanine Nucleotide-Binding Regulatory Protein

    NASA Astrophysics Data System (ADS)

    Komwatana, P.; Dinudom, A.; Young, J. A.; Cook, D. I.

    1996-07-01

    In tight Na+-absorbing epithelial cells, the rate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-β -S, pertussis toxin, and antibodies against the α -subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

  1. The archaeal feast/famine regulatory protein: Potential roles of its assembly forms for regulating transcription

    PubMed Central

    Koike, Hideaki; Ishijima, Sanae A.; Clowney, Lester; Suzuki, Masashi

    2004-01-01

    The classification feast/famine regulatory proteins (FFRPs) encompasses archaeal DNA-binding proteins with Escherichia coli transcription factors, the leucine-responsive regulatory protein and the asparagine synthase C gene product. In this paper, we describe two forms of the archaeal FFRP FL11 (pot0434017), both assembled from dimers. When crystallized, a helical cylinder is formed with six dimers per turn. In contrast, in solution, disks are formed, most likely consisting of four dimers each; an observation by cryoelectron microscopy. Whereas each dimer binds a 13-bp sequence, different forms will discriminate between promoters, based on the numbers of repeating 13-bp sequences, and types of linkers inserted between them, which are either of 7-8 or ≈18 bp. The amino acid sequences of these FFRPs are designed to form the same type of 3D structures, and the transition between their assembly forms is regulated by interaction with small molecules. These considerations lead us to propose a possible mechanism for regulating a number of genes by varying assembly forms and by combining different FFRPs into these assemblies, responding to environmental changes. PMID:14976242

  2. [The Regulatory Proteins of β-Adrenergic Receptor and Their Functions].

    PubMed

    Tian, Ai-ju; Li, Zi-jian

    2015-04-01

    Vascular diseases has become a top killer of human health, and cardiovascular receptors are pivotal in the occurrence, development, prevention and treatment of cardiovascular diseases. As for the important member of G protein-coupled receptor, β-adrenergic receptor is undoubtedly a most important target of cardiovascular drugs. Being the hot spot in the cardiovascular research and application, β- adrenergic receptor blocker has been considered as the greatest breakthrough for the prevention and cure of cardiovascular disease after digitalis. The 2012 Nobel Prize in chemistry was awarded again to the researchers on β-adrenergic receptors. Extensive researchs show that β-adrenergic receptors are precisely regulated by different regulatory proteins in cells in the transduction of different physiological and pathological signaling pathways. Based on these findings, function-selective ligands recently arise in the receptor research and will be the new chance of drug discovery. In this article we reviewed the related signal pathways and functions of β-adrenergic receptor regulatory proteins. PMID:26201103

  3. Cotyledon nuclear proteins bind to DNA fragments harboring regulatory elements of phytohemagglutinin genes.

    PubMed Central

    Riggs, C D; Voelker, T A; Chrispeels, M J

    1989-01-01

    The effects of deleting DNA sequences upstream from the phytohemagglutinin-L gene of Phaseolus vulgaris have been examined with respect to the level of gene product produced in the seeds of transgenic tobacco. Our studies indicate that several upstream regions quantitatively modulate expression. Between -1000 and -675, a negative regulatory element reduces expression approximately threefold relative to shorter deletion mutants that do not contain this region. Positive regulatory elements lie between -550 and -125 and, compared with constructs containing only 125 base pairs of upstream sequences (-125), the presence of these two regions can be correlated with a 25-fold and a 200-fold enhancement of phytohemagglutinin-L levels. These experiments were complemented by gel retardation assays, which demonstrated that two of the three regions bind cotyledon nuclear proteins from mid-mature seeds. One of the binding sites maps near a DNA sequence that is highly homologous to protein binding domains located upstream from the soybean seed lectin and Kunitz trypsin inhibitor genes. Competition experiments demonstrated that the upstream regions of a bean beta-phaseolin gene, the soybean seed lectin gene, and an oligonucleotide from the upstream region of the trypsin inhibitor gene can compete differentially for factor binding. We suggest that these legume genes may be regulated in part by evolutionarily conserved protein/DNA interactions. PMID:2535513

  4. The archaeal feast/famine regulatory protein: Potential roles of its assembly forms for regulating transcription

    NASA Astrophysics Data System (ADS)

    Koike, Hideaki; Ishijima, Sanae A.; Clowney, Lester; Suzuki, Masashi

    2004-03-01

    The classification feast/famine regulatory proteins (FFRPs) encompasses archaeal DNA-binding proteins with Escherichia coli transcription factors, the leucine-responsive regulatory protein and the asparagine synthase C gene product. In this paper, we describe two forms of the archaeal FFRP FL11 (pot0434017), both assembled from dimers. When crystallized, a helical cylinder is formed with six dimers per turn. In contrast, in solution, disks are formed, most likely consisting of four dimers each; an observation by cryoelectron microscopy. Whereas each dimer binds a 13-bp sequence, different forms will discriminate between promoters, based on the numbers of repeating 13-bp sequences, and types of linkers inserted between them, which are either of 7-8 or 18 bp. The amino acid sequences of these FFRPs are designed to form the same type of 3D structures, and the transition between their assembly forms is regulated by interaction with small molecules. These considerations lead us to propose a possible mechanism for regulating a number of genes by varying assembly forms and by combining different FFRPs into these assemblies, responding to environmental changes.

  5. Regulatory-auxiliary subunits of CLC chloride channel-transport proteins.

    PubMed

    Barrallo-Gimeno, Alejandro; Gradogna, Antonella; Zanardi, Ilaria; Pusch, Michael; Estévez, Raúl

    2015-09-15

    The CLC family of chloride channels and transporters is composed by nine members, but only three of them, ClC-Ka/b, ClC-7 and ClC-2, have been found so far associated with auxiliary subunits. These CLC regulatory subunits are small proteins that present few common characteristics among them, both structurally and functionally, and their effects on the corresponding CLC protein are different. Barttin, a protein with two transmembrane domains, is essential for the membrane localization of ClC-K proteins and their activity in the kidney and inner ear. Ostm1 is a protein with a single transmembrane domain and a highly glycosylated N-terminus. Unlike the other two CLC auxiliary subunits, Ostm1 shows a reciprocal relationship with ClC-7 for their stability. The subcellular localization of Ostm1 depends on ClC-7 and not the other way around. ClC-2 is active on its own, but GlialCAM, a transmembrane cell adhesion molecule with two extracellular immunoglobulin (Ig)-like domains, regulates its subcellular localization and activity in glial cells. The common theme for these three proteins is their requirement for a proper homeostasis, since their malfunction leads to distinct diseases. We will review here their properties and their role in normal chloride physiology and the pathological consequences of their improper function. PMID:25762128

  6. The protein kinase A regulatory subunit R1A (Prkar1a) plays critical roles in peripheral nerve development.

    PubMed

    Guo, Li; Lee, Audrey A; Rizvi, Tilat A; Ratner, Nancy; Kirschner, Lawrence S

    2013-11-13

    Signaling through cAMP has been implicated in Schwann cell (SC) proliferation and myelination, but the signaling pathway components downstream of cAMP required for SC function remain unknown. Protein kinase A (PKA) is a potential downstream effector of cAMP. Here, we induced loss of Prkar1a, the gene encoding the type 1A regulatory subunit of PKA, in SC to study its role in nerve development; loss of Prkar1a is predicted to elevate PKA activity. Conditional Prkar1a knock-out in mouse SC (Prkar1a-SCKO) resulted in a dramatic and persistent axonal sorting defect, and unexpectedly decreased SC proliferation in Prkar1a-SCKO nerves in vivo. Effects were cell autonomous as they were recapitulated in vitro in Prkar1a-SCKO SC, which showed elevated PKA activity. In the few SCs sorted into 1:1 relationships with axons in vivo, SC myelination was premature in Prkar1a-SCKO nerves, correlating with global increase in the cAMP-regulated transcription factor Oct-6 and expression of myelin basic protein. These data reveal a previously unknown role of PKA in axon sorting, an unexpected inhibitory role of PKA on SC cell proliferation in vivo and define the importance of Prkar1a in peripheral nerve development. PMID:24227708

  7. Myristoylated. cap alpha. subunits of guanine nucleotide-binding regulatory proteins

    SciTech Connect

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-11-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the ..cap alpha.. subunits of G/sub s/ (stimulatory) (..cap alpha../sub 45/ and ..cap alpha../sub 52/), a 41-kDa subunit of G/sub i/ (inhibitory) (..cap alpha../sub 41/), a 40-kDa protein (..cap alpha../sub 40/), and the 36-kDa ..beta.. subunit. No protein that comigrated with the ..cap alpha.. subunit of G/sup 0/ (unknown function) (..cap alpha../sub 39/) was detected. In cells grown in the presence of (/sup 3/H)myristic acid, ..cap alpha../sub 41/ and ..cap alpha../sub 40/ contained /sup 3/H label, while the ..beta.. subunit did not. Chemical analysis of lipids attached covalently to purified ..cap alpha../sub 41/ and ..cap alpha../sub 39/ from bovine brain also revealed myristic acid. Similar analysis of brain G protein ..beta.. and ..gamma.. subunits and of G/sub t/ (Transducin) subunits (..cap alpha.., ..beta.., and ..gamma..) failed to reveal fatty acids. The fatty acid associated with ..cap alpha../sub 41/ , ..cap alpha../sub 40/, and ..cap alpha../sub 39/ was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.

  8. Expanding the nitrogen regulatory protein superfamily: Homology detection at below random sequence identity.

    PubMed

    Kinch, Lisa N; Grishin, Nick V

    2002-07-01

    Nitrogen regulatory (PII) proteins are signal transduction molecules involved in controlling nitrogen metabolism in prokaryots. PII proteins integrate the signals of intracellular nitrogen and carbon status into the control of enzymes involved in nitrogen assimilation. Using elaborate sequence similarity detection schemes, we show that five clusters of orthologs (COGs) and several small divergent protein groups belong to the PII superfamily and predict their structure to be a (betaalphabeta)(2) ferredoxin-like fold. Proteins from the newly emerged PII superfamily are present in all major phylogenetic lineages. The PII homologs are quite diverse, with below random (as low as 1%) pairwise sequence identities between some members of distant groups. Despite this sequence diversity, evidence suggests that the different subfamilies retain the PII trimeric structure important for ligand-binding site formation and maintain a conservation of conservations at residue positions important for PII function. Because most of the orthologous groups within the PII superfamily are composed entirely of hypothetical proteins, our remote homology-based structure prediction provides the only information about them. Analogous to structural genomics efforts, such prediction gives clues to the biological roles of these proteins and allows us to hypothesize about locations of functional sites on model structures or rationalize about available experimental information. For instance, conserved residues in one of the families map in close proximity to each other on PII structure, allowing for a possible metal-binding site in the proteins coded by the locus known to affect sensitivity to divalent metal ions. Presented analysis pushes the limits of sequence similarity searches and exemplifies one of the extreme cases of reliable sequence-based structure prediction. In conjunction with structural genomics efforts to shed light on protein function, our strategies make it possible to detect

  9. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures

    PubMed Central

    Slinger, Betty L.; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M.

    2015-01-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition. PMID:26675164

  10. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures.

    PubMed

    Slinger, Betty L; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M

    2015-12-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition. PMID:26675164

  11. Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2.

    PubMed

    Tapia, Julio C; Bolanos-Garcia, Victor M; Sayed, Muhammed; Allende, Catherine C; Allende, Jorge E

    2004-04-01

    The protein kinase CK2 is constituted by two catalytic (alpha and/or alpha') and two regulatory (beta) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27(KIP1), which is a regulator of the cell cycle and a known inhibitor of cyclin-dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27(KIP1), but this phosphorylation occurs only in the presence of the regulatory beta subunit. The principal site of phosphorylation is serine-83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27(KIP1) interacts with the beta subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27(KIP1) phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27(KIP1) phosphorylation by CK2 probably involves a docking event mediated by the CK2beta subunit. The phosphorylation of p27(KIP1) by CK2 may affect its biological activity. PMID:15034923

  12. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

    PubMed

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  13. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

    PubMed Central

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  14. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    SciTech Connect

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. ); Pandey, S. )

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  15. ClpB N-terminal domain plays a regulatory role in protein disaggregation

    PubMed Central

    Rosenzweig, Rina; Farber, Patrick; Velyvis, Algirdas; Rennella, Enrico; Latham, Michael P.; Kay, Lewis E.

    2015-01-01

    ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB–substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD–substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD–substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD–substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation. PMID:26621746

  16. Possible regulatory function of the Saccharomyces cerevisiae Ty1 retrotransposon core protein.

    PubMed

    Roth, J F; Kingsman, S M; Kingsman, A J; Martin-Rendon, E

    2000-07-01

    The yeast Ty1 retrotransposon encodes proteins and RNA that assemble into virus-like particles (VLPs) as part of the life cycle of the retro-element. The Tya protein, which is equivalent to the retroviral Gag, is the major structural component of these particles. In this work, we demonstrate that Tya proteins fulfil other functions apart from their structural role. We show that Tya interacts in vitro with the Ty1 RNA domain required for RNA packaging, suggesting that this RNA-protein interaction may direct the packaging process. Furthermore, the overexpression of both Tya proteins, i.e. p1, the primary translation product, and p2, the mature form, increases endogenous Ty1 RNA levels in trans without increasing translation significantly. These observations suggest that Tya may exert a regulatory function during transposition. Interestingly, however, only p2, the mature form of Tya, trans-activates transposition of a marked genomic Ty element. This confirms that processing is required for transposition. PMID:10870103

  17. Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

    PubMed Central

    Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

    2014-01-01

    The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

  18. Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP.

    PubMed

    Maita, Nobuo; Okada, Kengo; Hatakeyama, Kazuyuki; Hakoshima, Toshio

    2002-02-01

    In the presence of phenylalanine, GTP cyclohydrolase I feedback regulatory protein (GFRP) forms a stimulatory 360-kDa complex with GTP cyclohydrolase I (GTPCHI), which is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. The crystal structure of the stimulatory complex reveals that the GTPCHI decamer is sandwiched by two GFRP homopentamers. Each GFRP pentamer forms a symmetrical five-membered ring similar to beta-propeller. Five phenylalanine molecules are buried inside each interface between GFRP and GTPCHI, thus enhancing the binding of these proteins. The complex structure suggests that phenylalanine-induced GTPCHI x GFRP complex formation enhances GTPCHI activity by locking the enzyme in the active state. PMID:11818540

  19. Downregulation of key regulatory proteins in androgen dependent prostate tumor cells by oncolytic reovirus.

    PubMed

    Gupta-Saraf, Pooja; Meseke, Tyler; Miller, Cathy L

    2015-11-01

    As prostate tumor cell growth depends on hormones, androgen ablation is an effective therapy for prostate cancer (PCa). However, progression of PCa cells to androgen independent growth (castrate resistant prostate cancer, CRPC) results in relapse and mortality. Hypoxia, a microenvironment of low oxygen that modifies the activity of PCa regulatory proteins including the androgen receptor (AR), plays a critical role in progression to CRPC. Therapies targeting hypoxia and the AR may lengthen the time to CRPC progression thereby increasing survival time of PCa patients. Mammalian Orthoreovirus (MRV) has shown promise for the treatment of prostate tumors in vitro and in vivo. In this study, we found that MRV infection induces downregulation of proteins implicated in CRPC progression, interferes with hypoxia-induced AR activity, and induces apoptosis in androgen dependent cells. This suggests MRV possesses traits that could be exploited to create novel therapies for the inhibition of progression to CRPC. PMID:26264969

  20. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    SciTech Connect

    Matsushita, Y. Murakawa, T. Shimamura, K. Oishi, M. Ohyama, T. Kurita, N.

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  1. Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart.

    PubMed

    Little, Sean C; Curran, Jerry; Makara, Michael A; Kline, Crystal F; Ho, Hsiang-Ting; Xu, Zhaobin; Wu, Xiangqiong; Polina, Iuliia; Musa, Hassan; Meadows, Allison M; Carnes, Cynthia A; Biesiadecki, Brandon J; Davis, Jonathan P; Weisleder, Noah; Györke, Sandor; Wehrens, Xander H; Hund, Thomas J; Mohler, Peter J

    2015-07-21

    Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α(+/-) myocytes resulted in reduced Ca(2+) waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca(2+) regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart. PMID:26198358

  2. Insulin accelerates global and mitochondrial protein synthesis rates in neonatal muscle during sepsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In neonatal pigs, sepsis decreases protein synthesis in skeletal muscle by decreasing translation initiation. However, insulin stimulates muscle protein synthesis despite persistent repression of translation initiation signaling. To determine whether the insulin-induced increase in global rates of m...

  3. The Positive Regulatory Roles of the TIFY10 Proteins in Plant Responses to Alkaline Stress

    PubMed Central

    Zhu, Dan; Li, Rongtian; Liu, Xin; Sun, Mingzhe; Wu, Jing; Zhang, Ning; Zhu, Yanming

    2014-01-01

    The TIFY family is a novel plant-specific protein family, and is characterized by a conserved TIFY motif (TIFF/YXG). Our previous studies indicated the potential roles of TIFY10/11 proteins in plant responses to alkaline stress. In the current study, we focused on the regulatory roles and possible physiological and molecular basis of the TIFY10 proteins in plant responses to alkaline stress. We demonstrated the positive function of TIFY10s in alkaline responses by using the AtTIFY10a and AtTIFY10b knockout Arabidopsis, as evidenced by the relatively lower germination rates of attify10a and attify10b mutant seeds under alkaline stress. We also revealed that ectopic expression of GsTIFY10a in Medicago sativa promoted plant growth, and increased the NADP-ME activity, citric acid content and free proline content but decreased the MDA content of transgenic plants under alkaline stress. Furthermore, expression levels of the stress responsive genes including NADP-ME, CS, H+-ppase and P5CS were also up-regulated in GsTIFY10a transgenic plants under alkaline stress. Interestingly, GsTIFY10a overexpression increased the jasmonate content of the transgenic alfalfa. In addition, we showed that neither GsTIFY10a nor GsTIFY10e exhibited transcriptional activity in yeast cells. However, through Y2H and BiFc assays, we demonstrated that GsTIFY10a, not GsTIFY10e, could form homodimers in yeast cells and in living plant cells. As expected, we also demonstrated that GsTIFY10a and GsTIFY10e could heterodimerize with each other in both yeast and plant cells. Taken together, our results provided direct evidence supporting the positive regulatory roles of the TIFY10 proteins in plant responses to alkaline stress. PMID:25375909

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

    PubMed Central

    2013-01-01

    Neurodevelopmental disorders are classified as diseases that cause abnormal functions of the brain or central nervous system. Children with neurodevelopmental disorders show impaired language and speech abilities, learning and memory damage, and poor motor skills. However, we still know very little about the molecular etiology of these disorders. Recent evidence implicates the bromodomain-containing proteins (BCPs) in the initiation and development of neurodevelopmental disorders. BCPs have a particular domain, the bromodomain (Brd), which was originally identified as specifically binding acetyl-lysine residues at the N-terminus of histone proteins in vitro and in vivo. Other domains of BCPs are responsible for binding partner proteins to form regulatory complexes. Once these complexes are assembled, BCPs alter chromosomal states and regulate gene expression. Some BCP complexes bind nucleosomes, are involved in basal transcription regulation, and influence the transcription of many genes. However, most BCPs are involved in targeting. For example, some BCPs function as a recruitment platform or scaffold through their Brds-binding targeting sites. Others are recruited to form a complex to bind the targeting sites of their partners. The regulation mediated by these proteins is especially critical during normal and abnormal development. Mutant BCPs or dysfunctional BCP-containing complexes are implicated in the initiation and development of neurodevelopmental disorders. However, the pathogenic molecular mechanisms are not fully understood. In this review, we focus on the roles of regulatory BCPs associated with neurodevelopmental disorders, including mental retardation, Fragile X syndrome (FRX), Williams syndrome (WS), Rett syndrome and Rubinstein-Taybi syndrome (RTS). A better understanding of the molecular pathogenesis, based upon the roles of BCPs, will lead to screening of targets for the treatment of neurodevelopmental disorders. PMID:23425632

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

    PubMed

    Li, Junlin; Zhao, Guifang; Gao, Xiaocai

    2013-01-01

    Neurodevelopmental disorders are classified as diseases that cause abnormal functions of the brain or central nervous system. Children with neurodevelopmental disorders show impaired language and speech abilities, learning and memory damage, and poor motor skills. However, we still know very little about the molecular etiology of these disorders. Recent evidence implicates the bromodomain-containing proteins (BCPs) in the initiation and development of neurodevelopmental disorders. BCPs have a particular domain, the bromodomain (Brd), which was originally identified as specifically binding acetyl-lysine residues at the N-terminus of histone proteins in vitro and in vivo. Other domains of BCPs are responsible for binding partner proteins to form regulatory complexes. Once these complexes are assembled, BCPs alter chromosomal states and regulate gene expression. Some BCP complexes bind nucleosomes, are involved in basal transcription regulation, and influence the transcription of many genes. However, most BCPs are involved in targeting. For example, some BCPs function as a recruitment platform or scaffold through their Brds-binding targeting sites. Others are recruited to form a complex to bind the targeting sites of their partners. The regulation mediated by these proteins is especially critical during normal and abnormal development. Mutant BCPs or dysfunctional BCP-containing complexes are implicated in the initiation and development of neurodevelopmental disorders. However, the pathogenic molecular mechanisms are not fully understood. In this review, we focus on the roles of regulatory BCPs associated with neurodevelopmental disorders, including mental retardation, Fragile X syndrome (FRX), Williams syndrome (WS), Rett syndrome and Rubinstein-Taybi syndrome (RTS). A better understanding of the molecular pathogenesis, based upon the roles of BCPs, will lead to screening of targets for the treatment of neurodevelopmental disorders. PMID:23425632

  6. GTP Cyclohydrolase I Expression, Protein, and Activity Determine Intracellular Tetrahydrobiopterin Levels, Independent of GTP Cyclohydrolase Feedback Regulatory Protein Expression

    PubMed Central

    Tatham, Amy L.; Crabtree, Mark J.; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J.; Channon, Keith M.

    2009-01-01

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r2 = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r2 = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  7. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  8. Identification of the binding sites of regulatory proteins in bacterial genomes

    PubMed Central

    Li, Hao; Rhodius, Virgil; Gross, Carol; Siggia, Eric D.

    2002-01-01

    We present an algorithm that extracts the binding sites (represented by position-specific weight matrices) for many different transcription factors from the regulatory regions of a genome, without the need for delineating groups of coregulated genes. The algorithm uses the fact that many DNA-binding proteins in bacteria bind to a bipartite motif with two short segments more conserved than the intervening region. It identifies all statistically significant patterns of the form W1NxW2, where W1 and W2 are two short oligonucleotides separated by x arbitrary bases, and groups them into clusters of similar patterns. These clusters are then used to derive quantitative recognition profiles of putative regulatory proteins. For a given cluster, the algorithm finds the matching sequences plus the flanking regions in the genome and performs a multiple sequence alignment to derive position-specific weight matrices. We have analyzed the Escherichia coli genome with this algorithm and found ≈1,500 significant patterns, which give rise to ≈160 distinct position-specific weight matrices. A fraction of these matrices match the binding sites of one-third of the ≈60 characterized transcription factors with high statistical significance. Many of the remaining matrices are likely to describe binding sites and regulons of uncharacterized transcription factors. The significance of these matrices was evaluated by their specificity, the location of the predicted sites, and the biological functions of the corresponding regulons, allowing us to suggest putative regulatory functions. The algorithm is efficient for analyzing newly sequenced bacterial genomes for which little is known about transcriptional regulation. PMID:12181488

  9. Heme binds to a short sequence that serves a regulatory function in diverse proteins.

    PubMed Central

    Zhang, L; Guarente, L

    1995-01-01

    Heme is a prosthetic group for numerous enzymes, cytochromes and globins, and it binds tightly, sometimes covalently, to these proteins. Interestingly, heme also potentiates binding of the yeast transcriptional activator HAP1 to DNA and inhibits mitochondrial import of the mammalian delta-aminolevulinate synthase (ALAS) and the catalytic activity of the reticulocyte kinase, HRI. All three of these proteins contain a short sequence, the heme regulatory motif (HRM), that occurs six times adjacent to the HAP1 DNA binding domain, twice in the leader targeting sequence of ALAS and twice near the catalytic domain of the HRI kinase. Here we show that a 10 amino acid peptide containing the HRM consensus binds to heme in the micromolar range, and shifts the heme absorption spectrum to a longer wavelength, a direction opposite to the change caused by cytochromes or globins. Further, we show that a single HRM regulates the acidic activation domains of HAP1 and GAL4 independently of regulation of DNA binding of the transcription factors. These findings thus establish a novel heme binding sequence which is structurally distinct from sequences in globins or cytochromes and which has a regulatory function. Images PMID:7835342

  10. Evolution of context dependent regulation by expansion of feast/famine regulatory proteins

    SciTech Connect

    Plaisier, Christopher L.; Lo, Fang -Yin; Ashworth, Justin; Brooks, Aaron N.; Beer, Karlyn D.; Kaur, Amardeep; Pan, Min; Reiss, David J.; Facciotti, Marc T.; Baliga, Nitin S.

    2014-11-14

    Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions that mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.

  11. Evolution of context dependent regulation by expansion of feast/famine regulatory proteins

    DOE PAGESBeta

    Plaisier, Christopher L.; Lo, Fang -Yin; Ashworth, Justin; Brooks, Aaron N.; Beer, Karlyn D.; Kaur, Amardeep; Pan, Min; Reiss, David J.; Facciotti, Marc T.; Baliga, Nitin S.

    2014-11-14

    Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions thatmore » mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.« less

  12. Theoretical investigations on the interactions of glucokinase regulatory protein with fructose phosphates.

    PubMed

    Ling, Baoping; Yan, Xueyuan; Sun, Min; Bi, Siwei

    2016-02-01

    Glucokinase (GK) plays a critical role in maintaining glucose homeostasis in the human liver and pancreas. In the liver, the activity of GK is modulated by the glucokinase regulatory protein (GKRP) which functions as a competitive inhibitor of glucose to bind to GK. Moreover, the inhibitory intensity of GKRP-GK is suppressed by fructose 1-phosphate (F1P), and reinforced by fructose 6-phosphate (F6P). Here, we employed a series of computational techniques to explore the interactions of fructose phosphates with GKRP. Calculation results reveal that F1P and F6P can bind to the same active site of GKRP with different binding modes, and electrostatic interaction provides a major driving force for the ligand binding. The presence of fructose phosphate severely influences the motions of protein and the conformational space, and the structural change of sugar phosphate influences its interactions with GKRP, leading to a large conformational rearrangement of loop2 in the SIS2 domain. In particular, the binding of F6P to GKRP facilitates the protruding loop2 contacting with GK to form the stable GK-GKRP complex. The conserved residues 179-184 of GKRP play a major role in the binding of phosphate group and maintaining the stability of GKRP. These results may provide deep insight into the regulatory mechanism of GKRP to the activity of GK. PMID:26629747

  13. Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

    PubMed

    Wang, Honglin; Sun, Yue; Chang, Jianhong; Zheng, Fangfang; Pei, Haixia; Yi, Yanjun; Chang, Caren; Dong, Chun-Hai

    2016-07-01

    Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling. PMID:27097903

  14. Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding.

    PubMed

    Korkuć, Paula; Walther, Dirk

    2016-05-01

    Phosphorylation is an important post-translational modification that regulates protein function by the attachment of negatively charged phosphate groups to phosphorylatable amino acid residues. As a mode of action, an influence of phosphorylation on the binding of compounds to proteins has been discussed and described for a number of proteins in the literature. However, a systematic statistical survey probing for enriched phosphorylation sites close to compound binding sites in support of this notion and with properly chosen random reference distributions has not been presented yet. Using high-resolution protein structures from the Protein Data Bank including their co-crystallized non-covalently bound compounds and experimentally determined phosphorylation sites, we analyzed the pairwise distance distributions of phosphorylation and compound binding sites on protein surfaces. We found that phosphorylation sites are indeed located at significantly closer distances to compounds than expected by chance holding true specifically also for the subset of compound binding sites serving as catalytic sites of metabolic reactions. This tendency was particularly evident when treating phosphorylation sites as collective sets supporting the relevance of phosphorylation hotspots. Interestingly, phosphorylation sites were found to be closer to negatively charged than to positively charged compounds suggesting a stronger modulation of the binding of negatively charged compounds in dependence on phosphorylation status than on positively charged compounds. The enrichment of phosphorylation sites near compound binding sites confirms a regulatory role of phosphorylation in compound binding and provides a solid statistical basis for the literature-reported selected events. Proteins 2016; 84:565-579. © 2016 Wiley Periodicals, Inc. PMID:26817627

  15. Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells

    PubMed Central

    Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J.; Alani, Sara; Bocchetta, Maurizio

    2015-01-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement—a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis. PMID:25283437

  16. Amyloid precursor protein (APP) affects global protein synthesis in dividing human cells.

    PubMed

    Sobol, Anna; Galluzzo, Paola; Liang, Shuang; Rambo, Brittany; Skucha, Sylvia; Weber, Megan J; Alani, Sara; Bocchetta, Maurizio

    2015-05-01

    Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis. PMID:25283437

  17. Assembly of the cysteine synthase complex and the regulatory role of protein-protein interactions.

    PubMed

    Kumaran, Sangaralingam; Yi, Hankuil; Krishnan, Hari B; Jez, Joseph M

    2009-04-10

    Macromolecular assemblies play critical roles in regulating cellular functions. The cysteine synthase complex (CSC), which is formed by association of serine O-acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), acts as a sensor and modulator of thiol metabolism by responding to changes in nutrient conditions. Here we examine the oligomerization and energetics of formation of the soybean CSC. Biophysical examination of the CSC by size exclusion chromatography and sedimentation ultracentrifugation indicates that this assembly (complex M(r) approximately 330,000) consists of a single SAT trimer (trimer M(r) approximately 110,000) and three OASS dimers (dimer M(r) approximately 70,000). Analysis of the SAT-OASS interaction by isothermal titration calorimetry reveals negative cooperativity with three distinct binding events during CSC formation with K(d) values of 0.3, 7.5, and 78 nm. The three binding events are also observed using surface plasmon resonance with comparable affinities. The stability of the CSC derives from rapid association and extremely slow dissociation of OASS with SAT and requires the C terminus of SAT for the interaction. Steady-state kinetic analysis shows that CSC formation enhances SAT activity and releases SAT from substrate inhibition and feedback inhibition by cysteine, the final product of the biosynthesis pathway. Cysteine inhibits SAT and the CSC with K(i) values of 2 and 70 microm, respectively. These results suggest a new model for the architecture of this regulatory complex and additional control mechanisms for biochemically controlling plant cysteine biosynthesis. Based on previous work and our results, we suggest that OASS acts as an enzyme chaperone of SAT in the CSC. PMID:19213732

  18. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    PubMed Central

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Hall, Traci M. Tanaka

    2009-01-01

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1–3 and 7–8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4–6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so. PMID:19901328

  19. Spectroscopic studies on peptides and proteins with cysteine-containing heme regulatory motifs (HRM).

    PubMed

    Schubert, Erik; Florin, Nicole; Duthie, Fraser; Henning Brewitz, H; Kühl, Toni; Imhof, Diana; Hagelueken, Gregor; Schiemann, Olav

    2015-07-01

    The role of heme as a cofactor in enzymatic reactions has been studied for a long time and in great detail. Recently it was discovered that heme can also serve as a signalling molecule in cells but so far only few examples of this regulation have been studied. In order to discover new potentially heme-regulated proteins, we screened protein sequence databases for bacterial proteins that contain sequence features like a Cysteine-Proline (CP) motif, which is known for its heme-binding propensity. Based on this search we synthesized a series of these potential heme regulatory motifs (HRMs). We used cw EPR spectroscopy to investigate whether these sequences do indeed bind to heme and if the spin state of heme is changed upon interaction with the peptides. The corresponding proteins of two potential HRMs, FeoB and GlpF, were expressed and purified and their interaction with heme was studied by cw EPR and UV-Visible (UV-Vis) spectroscopy. PMID:26050879

  20. ArsR arsenic-resistance regulatory protein from Cupriavidus metallidurans CH34.

    PubMed

    Zhang, Yian-Biao; Monchy, Sébastien; Greenberg, Bill; Mergeay, Max; Gang, Oleg; Taghavi, Safiyh; van der Lelie, Daniel

    2009-08-01

    The Cupriavidus metallidurans CH34 arsR gene, which is part of the arsRIC(2)BC(1)HP operon, and its putative arsenic-resistance regulatory protein were identified and characterized. The arsenic-induced transcriptome of C. metallidurans CH34 showed that the genes most upregulated in the presence of arsenate were all located within the ars operon, with none of the other numerous heavy metal resistance systems present in CH34 being induced. A transcriptional fusion between the luxCDABE operon and the arsR promoter/operator (P/O) region was used to confirm the in vivo induction of the ars operon by arsenite and arsenate. The arsR gene was cloned into expression vectors allowing for the overexpression of the ArsR protein as either his-tagged or untagged protein. The ability of the purified ArsR proteins to bind to the ars P/O region was analyzed in vitro by gel mobility shift assays. ArsR showed an affinity almost exclusively to its own ars P/O region. Dissociation of ArsR and its P/O region was metal dependent, and based on decreasing degrees of dissociation three groups of heavy metals could be distinguished: As(III), Bi(III), Co(II), Cu(II), Ni(II); Cd(II); Pb(II) and Zn(II), while no dissociation was observed in the presence of As(V). PMID:19238575

  1. ArsR arsenic-resistance regulatory protein from Cupriavidus metallidurans CH34

    SciTech Connect

    Zhang, Y.; van der Lelie, D.; Monchy, S.; Greenberg, B.; Gang, O.; Taghavi, S.

    2009-08-01

    The Cupriavidus metallidurans CH34 arsR gene, which is part of the arsRIC{sub 2}BC{sub 1}HP operon, and its putative arsenic-resistance regulatory protein were identified and characterized. The arsenic-induced transcriptome of C. metallidurans CH34 showed that the genes most upregulated in the presence of arsenate were all located within the ars operon, with none of the other numerous heavy metal resistance systems present in CH34 being induced. A transcriptional fusion between the luxCDABE operon and the arsR promoter/operator (P/O) region was used to confirm the in vivo induction of the ars operon by arsenite and arsenate. The arsR gene was cloned into expression vectors allowing for the overexpression of the ArsR protein as either his-tagged or untagged protein. The ability of the purified ArsR proteins to bind to the ars P/O region was analyzed in vitro by gel mobility shift assays. ArsR showed an affinity almost exclusively to its own ars P/O region. Dissociation of ArsR and its P/O region was metal dependent, and based on decreasing degrees of dissociation three groups of heavy metals could be distinguished: As(III), Bi(III), Co(II), Cu(II), Ni(II); Cd(II); Pb(II) and Zn(II), while no dissociation was observed in the presence of As(V).

  2. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues

    PubMed Central

    Suzuki, Takashi; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5′-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5′-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  3. The human actin-regulatory protein Cap G: Gene structure and chromosome location

    SciTech Connect

    Mishra, V.S.; Southwick, F.S.; Henske, E.P.; Kwiatkowski, D.J.

    1994-10-01

    Cap G (formerly called macrophage capping protein or gCap39) is a member of the gelsolin/villin family of actin-regulatory proteins. Unlike all other members of this family, Cap G caps the barbed ends of actin filaments, but does not sever them. This protein is half the molecular weight and contains half the number of repeat subunits (3 vs. 6) of gelsolin and villin, suggesting that these two proteins may have arisen by gene duplication of the Cap G gene. To investigate this possibility we have cloned and sequenced the human Cap G gene (gene symbol CAPG). The gene is 16.6 kb in size, contains 10 exons and 9 introns, and is located on the proximal short arm of chromosome 2. The open reading frame is 6.9 kb, having 9 exons and 8 introns. This region contains 3 splice sites that are nearly identical to the human gelsolin gene, but shares only one with villin, indicating that CAPG is more closely related to gelsolin. Further comparisons of these three genes, however, indicate that the evolutionary steps resulting in human gelsolin and villin are likely to have been more complex than a simple tandem duplication of the Cap G gene. 30 refs., 4 figs., 2 tabs.

  4. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    SciTech Connect

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M.

    2011-11-02

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  5. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues.

    PubMed

    Suzuki, Takashi; Swift, Larry L

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5'-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  6. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

    SciTech Connect

    Wang, Yeming; Opperman, Laura; Wickens, Marvin; Tanaka Hall, Traci M.

    2010-08-19

    Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

  7. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

    PubMed Central

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W.; Grubert, Fabian; Candille, Sophie I.; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L.; Tang, Hua; Ricci, Emiliano; Snyder, Michael P.

    2015-01-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy—many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. PMID:26297486

  8. [New regulatory protein isolated from the bovine eye lens and its action on the cataract development in rat in vitro].

    PubMed

    Krasnov, M S; Gurmizov, E P; Iamskova, V P; Gundorova, R A; Iamskov, I A

    2005-01-01

    The regulatory protein was isolated from the eye lens extract by using an early designed scheme including by means of salting-out of proteins by ammonium sulphate, isoelectrofocusing in pH gradient and electrophoresis in PAAG. A high-purity fraction of the regulatory protein was obtained. The localization of the regulatory protein in the rat-eye lens was investigated by means of primary rabbit antibodies obtained within the case study and by FITS-marked secondary antibodies. Cataractogenesis was induced, in vitro, in Wistar rat lenses through adding, to the cultivation medium, hydrogen peroxide (0.5 mM) or calcium chloride (15 mM). The regulatory protein isolated from the bovine eye lens was added alongside with damaging antibodies to the nutrition medium, concentration 10(-12) mg/ml. The lenses were cultivated for as long as 8 days at 37 degrees C. The degree of opacification of lenses was evaluated visually with the help of a lined substrate as well as by spectrophotometry. The studied protein was shown immunohistochemically to be localized in the intercellular space of the lens epithelium in the region of the basic membrane. The cataractogenesis-related research of the regulatory protein was made on rabbit eye lenses, which were cultivated as a whole for as long as 8 days in vitro. Their transparency and morphology were preserved in them in full since they were cultivated in a serum-free nutrition without admixture of any destructive agents. Opacification of lenses was induced in vitro by changing the concentration of calcium ions in the cultivation medium or through adding hydrogen peroxide to the medium. The valuations of the lens opacity degree as observed in different research series and made by visual observation well correlate with the results of spectrophotometry of lenses made after their cultivation. It can be stated that the studied regulatory protein, when added to the cultivation medium, enhances about two-fold the lens transparency versus the lenses

  9. Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

    SciTech Connect

    Shetty, Nishant D.; Reddy, Manchi C.M.; Palaninathan, Satheesh K.; Owen, Joshua L.; Sacchettini, James C.

    2010-10-11

    PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 {angstrom} resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T-loop of one subunit and the C-loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T-loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T-loop makes direct contact with the {gamma}-phosphate of the ATP molecule replacing the Mg{sup 2+} position seen in the Methanococcus jannaschii GlnK1 structure. The C-loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C-terminal 3{sub 10} helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.

  10. Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

    PubMed Central

    Shetty, Nishant D; Reddy, Manchi C M; Palaninathan, Satheesh K; Owen, Joshua L; Sacchettini, James C

    2010-01-01

    PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 Å resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T-loop of one subunit and the C-loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T-loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T-loop makes direct contact with the γ-phosphate of the ATP molecule replacing the Mg2+ position seen in the Methanococcus jannaschii GlnK1 structure. The C-loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C-terminal 310 helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway. PMID:20521335

  11. Experimental detection of short regulatory motifs in eukaryotic proteins: tips for good practice as well as for bad.

    PubMed

    Gibson, Toby J; Dinkel, Holger; Van Roey, Kim; Diella, Francesca

    2015-01-01

    It has become clear in outline though not yet in detail how cellular regulatory and signalling systems are constructed. The essential machines are protein complexes that effect regulatory decisions by undergoing internal changes of state. Subcomponents of these cellular complexes are assembled into molecular switches. Many of these switches employ one or more short peptide motifs as toggles that can move between one or more sites within the switch system, the simplest being on-off switches. Paradoxically, these motif modules (termed short linear motifs or SLiMs) are both hugely abundant but difficult to research. So despite the many successes in identifying short regulatory protein motifs, it is thought that only the "tip of the iceberg" has been exposed. Experimental and bioinformatic motif discovery remain challenging and error prone. The advice presented in this article is aimed at helping researchers to uncover genuine protein motifs, whilst avoiding the pitfalls that lead to reports of false discovery. PMID:26581338

  12. Unraveling the regulatory network in Streptococcus pyogenes: the global response regulator CovR represses rivR directly.

    PubMed

    Roberts, Samantha A; Churchward, Gordon G; Scott, June R

    2007-02-01

    The response regulator CovR acts as a master regulator of virulence in Streptococcus pyogenes by repressing transcription of approximately 15% of the group A streptococcus genome directly or indirectly. We demonstrate that phosphorylated CovR represses transcription of rivR directly by binding to conserved sequences located downstream from the promoter to block procession of RNA polymerase. This establishes the first link in a regulatory network where CovR interacts directly with other proteins that modulate gene expression. PMID:16963575

  13. The selective phosphorylation of a guanine nucleotide-binding regulatory protein

    SciTech Connect

    Carlson, K.E.

    1989-01-01

    Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the {alpha} subunit of G{sub i} and other G proteins in solution. However, the occurrence of the phosphorylation of G{sub 1} within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the {alpha} subunits of G{sub i} undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with ({gamma}{sup 32}P)ATP and ({sup 32}P)H{sub 3}PO{sub 4}, respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G{sub i{alpha}}-despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G{sub z{alpha}}, or antibodies for both G{sub z{alpha}} and G{sub i{alpha}}, precipitated a 40-kDa phosphoprotein.

  14. Signal regulatory protein α regulates the homeostasis of T lymphocytes in the spleen.

    PubMed

    Sato-Hashimoto, Miho; Saito, Yasuyuki; Ohnishi, Hiroshi; Iwamura, Hiroko; Kanazawa, Yoshitake; Kaneko, Tetsuya; Kusakari, Shinya; Kotani, Takenori; Mori, Munemasa; Murata, Yoji; Okazawa, Hideki; Ware, Carl F; Oldenborg, Per-Arne; Nojima, Yoshihisa; Matozaki, Takashi

    2011-07-01

    The molecular basis for formation of lymphoid follicle and its homeostasis in the secondary lymphoid organs remains unclear. Signal regulatory protein α (SIRPα), an Ig superfamily protein that is predominantly expressed in dendritic cells or macrophages, mediates cell-cell signaling by interacting with CD47, another Ig superfamily protein. In this study, we show that the size of the T cell zone as well as the number of CD4(+) T cells were markedly reduced in the spleen of mice bearing a mutant (MT) SIRPα that lacks the cytoplasmic region compared with those of wild-type mice. In addition, the expression of CCL19 and CCL21, as well as of IL-7, which are thought to be important for development or homeostasis of the T cell zone, was markedly decreased in the spleen of SIRPα MT mice. By the use of bone marrow chimera, we found that hematopoietic SIRPα is important for development of the T cell zone as well as the expression of CCL19 and CCL21 in the spleen. The expression of lymphotoxin and its receptor, lymphotoxin β receptor, as well as the in vivo response to lymphotoxin β receptor stimulation were also decreased in the spleen of SIRPα MT mice. CD47-deficient mice also manifested phenotypes similar to SIRPα MT mice. These data suggest that SIRPα as well as its ligand CD47 are thus essential for steady-state homeostasis of T cells in the spleen. PMID:21632712

  15. Protein Phosphatase 2A in the Regulatory Network Underlying Biotic Stress Resistance in Plants.

    PubMed

    Durian, Guido; Rahikainen, Moona; Alegre, Sara; Brosché, Mikael; Kangasjärvi, Saijaliisa

    2016-01-01

    Biotic stress factors pose a major threat to plant health and can significantly deteriorate plant productivity by impairing the physiological functions of the plant. To combat the wide range of pathogens and insect herbivores, plants deploy converging signaling pathways, where counteracting activities of protein kinases and phosphatases form a basic mechanism for determining appropriate defensive measures. Recent studies have identified Protein Phosphatase 2A (PP2A) as a crucial component that controls pathogenesis responses in various plant species. Genetic, proteomic and metabolomic approaches have underscored the versatile nature of PP2A, which contributes to the regulation of receptor signaling, organellar signaling, gene expression, metabolic pathways, and cell death, all of which essentially impact plant immunity. Associated with this, various PP2A subunits mediate post-translational regulation of metabolic enzymes and signaling components. Here we provide an overview of protein kinase/phosphatase functions in plant immunity signaling, and position the multifaceted functions of PP2A in the tightly inter-connected regulatory network that controls the perception, signaling and responding to biotic stress agents in plants. PMID:27375664

  16. Role of Signal Regulatory Protein α in Arsenic Trioxide-induced Promyelocytic Leukemia Cell Apoptosis.

    PubMed

    Pan, Chaoyun; Zhu, Dihan; Zhuo, Jianjiang; Li, Limin; Wang, Dong; Zhang, Chen-Yu; Liu, Yuan; Zen, Ke

    2016-01-01

    Signal regulatory protein α (SIRPα) has been shown to operate as a negative regulator in cancer cell survival. The mechanism underneath such function, however, remains poorly defined. In the present study, we demonstrate that overexpression of SIRPα in acute promyelocytic leukemia (APL) cells results in apoptosis possibly via inhibiting the β-catenin signaling pathway and upregulating Foxo3a. Pharmacological activation of β-catenin signal pathway attenuates apoptosis caused by SIRPα. Interestingly, we also find that the pro-apoptotic effect of SIRPα plays an important role in arsenic trioxide (ATO)-induced apoptosis in APL cells. ATO treatment induces the SIRPα protein expression in APL cells and abrogation of SIRPα induction by lentivirus-mediated SIRPα shRNA significantly reduces the ATO-induced apoptosis. Mechanistic study further shows that induction of SIRPα protein in APL cells by ATO is mediated through suppression of c-Myc, resulting in reduction of three SIRPα-targeting microRNAs: miR-17, miR-20a and miR-106a. In summary, our results demonstrate that SIRPα inhibits tumor cell survival and significantly contributes to ATO-induced APL cell apoptosis. PMID:27010069

  17. Role of Signal Regulatory Protein α in Arsenic Trioxide-induced Promyelocytic Leukemia Cell Apoptosis

    PubMed Central

    Pan, Chaoyun; Zhu, Dihan; Zhuo, Jianjiang; Li, Limin; Wang, Dong; Zhang, Chen-Yu; Liu, Yuan; Zen, Ke

    2016-01-01

    Signal regulatory protein α (SIRPα) has been shown to operate as a negative regulator in cancer cell survival. The mechanism underneath such function, however, remains poorly defined. In the present study, we demonstrate that overexpression of SIRPα in acute promyelocytic leukemia (APL) cells results in apoptosis possibly via inhibiting the β-catenin signaling pathway and upregulating Foxo3a. Pharmacological activation of β-catenin signal pathway attenuates apoptosis caused by SIRPα. Interestingly, we also find that the pro-apoptotic effect of SIRPα plays an important role in arsenic trioxide (ATO)-induced apoptosis in APL cells. ATO treatment induces the SIRPα protein expression in APL cells and abrogation of SIRPα induction by lentivirus-mediated SIRPα shRNA significantly reduces the ATO-induced apoptosis. Mechanistic study further shows that induction of SIRPα protein in APL cells by ATO is mediated through suppression of c-Myc, resulting in reduction of three SIRPα-targeting microRNAs: miR-17, miR-20a and miR-106a. In summary, our results demonstrate that SIRPα inhibits tumor cell survival and significantly contributes to ATO-induced APL cell apoptosis. PMID:27010069

  18. Structural Basis of Reversible Phosphorylation by Maize Pyruvate Orthophosphate Dikinase Regulatory Protein1[OPEN

    PubMed Central

    Jiang, Lun; Chen, Yi-bo; Zheng, Jiangge; Chen, Zhenhang; Liu, Yujie; Tao, Ye; Wu, Wei; Wang, Bai-chen

    2016-01-01

    Pyruvate orthophosphate dikinase (PPDK) is one of the most important enzymes in C4 photosynthesis. PPDK regulatory protein (PDRP) regulates the inorganic phosphate-dependent activation and ADP-dependent inactivation of PPDK by reversible phosphorylation. PDRP shares no significant sequence similarity with other protein kinases or phosphatases. To investigate the molecular mechanism by which PDRP carries out its dual and competing activities, we determined the crystal structure of PDRP from maize (Zea mays). PDRP forms a compact homo-dimer in which each protomer contains two separate N-terminal (NTD) and C-terminal (CTD) domains. The CTD includes several key elements for performing both phosphorylation and dephosphorylation activities: the phosphate binding loop (P-loop) for binding the ADP and inorganic phosphate substrates, residues Lys-274 and Lys-299 for neutralizing the negative charge, and residue Asp-277 for protonating and deprotonating the target Thr residue of PPDK to promote nucleophilic attack. Surprisingly, the NTD shares the same protein fold as the CTD and also includes a putative P-loop with AMP bound but lacking enzymatic activities. Structural analysis indicated that this loop may participate in the interaction with and regulation of PPDK. The NTD has conserved intramolecular and intermolecular disulfide bonds for PDRP dimerization. Moreover, PDRP is the first structure of the domain of unknown function 299 enzyme family reported. This study provides a structural basis for understanding the catalytic mechanism of PDRP and offers a foundation for the development of selective activators or inhibitors that may regulate photosynthesis. PMID:26620526

  19. Protein Phosphatase 2A in the Regulatory Network Underlying Biotic Stress Resistance in Plants

    PubMed Central

    Durian, Guido; Rahikainen, Moona; Alegre, Sara; Brosché, Mikael; Kangasjärvi, Saijaliisa

    2016-01-01

    Biotic stress factors pose a major threat to plant health and can significantly deteriorate plant productivity by impairing the physiological functions of the plant. To combat the wide range of pathogens and insect herbivores, plants deploy converging signaling pathways, where counteracting activities of protein kinases and phosphatases form a basic mechanism for determining appropriate defensive measures. Recent studies have identified Protein Phosphatase 2A (PP2A) as a crucial component that controls pathogenesis responses in various plant species. Genetic, proteomic and metabolomic approaches have underscored the versatile nature of PP2A, which contributes to the regulation of receptor signaling, organellar signaling, gene expression, metabolic pathways, and cell death, all of which essentially impact plant immunity. Associated with this, various PP2A subunits mediate post-translational regulation of metabolic enzymes and signaling components. Here we provide an overview of protein kinase/phosphatase functions in plant immunity signaling, and position the multifaceted functions of PP2A in the tightly inter-connected regulatory network that controls the perception, signaling and responding to biotic stress agents in plants. PMID:27375664

  20. Regulation of the endogenous VEGF-A gene by exogenous designed regulatory proteins

    PubMed Central

    Tachikawa, Kiyoshi; Schröder, Oliver; Frey, Gerhard; Briggs, Steven P.; Sera, Takashi

    2004-01-01

    We describe a facile method to activate or repress transcription of endogenous genes in a quantitative and specific manner by treatment with designed regulatory proteins (DRPs), in which artificial transcription factors (ATFs) are fused to cell-penetrating peptides (CPPs). Penetration of DRPs into cells is mediated by an N-terminal CPP fused to a nuclear localization signal; a DNA-binding domain and a transactivation domain follow. The DNA-binding domain was targeted to the vascular endothelial growth factor (VEGF)-A gene. An agonist DRP was rapidly taken up by cells and transported to the nucleus; soon after, the cells began transcribing the gene and secreting VEGF-A protein in a dose-dependent manner. Multiple copies of a short oligopeptide derived from a minimal transactivation domain of human β-catenin was stronger than VP-16. The SRDX domain from the plant transcription factor, SUPERMAN, changed the DRP to a hypoxia-induced antagonist of VEGF-A. DRPs combine many of the potential benefits of transgenes with those of recombinant proteins. PMID:15475575

  1. A direct role for murine Cdx proteins in the trunk neural crest gene regulatory network.

    PubMed

    Sanchez-Ferras, Oraly; Bernas, Guillaume; Farnos, Omar; Touré, Aboubacrine M; Souchkova, Ouliana; Pilon, Nicolas

    2016-04-15

    Numerous studies in chordates and arthropods currently indicate that Cdx proteins have a major ancestral role in the organization of post-head tissues. In urochordate embryos, Cdx loss-of-function has been shown to impair axial elongation, neural tube (NT) closure and pigment cell development. Intriguingly, in contrast to axial elongation and NT closure, a Cdx role in neural crest (NC)-derived melanocyte/pigment cell development has not been reported in any other chordate species. To address this, we generated a new conditional pan-Cdx functional knockdown mouse model that circumvents Cdx functional redundancy as well as the early embryonic lethality of Cdx mutants. Through directed inhibition in the neuroectoderm, we providein vivoevidence that murine Cdx proteins impact melanocyte and enteric nervous system development by, at least in part, directly controlling the expression of the key early regulators of NC ontogenesisPax3,Msx1andFoxd3 Our work thus reveals a novel role for Cdx proteins at the top of the trunk NC gene regulatory network in the mouse, which appears to have been inherited from their ancestral ortholog. PMID:26952979

  2. PreImplantation factor (PIF*) regulates systemic immunity and targets protective regulatory and cytoskeleton proteins.

    PubMed

    Barnea, Eytan R; Hayrabedyan, Soren; Todorova, Krassimira; Almogi-Hazan, Osnat; Or, Reuven; Guingab, Joy; McElhinney, James; Fernandez, Nelson; Barder, Timothy

    2016-07-01

    Secreted by viable embryos, PIF is expressed by the placenta and found in maternal circulation. It promotes implantation and trophoblast invasion, achieving systemic immune homeostasis. Synthetic PIF successfully transposes endogenous PIF features to non-pregnant immune and transplant models. PIF affects innate and activated PBMC cytokines and genes expression. We report that PIF targets similar proteins in CD14+, CD4+ and CD8+ cells instigating integrated immune regulation. PIF-affinity chromatography followed by mass-spectrometry, pathway and heatmap analysis reveals that SET-apoptosis inhibitor, vimentin, myosin-9 and calmodulin are pivotal for immune regulation. PIF acts on macrophages down-stream of LPS (lipopolysaccharide-bacterial antigen) CD14/TLR4/MD2 complex, targeting myosin-9, thymosin-α1 and 14-3-3eta. PIF mainly targets platelet aggregation in CD4+, and skeletal proteins in CD8+ cells. Pathway analysis demonstrates that PIF targets and regulates SET, tubulin, actin-b, and S100 genes expression. PIF targets systemic immunity and has a short circulating half-life. Collectively, PIF targets identified; protective, immune regulatory and cytoskeleton proteins reveal mechanisms involved in the observed efficacy against immune disorders. PMID:26944449

  3. Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1

    PubMed Central

    2010-01-01

    Background Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp)-homologues. The function of two of them, Irp (OE3923F) and lrpA1 (OE2621R), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays. Results It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter. Conclusion The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3. PMID:20509863

  4. Arthritis protective regulatory potential of self–heat shock protein cross-reactive T cells

    PubMed Central

    van Eden, Willem; Wendling, Uwe; Paul, Liesbeth; Prakken, Berent; van Kooten, Peter; van der Zee, Ruurd

    2000-01-01

    Immunization with heat shock proteins has protective effects in models of induced arthritis. Analysis has shown a reduced synovial inflammation in such protected animals. Adoptive transfer and immunization with selected T cell epitopes (synthetic peptides) have indicated the protection to be mediated by T cells directed to conserved hsp epitopes. This was shown first for mycobacterial hsp60 and later for mycobacterial hsp70. Fine specificity analysis showed that such T cells were cross-reactive with the homologous self hsp. Therefore protection by microbial hsp reactive T cells can be by cross-recognition of self hsp overexpressed in the inflamed tissue. Preimmunization with hsp leads to a relative expansion of such self hsp cross-responsive T cells. The regulatory nature of such T cells may originate from mucosal tolerance maintained by commensal flora derived hsp or from partial activation through recognition of self hsp as a partial agonist (Altered Peptide Ligand) or in the absence of proper costimulation. Recently, we reported the selective upregulation of B7.2 on microbial hsp60 specific T cells in response to self hsp60. Through a preferred interaction with CTLA-4 on proinflammatory T cells this may constitute an effector mechanism of regulation. Also, regulatory T cells produced IL10. PMID:11189451

  5. Different expression of protein kinase A (PKA) regulatory subunits in normal and neoplastic thyroid tissues.

    PubMed

    Ferrero, Stefano; Vaira, Valentina; Del Gobbo, Alessandro; Vicentini, Leonardo; Bosari, Silvano; Beck-Peccoz, Paolo; Mantovani, Giovanna; Spada, Anna; Lania, Andrea G

    2015-04-01

    The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in both cell growth and cell differentiation control. Mutations of the PRKAR1A gene have been found in patients with Carney complex and in a minority of sporadic anaplastic thyroid carcinomas. The aim of the study was to retrospectively evaluate the expression of different PKA regulatory subunits in benign and non benign human thyroid tumours and to correlate their expression with clinical phenotype. Immunohistochemistry demonstrated a significant increase in PRKAR2B expression in both differentiated and undifferentiated (anaplastic) thyroid tumors in comparison with normal thyroid tissues. Conversely, a significant increase in PRKAR1A expression was only demonstrated in undifferentiated thyroid carcinomas in comparison with normal thyroid tissue and differentiated thyroid tumors. In thyroid cancers without lymph nodal metastases PRKAR1A expression was higher in tumours of more than 2 cm in size (T2 and T3) compared to smaller ones (T1). In conclusion, our data shows that an increased PRKAR1A expression is associated with aggressive and undifferentiated thyroid tumors. PMID:25393625

  6. Subcelluar compartmentalization of cAMP-dependent protein kinase regulatory subunits during palate ontogeny

    SciTech Connect

    Linask, K.K.; Greene, R.M. )

    1989-01-01

    Mammalian palatal ontogeny involves epithelial-mesenchymal interactions, cell differentiation, and cell movement. These events occur on days 12, 13, and 14 of gestation in the C57BL/6J mouse embryo. During this period intracellular cAMP levels and cAMP-dependent protein kinase (cAMP-dPK) levels in the palate transiently elevate. Cyclic AMP activates cAMP-dPK by binding primarily to two types of regulatory subunits of this enzyme, designated as R{sub I} and R{sub II}. To assess whether differential compartmentalization of the regulatory subunits occurs during palatal ontogeny, cytosolic, nuclear, and particulate fractions were prepared from day 12, 13, and 14 embryonic maxillary and palatal tissue. After photo-affinity labeling of each fraction with 8-azido ({sup 32}P) cAMP, SDS-PAGE, and autoradiography, autoradiograms were analyzed densitometrically. The R{sub I} isoform predominated in the nuclear and particulate fractions on all three developmental days; whereas R{sub II} predominated in the cytosolic fractions. Thus, differential compartmentalization of cAMP-dPK may be a means by which cAMP dependent responses are regulated during palatogenesis.

  7. Protein SUMOylation Is Required for Regulatory T Cell Expansion and Function.

    PubMed

    Ding, Xiao; Wang, Aibo; Ma, Xiaopeng; Demarque, Maud; Jin, Wei; Xin, Huawei; Dejean, Anne; Dong, Chen

    2016-07-26

    Foxp3-expressing regulatory T (Treg) cells are essential for immune tolerance; however, the molecular mechanisms underlying Treg cell expansion and function are still not well understood. SUMOylation is a protein post-translational modification characterized by covalent attachment of SUMO moieties to lysines. UBC9 is the only E2 conjugating enzyme involved in this process, and loss of UBC9 completely abolishes the SUMOylation pathway. Here, we report that selective deletion of Ubc9 within the Treg lineage results in fatal early-onset autoimmunity similar to Foxp3 mutant mice. Ubc9-deficient Treg cells exhibit severe defects in TCR-driven homeostatic proliferation, accompanied by impaired activation and compromised suppressor function. Importantly, TCR ligation enhanced SUMOylation of IRF4, a critical regulator of Treg cell function downstream of TCR signals, which regulates its stability in Treg cells. Our data thus have demonstrated an essential role of SUMOylation in the expansion and function of Treg cells. PMID:27425617

  8. Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Richardson, Dale N.; Wiehe, Thomas

    Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

  9. The reduced soluble fibrinogen-like protein 2 and regulatory T cells in acute coronary syndrome.

    PubMed

    Liu, Kun; Li, Ting; Huang, Shiyuan; Long, Rui; You, Ya; Liu, Jinping; Wang, Zhaohui

    2016-02-01

    Soluble fibrinogen-like protein 2, sfgl2, is the new effector of CD4(+)CD25(+)FOXP3(+) regulatory T cell (Treg) and exerts immunosuppressive activity. We design this study to investigate the possible role of sfgl2 in atherosclerosis. A total of 58 acute coronary syndrome (ACS) patients, together with 22 stable angina (SA) patients and 31 normal coronary artery (NCA) people were enrolled in our study. Serum level of sfgl2 and plasma level of Treg were respectively measured. In line with the change of Treg, serum level of sfgl2 in ACS (8.70 ng/mL) was significantly decreased (P = 0.003), compared with that in SA (11.86 ng/mL) and NCA (17.55 ng/mL). Both sfgl2 and Treg level were obviously decreased in ACS; Sfgl2 may play a protective role in atherosclerosis. PMID:26515143

  10. Mitochondrial Protein Phosphorylation as a Regulatory Modality: Implications for Mitochondrial Dysfunction in Heart Failure

    PubMed Central

    O’Rourke, Brian; Van Eyk, Jennifer E.; Foster, D. Brian

    2014-01-01

    Phosphorylation of mitochondrial proteins has been recognized for decades, and the regulation of pyruvate- and branched-chain α-ketoacid dehydrogenases by an atypical kinase/phosphatase cascade is well established. More recently, the development of new mass spectrometry-based technologies has led to the discovery of many novel phosphorylation sites on a variety of mitochondrial targets. The evidence suggests that the major classes of kinase and several phosphatases may be present at the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix, but many questions remain to be answered as to the location, timing, and reversibility of these phosphorylation events and whether they are functionally relevant. The authors review phosphorylation as a mitochondrial regulatory strategy and highlight its possible role in the pathophysiology of cardiac hypertrophy and failure. PMID:22103918

  11. Regulation of Phagocyte Migration by Signal Regulatory Protein-Alpha Signaling

    PubMed Central

    Alvarez-Zarate, Julian; Matlung, Hanke L.; Matozaki, Takashi; Kuijpers, Taco W.; Maridonneau-Parini, Isabelle; van den Berg, Timo K.

    2015-01-01

    Signaling through the inhibitory receptor signal regulatory protein-alpha (SIRPα) controls effector functions in phagocytes. However, there are also indications that interactions between SIRPα and its ligand CD47 are involved in phagocyte transendothelial migration. We have investigated the involvement of SIRPα signaling in phagocyte migration in vitro and in vivo using mice that lack the SIRPα cytoplasmic tail. During thioglycolate-induced peritonitis in SIRPα mutant mice, both neutrophil and macrophage influx were found to occur, but to be significantly delayed. SIRPα signaling appeared to be essential for an optimal transendothelial migration and chemotaxis, and for the amoeboid type of phagocyte migration in 3-dimensional environments. These findings demonstrate, for the first time, that SIRPα signaling can directly control phagocyte migration, and this may contribute to the impaired inflammatory phenotype that has been observed in the absence of SIRPα signaling. PMID:26057870

  12. Role of the steroidogenic acute regulatory protein in health and disease.

    PubMed

    Manna, Pulak R; Stetson, Cloyce L; Slominski, Andrzej T; Pruitt, Kevin

    2016-01-01

    Steroid hormones are an important class of regulatory molecules that are synthesized in steroidogenic cells of the adrenal, ovary, testis, placenta, brain, and skin, and influence a spectrum of developmental and physiological processes. The steroidogenic acute regulatory protein (STAR) predominantly mediates the rate-limiting step in steroid biosynthesis, i.e., the transport of the substrate of all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane. At the inner membrane, cytochrome P450 cholesterol side chain cleavage enzyme cleaves the cholesterol side chain to form the first steroid, pregnenolone, which is converted by a series of enzymes to various steroid hormones in specific tissues. Both basic and clinical evidence have demonstrated the crucial involvement of the STAR protein in the regulation of steroid biosynthesis. Multiple levels of regulation impinge on STAR action. Recent findings demonstrate that hormone-sensitive lipase, through its action on the hydrolysis of cholesteryl esters, plays an important role in regulating STAR expression and steroidogenesis which involve the liver X receptor pathway. Activation of the latter influences macrophage cholesterol efflux that is a key process in the prevention of atherosclerotic cardiovascular disease. Appropriate regulation of steroid hormones is vital for proper functioning of many important biological activities, which are also paramount for geriatric populations to live longer and healthier. This review summarizes the current level of understanding on tissue-specific and hormone-induced regulation of STAR expression and steroidogenesis, and provides insights into a number of cholesterol and/or steroid coupled physiological and pathophysiological consequences. PMID:26271515

  13. Evolutionary Adaptation of an AraC-Like Regulatory Protein in Citrobacter rodentium and Escherichia Species

    PubMed Central

    Tan, Aimee; Petty, Nicola K.; Hocking, Dianna; Bennett-Wood, Vicki; Wakefield, Matthew; Praszkier, Judyta; Tauschek, Marija; Yang, Ji

    2015-01-01

    The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogen Citrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and the grlRA operon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genus Escherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated that regA has been horizontally transferred to Escherichia spp. and C. rodentium. Comparative studies of two RegA homologues, namely, those from C. rodentium and E. coli SMS-3-5, a multiresistant environmental strain of E. coli, showed that the two regulators acted similarly in vitro but differed in terms of their abilities to activate the virulence of C. rodentium in vivo, which evidently was due to their differential activation of grlRA. Our data indicate that RegA from C. rodentium has strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence by C. rodentium and on the evolution of virulence-regulatory genes of bacterial pathogens in general. PMID:25624355

  14. Biosafety, biosecurity and internationally mandated regulatory regimes: compliance mechanisms for education and global health security

    PubMed Central

    Sture, Judi; Whitby, Simon; Perkins, Dana

    2015-01-01

    This paper highlights the biosafety and biosecurity training obligations that three international regulatory regimes place upon states parties. The duty to report upon the existence of such provisions as evidence of compliance is discussed in relation to each regime. We argue that such mechanisms can be regarded as building blocks for the development and delivery of complementary biosafety and biosecurity teaching and training materials. We show that such building blocks represent foundations upon which life and associated scientists – through greater awareness of biosecurity concerns – can better fulfil their responsibilities to guard their work from misuse in the future. PMID:24494580

  15. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  16. 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

  17. Global risk assessment of aflatoxins in maize and peanuts: are regulatory standards adequately protective?

    PubMed

    Wu, Felicia; Stacy, Shaina L; Kensler, Thomas W

    2013-09-01

    The aflatoxins are a group of fungal metabolites that contaminate a variety of staple crops, including maize and peanuts, and cause an array of acute and chronic human health effects. Aflatoxin B1 in particular is a potent liver carcinogen, and hepatocellular carcinoma (HCC) risk is multiplicatively higher for individuals exposed to both aflatoxin and chronic infection with hepatitis B virus (HBV). In this work, we sought to answer the question: do current aflatoxin regulatory standards around the world adequately protect human health? Depending upon the level of protection desired, the answer to this question varies. Currently, most nations have a maximum tolerable level of total aflatoxins in maize and peanuts ranging from 4 to 20ng/g. If the level of protection desired is that aflatoxin exposures would not increase lifetime HCC risk by more than 1 in 100,000 cases in the population, then most current regulatory standards are not adequately protective even if enforced, especially in low-income countries where large amounts of maize and peanuts are consumed and HBV prevalence is high. At the protection level of 1 in 10,000 lifetime HCC cases in the population, however, almost all aflatoxin regulations worldwide are adequately protective, with the exception of several nations in Africa and Latin America. PMID:23761295

  18. Global Risk Assessment of Aflatoxins in Maize and Peanuts: Are Regulatory Standards Adequately Protective?

    PubMed Central

    Wu, Felicia

    2013-01-01

    The aflatoxins are a group of fungal metabolites that contaminate a variety of staple crops, including maize and peanuts, and cause an array of acute and chronic human health effects. Aflatoxin B1 in particular is a potent liver carcinogen, and hepatocellular carcinoma (HCC) risk is multiplicatively higher for individuals exposed to both aflatoxin and chronic infection with hepatitis B virus (HBV). In this work, we sought to answer the question: do current aflatoxin regulatory standards around the world adequately protect human health? Depending upon the level of protection desired, the answer to this question varies. Currently, most nations have a maximum tolerable level of total aflatoxins in maize and peanuts ranging from 4 to 20ng/g. If the level of protection desired is that aflatoxin exposures would not increase lifetime HCC risk by more than 1 in 100,000 cases in the population, then most current regulatory standards are not adequately protective even if enforced, especially in low-income countries where large amounts of maize and peanuts are consumed and HBV prevalence is high. At the protection level of 1 in 10,000 lifetime HCC cases in the population, however, almost all aflatoxin regulations worldwide are adequately protective, with the exception of several nations in Africa and Latin America. PMID:23761295

  19. Systematic identification of regulatory proteins critical for T-cell activation

    PubMed Central

    Chu, Peter; Pardo, Jorge; Zhao, Haoran; Li, Connie C; Pali, Erlina; Shen, Mary M; Qu, Kunbin; Yu, Simon X; Huang, Betty CB; Yu, Peiwen; Masuda, Esteban S; Molineaux, Susan M; Kolbinger, Frank; Aversa, Gregorio; de Vries, Jan; Payan, Donald G; Liao, X Charlene

    2003-01-01

    Background The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. Results In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10Rα and integrin α2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. Conclusions This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings. PMID:12974981

  20. Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis

    PubMed Central

    Elsholz, Alexander K. W.; Turgay, Kürşad; Michalik, Stephan; Hessling, Bernd; Gronau, Katrin; Oertel, Dan; Mäder, Ulrike; Bernhardt, Jörg; Becher, Dörte; Hecker, Michael; Gerth, Ulf

    2012-01-01

    Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria. PMID:22517742

  1. Retinoid regulated macrophage cholesterol efflux involves the steroidogenic acute regulatory protein

    PubMed Central

    Manna, Pulak R.

    2016-01-01

    Elimination of excess cholesteryl esters from macrophage-derived foam cells is known to be a key process in limiting plaque stability and progression of atherosclerotic lesions. We have recently demonstrated that regulation of retinoid mediated cholesterol efflux is influenced by liver X receptor (LXR) signaling in mouse macrophages (Manna, P.R. et al., 2015, Biochem. Biophys. Res. Commun., 464:312-317). The data presented in this article evaluate the importance of the steroidogenic acute regulatory protein (StAR) in retinoid mediated macrophage cholesterol efflux. Overexpression of StAR in mouse RAW 264.7 macrophages increased the effects of both all-trans retinoic acid (atRA) and 9-cis RA on cholesterol efflux, suggesting StAR enhances the efficacy of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) ligands. Additional data revealed that atRA enhances (Bu)2cAMP induced StAR and ATP-binding cassette transporter A1 protein levels. Treatment of macrophages transfected with an LXRE reporter plasmid (pLXREx3-Luc) was found to induce the effects of RAR and RXR analogs on LXR activity. PMID:27081671

  2. Retinoid regulated macrophage cholesterol efflux involves the steroidogenic acute regulatory protein.

    PubMed

    Manna, Pulak R

    2016-06-01

    Elimination of excess cholesteryl esters from macrophage-derived foam cells is known to be a key process in limiting plaque stability and progression of atherosclerotic lesions. We have recently demonstrated that regulation of retinoid mediated cholesterol efflux is influenced by liver X receptor (LXR) signaling in mouse macrophages (Manna, P.R. et al., 2015, Biochem. Biophys. Res. Commun., 464:312-317). The data presented in this article evaluate the importance of the steroidogenic acute regulatory protein (StAR) in retinoid mediated macrophage cholesterol efflux. Overexpression of StAR in mouse RAW 264.7 macrophages increased the effects of both all-trans retinoic acid (atRA) and 9-cis RA on cholesterol efflux, suggesting StAR enhances the efficacy of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) ligands. Additional data revealed that atRA enhances (Bu)2cAMP induced StAR and ATP-binding cassette transporter A1 protein levels. Treatment of macrophages transfected with an LXRE reporter plasmid (pLXREx3-Luc) was found to induce the effects of RAR and RXR analogs on LXR activity. PMID:27081671

  3. Age-related changes in red blood cell complement regulatory proteins and susceptibility to severe malaria.

    PubMed

    Waitumbi, John N; Donvito, Béatrice; Kisserli, Aymric; Cohen, Jacques H M; Stoute, José A

    2004-09-15

    Severe malaria-associated anemia and cerebral malaria are life-threatening complications of Plasmodium falciparum infection. Red blood cell (RBC) complement regulatory proteins (CRPs) have been implicated in the pathogenesis of both. We sought to determine whether there are age-related changes in the expression of CRPs that could explain the susceptibility to severe malaria-associated anemia in young children and the susceptibility to cerebral malaria in older children and adults. In cross-sectional surveys in malaria-endemic and -nonendemic areas of Kenya and in Reims, France, the level of RBC CRPs was lowest in young children and increased into adulthood. In case-control studies, patients with cerebral malaria and matched control subjects had higher levels of RBC CRPs than did patients with severe anemia and matched control subjects, especially during convalescence. We conclude that RBC CRP levels vary with age and that the lower levels of these proteins in young children in areas of high transmission, such as western Kenya, may place these children at greater risk of severe malaria-associated anemia than cerebral malaria. PMID:15319870

  4. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

    PubMed Central

    Garbers, C; DeLong, A; Deruére, J; Bernasconi, P; Söll, D

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis. Images PMID:8641277

  5. Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

    PubMed

    Werner, Ernst R; Bahrami, Soheyl; Heller, Regine; Werner-Felmayer, Gabriele

    2002-03-22

    GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine. PMID:11799107

  6. GTP cyclohydrolase I feedback regulatory protein-dependent and -independent inhibitors of GTP cyclohydrolase I.

    PubMed

    Yoneyama, T; Wilson, L M; Hatakeyama, K

    2001-04-01

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates the feedback inhibition of GTP cyclohydrolase I activity by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) through protein complex formation. Since guanine and BH4 have a common pyrimidine ring structure, we examined the inhibitory effect of guanine and its analogs on the enzyme activity. Guanine, 8-hydroxyguanine, 8-methylguanine, and 8-bromoguanine inhibited the enzyme activity in a GFRP-dependent and pH-dependent manner and induced complex formation between GTP cyclohydrolase I and GFRP. The type of inhibition by this group is a mixed type. All these properties were shared with BH4. In striking contrast, inhibition by 8-azaguanine and 8-mercaptoguanine was GFRP-independent and pH-independent. The type of inhibition by 8-azaguanine and 8-mercaptoguanine was a competitive type. The two compounds did not induce complex formation between the enzyme and GFRP. These results demonstrate that guanine compounds of the first group bind to the BH4-binding site of the GTP cyclohydrolase I/GFRP complex, whereas 8-azaguanine and 8-mercaptoguanine bind to the active site of the enzyme. Finally, the possible implications in Lesch-Nyhan syndrome and Parkinson diseases of the inhibition of GTP cyclohydrolase I by guanine and 8-hydroxyguanine are discussed. PMID:11361142

  7. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  8. Proteins that bind regulatory regions identified by histone modification chromatin immunoprecipitations and mass spectrometry

    PubMed Central

    Engelen, Erik; Brandsma, Johannes H.; Moen, Maaike J.; Signorile, Luca; Dekkers, Dick H. W.; Demmers, Jeroen; Kockx, Christel E. M.; Ozgür, Zehila; van IJcken, Wilfred F. J.; van den Berg, Debbie L. C.; Poot, Raymond A.

    2015-01-01

    The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provides a detailed read-out of the transcriptional landscape representative of the investigated cell type. PMID:25990348

  9. The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

    PubMed Central

    Brownlee, Christopher W.; Klebba, Joey E.; Buster, Daniel W.

    2011-01-01

    Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. In this paper, we show that PP2A (Protein Phosphatase 2ATwins) counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A’s regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification. PMID:21987638

  10. Complement regulatory proteins are incorporated into lentiviral vectors and protect particles against complement inactivation.

    PubMed

    Schauber-Plewa, C; Simmons, A; Tuerk, M J; Pacheco, C D; Veres, G

    2005-02-01

    Lentiviral vectors pseudotyped with G glycoprotein from vesicular stomatitis virus (VSV-G) and baculovirus gp64 are inactivated by human complement. The extent of vector inactivation in serum from individual donors was examined and results showed wide donor-dependent variation in complement sensitivity for VSV-G-pseudotyped lentivectors. Amphotropic envelope (Ampho)-pseudotyped vectors were generally resistant to serum from all donors, while gp64-pseudotyped vectors were inactivated but showed less donor-to-donor variation than VSV-G. In animal sera, the vectors were mostly resistant to inactivation by rodent complement, whereas canine complement caused a moderate reduction in titer. In a novel advance for the lentiviral vector system, human complement-resistant-pseudotyped lentivector particles were produced through incorporation of complement regulatory proteins (CRPs). Decay accelerating factor (DAF)/CD55 provided the most effective protection using this method, while membrane cofactor protein (MCP)/CD46 showed donor-dependent protection and CD59 provided little or no protection against complement inactivation. Unlike previous approaches using CRPs to produce complement-resistant viral vectors, CRP-containing lentivectors particles were generated for this study without engineering the CRP molecules. Thus, through overexpression of native DAF/CD55 in the viral producer cell, an easy method was developed for generation of lentiviral vectors that are almost completely resistant to inactivation by human complement. Production of complement-resistant lentiviral particles is a critical step toward use of these vectors for in vivo gene therapy applications. PMID:15550926

  11. Activation of Interferon Regulatory Factor 3 Is Inhibited by the Influenza A Virus NS1 Protein

    PubMed Central

    Talon, Julie; Horvath, Curt M.; Polley, Rosalind; Basler, Christopher F.; Muster, Thomas; Palese, Peter; García-Sastre, Adolfo

    2000-01-01

    We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-α/β) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral interferon response by inhibiting the activation of interferon regulatory factor 3 (IRF-3), a key regulator of IFN-α/β gene expression. IRF-3 activation and, as a consequence, IFN-β mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the interferon signaling pathway. Inhibition of IRF-3 activation can be achieved by the expression of wild-type NS1 in trans, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-3 activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses. PMID:10933707

  12. Dynamic Localization of Glucokinase and Its Regulatory Protein in Hypothalamic Tanycytes

    PubMed Central

    Ordenes, Patricio; Millán, Carola; Yañez, María José; Llanos, Paula; Villagra, Marcos; Elizondo-Vega, Roberto; Martínez, Fernando; Nualart, Francisco; Uribe, Elena; de los Angeles García-Robles, María

    2014-01-01

    Glucokinase (GK), the hexokinase involved in glucose sensing in pancreatic β cells, is also expressed in hypothalamic tanycytes, which cover the ventricular walls of the basal hypothalamus and are implicated in an indirect control of neuronal activity by glucose. Previously, we demonstrated that GK was preferentially localized in tanycyte nuclei in euglycemic rats, which has been reported in hepatocytes and is suggestive of the presence of the GK regulatory protein, GKRP. In the present study, GK intracellular localization in hypothalamic and hepatic tissues of the same rats under several glycemic conditions was compared using confocal microscopy and Western blot analysis. In the hypothalamus, increased GK nuclear localization was observed in hyperglycemic conditions; however, it was primarily localized in the cytoplasm in hepatic tissue under the same conditions. Both GK and GKRP were next cloned from primary cultures of tanycytes. Expression of GK by Escherichia coli revealed a functional cooperative protein with a S0.5 of 10 mM. GKRP, expressed in Saccharomyces cerevisiae, inhibited GK activity in vitro with a Ki 0.2 µM. We also demonstrated increased nuclear reactivity of both GK and GKRP in response to high glucose concentrations in tanycyte cultures. These data were confirmed using Western blot analysis of nuclear extracts. Results indicate that GK undergoes short-term regulation by nuclear compartmentalization. Thus, in tanycytes, GK can act as a molecular switch to arrest cellular responses to increased glucose. PMID:24739934

  13. Inhibition of ovarian cancer cell proliferation in vivo and incorporation of /sup 3/H-thymidine in vitro after follicle regulatory protein administration

    SciTech Connect

    Rodgers, K.E.; Montz, F.J.; Scott, L.; Condon, S.; Fujimori, K.; diZerega, G.S.

    1989-01-01

    Follicle regulatory protein immunoreactivity and biologic activity were measured in ascites from a patient with juvenile granulosa cell tumor. Microscopic examination of immunohistochemical staining of a juvenile granulosa cell tumor with anti-follicle regulatory protein antisera showed homogeneous cytosolic expression of follicle regulatory protein throughout the tumor. Tumor cells were injected subcutaneously into nude mice. Partially purified follicle regulatory protein (50 micrograms/day) was then injected daily for 10 days, or for 25 days once the tumor became palpable. Treatment with follicle regulatory protein significantly slowed the rate of tumor growth with both treatments. To test the tissue specificity of the effect, a metastatic, well-differentiated endometrial adenocarcinoma was also grown in nude mice. Follicle regulatory protein treatment did not alter the rate of tumor growth. An in vitro clonigenic assay confirmed these in vivo results. Partially purified follicle regulatory protein had a biphasic effect on the proliferation of juvenile granulosa tumor cell but did not affect the proliferation of endometrial adenocarcinoma cells. Clonigenic assays were performed on five ovarian adenocarcinomas passaged in vitro, and these tumor cells exhibited a biphasic response to follicle regulatory protein. Immunoneutralization studies showed that this biphasic response was due to impurities in the follicle regulatory protein preparations. The longer the exposure of the tumor cells to follicle regulatory protein, the greater the degree of inhibition of proliferation. In summary, administration of follicle regulatory protein slowed tumor growth through a direct effect on the tumor cell rather than an indirect effect on the hormonal or immune status of the host.

  14. Global trade and assisted reproductive technologies: regulatory challenges in international surrogacy.

    PubMed

    Nelson, Erin

    2013-01-01

    International surrogacy is an increasingly common phenomenon and an important global health challenge. Legal rules are a key consideration for the participants in international surrogacy arrangements. In some cases the law can help to resolve the complex issues that arise in this context, but it is important to consider the role played by law in contributing to the complex conflicts that such arrangements can generate. PMID:23581668

  15. Influence of energy supply on expression of genes encoding for lipogenic enzymes and regulatory proteins in growing beef steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty crossbred beef steers were used to determine the effects metabolizable energy (ME) intake and of site and complexity of carbohydrate (CHO) infusion on expression of genes encoding lipogenic enzymes and regulatory proteins in subcutaneous (SC), mesenteric (MES) and omental (OM) adipose. Treatm...

  16. Extracellular Superoxide Dismutase Regulates the Expression of Small GTPase Regulatory Proteins GEFs, GAPs, and GDI

    PubMed Central

    Laukkanen, Mikko O.; Cammarota, Francesca; Esposito, Tiziana; Salvatore, Marco; Castellone, Maria D.

    2015-01-01

    Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3–induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3–driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme. PMID:25751262

  17. Extracellular superoxide dismutase regulates the expression of small gtpase regulatory proteins GEFs, GAPs, and GDI.

    PubMed

    Laukkanen, Mikko O; Cammarota, Francesca; Esposito, Tiziana; Salvatore, Marco; Castellone, Maria D

    2015-01-01

    Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3-induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3-driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme. PMID:25751262

  18. Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

    PubMed

    Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

    2016-03-01

    The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators. PMID:26552797

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

    PubMed

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

    2006-12-01

    RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-gamma-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1-DNA and STAT-DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression. PMID:16886906

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

    PubMed Central

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

    2006-01-01

    RNA-binding activity of IRP1 (iron regulatory protein 1) is regulated by the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule. NO (nitic oxide), whose ability to activate IRP1 by removing its [4Fe-4S] cluster is well known, has also been shown to down-regulate expression of the IRP1 gene. In the present study, we examine whether this regulation occurs at the transcriptional level. Analysis of the mouse IRP1 promoter sequence revealed two conserved putative binding sites for transcription factor(s) regulated by NO and/or changes in intracellular iron level: Sp1 (promoter-selective transcription factor 1) and MTF1 (metal transcription factor 1), plus GAS (interferon-γ-activated sequence), a binding site for STAT (signal transducer and activator of transcription) proteins. In order to define the functional activity of these sequences, reporter constructs were generated through the insertion of overlapping fragments of the mouse IRP1 promoter upstream of the luciferase gene. Transient expression assays following transfection of HuH7 cells with these plasmids revealed that while both the Sp1 and GAS sequences are involved in basal transcriptional activity of the IRP1 promoter, the role of the latter is predominant. Analysis of protein binding to these sequences in EMSAs (electrophoretic mobility-shift assays) using nuclear extracts from mouse RAW 264.7 macrophages stimulated to synthesize NO showed a significant decrease in the formation of Sp1–DNA and STAT–DNA complexes, compared with controls. We have also demonstrated that the GAS sequence is involved in NO-dependent down-regulation of IRP1 transcription. Further analysis revealed that levels of STAT5a and STAT5b in the nucleus and cytosol of NO-producing macrophages are substantially lower than in control cells. These findings provide evidence that STAT5 proteins play a role in NO-mediated down-regulation of IRP1 gene expression. PMID:16886906

  1. GTP cyclohydrolase I feedback regulatory protein is expressed in serotonin neurons and regulates tetrahydrobiopterin biosynthesis.

    PubMed

    Kapatos, G; Hirayama, K; Shimoji, M; Milstien, S

    1999-02-01

    Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalanine, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit, the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L-phenylalanine specifically stimulates tetrahydrobiopterin synthesis by displacing tetrahydrobiopterin from the GTPCH-GFRP complex. To explore the role of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double-label in situ hybridization. GFRP mRNA expression was abundant in serotonin neurons of the dorsal raphe nucleus but was undetectable in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrain. Two species of GFRP mRNA differing by approximately 20 nucleotides in length were detected in brainstem but not in other tissues, with the longer, more abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mRNA, although GTPCH mRNA was abundant in both. Primary neuronal cultures were used to examine the role of GFRP-mediated regulation of GTPCH on tetrahydrobiopterin synthesis within brainstem serotonin neurons and midbrain dopamine neurons. L-Phenylalanine increased tetrahydrobiopterin levels in serotonin neurons to a maximum of twofold in a concentration-dependent manner, whereas D-phenylalanine and L-tryptophan were without effect. In contrast, tetrahydrobiopterin levels within cultured dopamine neurons were not altered by L-phenylalanine. The time course of this effect was very rapid, with a maximal response observed within 60 min. Inhibitors of tetrahydrobiopterin biosynthesis prevented the L

  2. Identification of a regulatory subunit of protein phosphatase 1 which mediates blue light signaling for stomatal opening.

    PubMed

    Takemiya, Atsushi; Yamauchi, Shota; Yano, Takayuki; Ariyoshi, Chie; Shimazaki, Ken-ichiro

    2013-01-01

    Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase comprised of a catalytic subunit (PP1c) and a regulatory subunit that modulates catalytic activity, subcellular localization and substrate specificity. PP1c positively regulates stomatal opening through blue light signaling between phototropins and the plasma membrane H(+)-ATPase in guard cells. However, the regulatory subunit functioning in this process is unknown. We identified Arabidopsis PRSL1 (PP1 regulatory subunit2-like protein1) as a regulatory subunit of PP1c. Tautomycin, a selective inhibitor of PP1c, inhibited blue light responses of stomata in the single mutants phot1 and phot2, supporting the idea that signals from phot1 and phot2 converge on PP1c. We obtained PRSL1 based on the sequence similarity to Vicia faba PRS2, a PP1c-binding protein isolated by a yeast two-hybrid screen. PRSL1 bound to Arabidopsis PP1c through its RVxF motif, a consensus PP1c-binding sequence. Arabidopsis prsl1 mutants were impaired in blue light-dependent stomatal opening, H(+) pumping and phosphorylation of the H(+)-ATPase, but showed normal phototropin activities. PRSL1 complemented the prsl1 phenotype, but not if the protein carried a mutation in the RVxF motif, suggesting that PRSL1 functions through binding PP1c via the RVxF motif. PRSL1 did not affect the catalytic activity of Arabidopsis PP1c but it stimulated the localization of PP1c in the cytoplasm. We conclude that PRSL1 functions as a regulatory subunit of PP1 and regulates blue light signaling in stomata. PMID:22585556

  3. A Common Missense Variant in the Glucokinase Regulatory Protein Gene (GCKR) Is Associated with Increased Plasma Triglyceride and C-Reactive Protein but Lower Fasting Glucose Concentrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVE-Using the genome-wide-association approach, we recently identified the glucokinase regulatory protein gene (GCKR, rs780094) region as a novel quantitative trait locus for plasma triglyceride concentration in Europeans. Here, we sought to study the association of GCKR variants with metaboli...

  4. In Bacillus subtilis LutR is part of the global complex regulatory network governing the adaptation to the transition from exponential growth to stationary phase.

    PubMed

    Irigül-Sönmez, Öykü; Köroğlu, Türkan E; Öztürk, Büşra; Kovács, Ákos T; Kuipers, Oscar P; Yazgan-Karataş, Ayten

    2014-02-01

    The lutR gene, encoding a product resembling a GntR-family transcriptional regulator, has previously been identified as a gene required for the production of the dipeptide antibiotic bacilysin in Bacillus subtilis. To understand the broader regulatory roles of LutR in B. subtilis, we studied the genome-wide effects of a lutR null mutation by combining transcriptional profiling studies using DNA microarrays, reverse transcription quantitative PCR, lacZ fusion analyses and gel mobility shift assays. We report that 65 transcriptional units corresponding to 23 mono-cistronic units and 42 operons show altered expression levels in lutR mutant cells, as compared with lutR(+) wild-type cells in early stationary phase. Among these, 11 single genes and 25 operons are likely to be under direct control of LutR. The products of these genes are involved in a variety of physiological processes associated with the onset of stationary phase in B. subtilis, including degradative enzyme production, antibiotic production and resistance, carbohydrate utilization and transport, nitrogen metabolism, phosphate uptake, fatty acid and phospholipid biosynthesis, protein synthesis and translocation, cell-wall metabolism, energy production, transfer of mobile genetic elements, induction of phage-related genes, sporulation, delay of sporulation and cannibalism, and biofilm formation. Furthermore, an electrophoretic mobility shift assay performed in the presence of both SinR and LutR revealed a close overlap between the LutR and SinR targets. Our data also revealed a significant overlap with the AbrB regulon. Together, these findings reveal that LutR is part of the global complex, interconnected regulatory systems governing adaptation of bacteria to the transition from exponential growth to stationary phase. PMID:24196425

  5. Global identification of the genetic networks and cis-regulatory elements of the cold response in zebrafish

    PubMed Central

    Hu, Peng; Liu, Mingli; Zhang, Dong; Wang, Jinfeng; Niu, Hongbo; Liu, Yimeng; Wu, Zhichao; Han, Bingshe; Zhai, Wanying; Shen, Yu; Chen, Liangbiao

    2015-01-01

    The transcriptional programs of ectothermic teleosts are directly influenced by water temperature. However, the cis- and trans-factors governing cold responses are not well characterized. We profiled transcriptional changes in eight zebrafish tissues exposed to mildly and severely cold temperatures using RNA-Seq. A total of 1943 differentially expressed genes (DEGs) were identified, from which 34 clusters representing distinct tissue and temperature response expression patterns were derived using the k-means fuzzy clustering algorithm. The promoter regions of the clustered DEGs that demonstrated strong co-regulation were analysed for enriched cis-regulatory elements with a motif discovery program, DREME. Seventeen motifs, ten known and seven novel, were identified, which covered 23% of the DEGs. Two motifs predicted to be the binding sites for the transcription factors Bcl6 and Jun, respectively, were chosen for experimental verification, and they demonstrated the expected cold-induced and cold-repressed patterns of gene regulation. Protein interaction modeling of the network components followed by experimental validation suggested that Jun physically interacts with Bcl6 and might be a hub factor that orchestrates the cold response in zebrafish. Thus, the methodology used and the regulatory networks uncovered in this study provide a foundation for exploring the mechanisms of cold adaptation in teleosts. PMID:26227973

  6. The US regulatory and pharmacopeia response to the global heparin contamination crisis.

    PubMed

    Szajek, Anita Y; Chess, Edward; Johansen, Kristian; Gratzl, Gyöngyi; Gray, Elaine; Keire, David; Linhardt, Robert J; Liu, Jian; Morris, Tina; Mulloy, Barbara; Nasr, Moheb; Shriver, Zachary; Torralba, Pearle; Viskov, Christian; Williams, Roger; Woodcock, Janet; Workman, Wesley; Al-Hakim, Ali

    2016-06-01

    The contamination of the widely used lifesaving anticoagulant drug heparin in 2007 has drawn renewed attention to the challenges that are associated with the characterization, quality control and standardization of complex biological medicines from natural sources. Heparin is a linear, highly sulfated polysaccharide consisting of alternating glucosamine and uronic acid monosaccharide residues. Heparin has been used successfully as an injectable antithrombotic medicine since the 1930s, and its isolation from animal sources (primarily porcine intestine) as well as its manufacturing processes have not changed substantially since its introduction. The 2007 heparin contamination crisis resulted in several deaths in the United States and hundreds of adverse reactions worldwide, revealing the vulnerability of a complex global supply chain to sophisticated adulteration. This Perspective discusses how the US Food and Drug Administration (FDA), the United States Pharmacopeial Convention (USP) and international stakeholders collaborated to redefine quality expectations for heparin, thus making an important natural product better controlled and less susceptible to economically motivated adulteration. PMID:27281424

  7. Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP.

    PubMed

    Milstien, S; Jaffe, H; Kowlessur, D; Bonner, T I

    1996-08-16

    The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and -phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern

  8. Bayesian Proteoform Modeling Improves Protein Quantification of Global Proteomic Measurements

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Datta, Susmita; Payne, Samuel H.; Kang, Jiyun; Bramer, Lisa M.; Nicora, Carrie D.; Shukla, Anil K.; Metz, Thomas O.; Rodland, Karin D.; Smith, Richard D.; Tardiff, Mark F.; McDermott, Jason E.; Pounds, Joel G.; Waters, Katrina M.

    2014-12-01

    As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statistical inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.

  9. The Staphylococcus aureus Global Regulator MgrA Modulates Clumping and Virulence by Controlling Surface Protein Expression

    PubMed Central

    Crosby, Heidi A.; Schlievert, Patrick M.; Merriman, Joseph A.; King, Jessica M.; Salgado-Pabón, Wilmara; Horswill, Alexander R.

    2016-01-01

    Staphylococcus aureus is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of S. aureus is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking mgrA fail to clump in the presence of fibrinogen, and clumping can be restored to an arlRS mutant by overexpressing either arlRS or mgrA, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses ebh, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD). EMSA analysis showed that MgrA directly represses expression of ebh and sraP. Clumping can be restored to an mgrA mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that mgrA mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins ebh, sraP, and sasG. While mgrA mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins. PMID:27144398

  10. Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state.

    PubMed

    Launay, Hélène; Barré, Patrick; Puppo, Carine; Manneville, Stéphanie; Gontero, Brigitte; Receveur-Bréchot, Véronique

    2016-08-12

    The redox switch protein CP12 is a key player of the regulation of the Benson-Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold. PMID:27268235

  11. The physiological functions of iron regulatory proteins in iron homeostasis - an update

    PubMed Central

    Zhang, De-Liang; Ghosh, Manik C.; Rouault, Tracey A.

    2014-01-01

    Iron regulatory proteins (IRPs) regulate the expression of genes involved in iron metabolism by binding to RNA stem-loop structures known as iron responsive elements (IREs) in target mRNAs. IRP binding inhibits the translation of mRNAs that contain an IRE in the 5′untranslated region of the transcripts, and increases the stability of mRNAs that contain IREs in the 3′untranslated region of transcripts. By these mechanisms, IRPs increase cellular iron absorption and decrease storage and export of iron to maintain an optimal intracellular iron balance. There are two members of the mammalian IRP protein family, IRP1 and IRP2, and they have redundant functions as evidenced by the embryonic lethality of the mice that completely lack IRP expression (Irp1-/-/Irp2-/- mice), which contrasts with the fact that Irp1-/- and Irp2-/- mice are viable. In addition, Irp2-/- mice also display neurodegenerative symptoms and microcytic hypochromic anemia, suggesting that IRP2 function predominates in the nervous system and erythropoietic homeostasis. Though the physiological significance of IRP1 had been unclear since Irp1-/- animals were first assessed in the early 1990s, recent studies indicate that IRP1 plays an essential function in orchestrating the balance between erythropoiesis and bodily iron homeostasis. Additionally, Irp1-/- mice develop pulmonary hypertension, and they experience sudden death when maintained on an iron-deficient diet, indicating that IRP1 has a critical role in the pulmonary and cardiovascular systems. This review summarizes recent progress that has been made in understanding the physiological roles of IRP1 and IRP2, and further discusses the implications for clinical research on patients with idiopathic polycythemia, pulmonary hypertension, and neurodegeneration. PMID:24982634

  12. The COP9 Signalosome Interacts with and Regulates Interferon Regulatory Factor 5 Protein Stability

    PubMed Central

    Korczeniewska, Justyna

    2013-01-01

    The transcription factor interferon regulatory factor 5 (IRF5) exerts crucial functions in the regulation of host immunity against extracellular pathogens, DNA damage-induced apoptosis, death receptor signaling, and macrophage polarization. Tight regulation of IRF5 is thus warranted for an efficient response toward extracellular stressors and for limiting autoimmune and inflammatory responses. Here we report that the COP9 signalosome (CSN), a general modulator of diverse cellular and developmental processes, associates constitutively with IRF5 and promotes its protein stability. The constitutive CSN/IRF5 interaction was identified using proteomics and confirmed by endogenous immunoprecipitations. The CSN/IRF5 interaction occurred on the carboxyl and amino termini of IRF5; a single internal deletion from amino acids 455 to 466 (Δ455-466) was found to significantly reduce IRF5 protein stability. CSN subunit 3 (CSN3) was identified as a direct interacting partner of IRF5, and knockdown of this subunit with small interfering RNAs resulted in enhanced degradation. Degradation was further augmented by knockdown of CSN1 and CSN3 together. The ubiquitin E1 inhibitor UBEI-41 or the proteasome inhibitor MG132 prevented IRF5 degradation, supporting the idea that its stability is regulated by the ubiquitin-proteasome system. Importantly, activation of IRF5 by the death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in enhanced degradation via loss of the CSN/IRF5 interaction. This study defines CSN to be a new interacting partner of IRF5 that controls its stability. PMID:23275442

  13. Sterol regulatory element-binding proteins are transcriptional regulators of the thyroglobulin gene in thyroid cells.

    PubMed

    Wen, Gaiping; Eder, Klaus; Ringseis, Robert

    2016-08-01

    The genes encoding sodium/iodide symporter (NIS) and thyroid peroxidase (TPO), both of which are essential for thyroid hormone (TH) synthesis, were shown to be regulated by sterol regulatory element-binding proteins (SREBP)-1c and -2. In the present study we tested the hypothesis that transcription of a further gene essential for TH synthesis, the thyroglobulin (TG) gene, is under the control of SREBP. To test this hypothesis, we studied the influence of inhibition of SREBP maturation and SREBP knockdown on TG expression in FRTL-5 thyrocytes and explored transcriptional regulation of the TG promoter by reporter gene experiments in FRTL-5 and HepG2 cells, gel shift assays and chromatin immunoprecipitation. Inhibition of SREBP maturation by 25-hydroxycholesterol and siRNA-mediated knockdown of either SREBP-1c or SREBP-2 decreased mRNA and protein levels of TG in FRTL-5 thyrocytes. Reporter gene assays with wild-type and mutated TG promoter reporter truncation constructs revealed that the rat TG promoter is transcriptionally activated by nSREBP-1c and nSREBP-2. DNA-binding assays and chromatin immunoprecipitation assays showed that both nSREBP-1c and nSREBP-2 bind to a SREBP binding motif with characteristics of an E-box SRE at position -63 in the rat TG promoter. In connection with recent findings that NIS and TPO are regulated by SREBP in thyrocytes the present findings support the view that SREBP are regulators of essential steps of TH synthesis in the thyroid gland such as iodide uptake, iodide oxidation and iodination of tyrosyl residues of TG. This moreover suggests that SREBP may be molecular targets for pharmacological modulation of TH synthesis. PMID:27321819

  14. The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3

    PubMed Central

    Basler, Christopher F.; Mikulasova, Andrea; Martinez-Sobrido, Luis; Paragas, Jason; Mühlberger, Elke; Bray, Mike; Klenk, Hans-Dieter; Palese, Peter; García-Sastre, Adolfo

    2003-01-01

    The Ebola virus VP35 protein was previously found to act as an interferon (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-β) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP35 inhibits virus infection-induced transcriptional activation of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and that VP35 does not block signaling from the IFN-α/β receptor. The ability of VP35 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-3, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP35 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-3, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent activation of the IFN-α4 promoter in response to viral infection. The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation. VP35 blocks virus-induced IRF-3 phosphorylation and subsequent IRF-3 dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that in Ebola virus-infected cells, VP35 inhibits the induction of antiviral genes, including the IFN-β gene, by blocking IRF-3 activation. PMID:12829834

  15. Regulatory roles of RNA binding proteins in the nervous system of C. elegans

    PubMed Central

    Sharifnia, Panid; Jin, Yishi

    2015-01-01

    Neurons have evolved to employ many factors involved in the regulation of RNA processing due to their complex cellular compartments. RNA binding proteins (RBPs) are key regulators in transcription, translation, and RNA degradation. Increasing studies have shown that regulatory RNA processing is critical for the establishment, functionality, and maintenance of neural circuits. Recent advances in high-throughput transcriptomics have rapidly expanded our knowledge of the landscape of RNA regulation, but also raised the challenge for mechanistic dissection of the specific roles of RBPs in complex tissues such as the nervous system. The C. elegans genome encodes many RBPs conserved throughout evolution. The rich analytic tools in molecular genetics and simple neural anatomy of C. elegans offer advantages to define functions of genes in vivo at the level of a single cell. Notably, the discovery of microRNAs has had transformative effects to the understanding of neuronal development, circuit plasticity, and neurological diseases. Here we review recent studies unraveling diverse roles of RBPs in the development, function, and plasticity of C. elegans nervous system. We first summarize the general technologies for studying RBPs in C. elegans. We then focus on the roles of several RBPs that control gene- and cell-type specific production of neuronal transcripts. PMID:25628531

  16. Generation of novel bacterial regulatory proteins that detect priority pollutant phenols

    SciTech Connect

    Wise, A.A.; Kuske, C.R.

    2000-01-01

    The genetic systems of bacteria that have the ability to use organic pollutants as carbon and energy sources can be adapted to create bacterial biosensors for the detection of industrial pollution. The creation of bacterial biosensors is hampered by a lack of information about the genetic systems that control production of bacterial enzymes that metabolize pollutants. The authors have attempted to overcome this problem through modification of DmpR, a regulatory protein for the phenol degradation pathway of Pseudomonas sp. strain CF600. The phenol detection capacity of DmpR was altered by using mutagenic PCR targeted to the DmpR sensor domain. DmpR mutants were identified that both increased sensitivity to the phenolic effectors of wild-type DmpR and increased the range of molecules detected. The phenol detection characteristics of seven DmpR mutants were demonstrated through their ability to activate transcription of a lacZ reporter gene. Effectors of the DmpR derivatives included phenol, 2-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4-dimethylphenol, 2-nitrophenol, and 4-nitrophenol.

  17. Recognition of the small regulatory RNA RydC by the bacterial Hfq protein

    PubMed Central

    Dimastrogiovanni, Daniela; Fröhlich, Kathrin S; Bandyra, Katarzyna J; Bruce, Heather A; Hohensee, Susann; Vogel, Jörg; Luisi, Ben F

    2014-01-01

    Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a ‘seed’ region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at a 3.48 Å resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein–RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target. DOI: http://dx.doi.org/10.7554/eLife.05375.001 PMID:25551292

  18. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ravikumar, Vaishnavi; Dobrinic, Paula; Macek, Boris; Franjevic, Damjan; Noirot-Gros, Marie-Francoise; Mijakovic, Ivan

    2014-01-01

    Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells. PMID:25278935

  19. Enzymatic changes in myosin regulatory proteins may explain vasoplegia in terminally ill patients with sepsis

    PubMed Central

    Zheng, Wentao; Kou, Yong; Gao, Feng-lan; Ouyang, Xiu-he

    2016-01-01

    The current study was conducted with the hypothesis that failure of maintenance of the vascular tone may be central to failure of the peripheral circulation and spiralling down of blood pressure in sepsis. Namely, we examined the balance between expression of myosin light chain (MLC) phosphatase and kinase, enzymes that regulate MLCs dephosphorylation and phosphorylation with a direct effect on pharmacomechanical coupling for smooth muscle relaxation and contraction respectively. Mechanical recordings and enzyme immunoassays of vascular smooth muscle lysates were used as the major methods to examine arterial biopsy samples from terminally ill sepsis patients. The results of the present study provide evidence that genomic alteration of expression of key regulatory proteins in vascular smooth muscles may be responsible for the relentless downhill course in sepsis. Down-regulation of myosin light chain kinase (MLCK) and up-regulation of MLCK may explain the loss of tone and failure to mount contractile response in vivo during circulation. The mechanical studies demonstrated the inability of the arteries to develop tone when stimulated by phenylephrine in vitro. The results of our study provide indirect hint that control of inflammation is a major therapeutic approach in sepsis, and may facilitate to ameliorate the progressive cardiovascular collapse. PMID:26772992

  20. Exchange Protein Directly Activated by cAMP Modulates Regulatory T-Cell-Mediated Immunosuppression

    PubMed Central

    Almahariq, Muayad; Mei, Fang C.; Wang, Hui; Cao, Anthony T.; Yao, Suxia; Soong, Lynn; Sun, Jiaren; Cong, Yingzi; Chen, Ju; Cheng, Xiaodong

    2016-01-01

    The cyclic adenosine monophosphate (cAMP) signaling pathway plays an essential role in immune functions. In this study we examined the role of the cAMP/EPAC1 (exchange protein directly activated by cAMP) axis in regulatory T-cell (Treg)-mediated immune suppression using genetic and pharmacologic approaches. Genetic deletion of EPAC1 in Treg and effector T-cells (Teff) synergistically attenuated Treg-mediated suppression of Teff. Mechanistically, EPAC1 inhibition enhanced activation of the transcription factor STAT3 and up-regulated SMAD7 expression while down-regulating expression of SMAD4. Consequently, CD4+T-cells were desensitized to TGF-β1, a cytokine employed by Treg cells to exert a broad inhibitory function within the immune system. Furthermore, deletion of EPAC1 led to production of significant levels of OVA-IgG antibodies in a low dose oral tolerance mouse mode. These in vivo observations are consistent with the finding that EPAC1 plays an important role in Treg-mediated suppression. More importantly, pharmacological inhibition of EPAC1 using an EPAC specific inhibitor recapitulates the EPAC1 deletion phenotype both in vivo and in vitro. Our results show that EPAC1 boosts Treg-mediated suppression, and identify EPAC1 as a target with broad therapeutic potential since Treg cells are involved in numerous pathologies including autoimmunity, infections, and a wide range of cancers. PMID:25339598

  1. Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

    SciTech Connect

    Stein, J.C.

    1985-01-01

    Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several /sup 37/P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the /sup 32/P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex.

  2. Acetylation of glucokinase regulatory protein decreases glucose metabolism by suppressing glucokinase activity

    PubMed Central

    Park, Joo-Man; Kim, Tae-Hyun; Jo, Seong-Ho; Kim, Mi-Young; Ahn, Yong-Ho

    2015-01-01

    Glucokinase (GK), mainly expressed in the liver and pancreatic β-cells, is critical for maintaining glucose homeostasis. GK expression and kinase activity, respectively, are both modulated at the transcriptional and post-translational levels. Post-translationally, GK is regulated by binding the glucokinase regulatory protein (GKRP), resulting in GK retention in the nucleus and its inability to participate in cytosolic glycolysis. Although hepatic GKRP is known to be regulated by allosteric mechanisms, the precise details of modulation of GKRP activity, by post-translational modification, are not well known. Here, we demonstrate that GKRP is acetylated at Lys5 by the acetyltransferase p300. Acetylated GKRP is resistant to degradation by the ubiquitin-dependent proteasome pathway, suggesting that acetylation increases GKRP stability and binding to GK, further inhibiting GK nuclear export. Deacetylation of GKRP is effected by the NAD+-dependent, class III histone deacetylase SIRT2, which is inhibited by nicotinamide. Moreover, the livers of db/db obese, diabetic mice also show elevated GKRP acetylation, suggesting a broader, critical role in regulating blood glucose. Given that acetylated GKRP may affiliate with type-2 diabetes mellitus (T2DM), understanding the mechanism of GKRP acetylation in the liver could reveal novel targets within the GK-GKRP pathway, for treating T2DM and other metabolic pathologies. PMID:26620281

  3. Serotonin transporter protein overexpression and association to Th17 and T regulatory cells in lupoid leishmaniasis.

    PubMed

    Mashayekhi Goyonlo, Vahid; Elnour, Husameldin; Nordlind, Klas

    2014-03-01

    The immunopathogenesis of chronic non-healing Old World cutaneous leishmaniasis is challenging. There is a bidirectional communication between the nervous and immune systems, serotonin being an important mediator in this respect. Our aim was to study the role of the serotonin transporter protein (SERT) and its relation to T cell-related immune responses in lupoid leishmaniasis. Paraffin-embedded skin biopsies of 12 cases of lupoid and 12 cases of usual types of cutaneous leishmaniasis were investigated using immunohistochemistry regarding expression of SERT, Th1, Th2, Th17 and T regulatory cell (Treg) markers. SERT as well as Tregs and interleukin (IL)-17 positive cells were more prevalent while IL-5 (Th2) and interferon (IFN)-γ (Th1) expressing cells were less numerous in the lupoid tissue compared to those from the usual type of leishmaniasis. The majority of the SERT(+) cells were also tryptase(+) (mast cells). There was a positive correlation between a higher number of SERT(+) and IL-17(+) cells in the lupoid type, while lower numbers of SERT(+) cells were significantly related to lower percentages of CD25(+) cells in the usual type of leishmaniasis. These results might indicate a role for SERT, Th17 and Tregs in the pathogenesis of lupoid leishmaniasis. PMID:23989888

  4. Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*

    PubMed Central

    Larance, Mark; Kirkwood, Kathryn J.; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A. J.; Lamond, Angus I.

    2016-01-01

    We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452

  5. Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling.

    PubMed

    Larance, Mark; Kirkwood, Kathryn J; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A J; Lamond, Angus I

    2016-07-01

    We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452

  6. Global Analysis of mRNA, Translation, and Protein Localization: Local Translation Is a Key Regulator of Cell Protrusions

    PubMed Central

    Mardakheh, Faraz K.; Paul, Angela; Kümper, Sandra; Sadok, Amine; Paterson, Hugh; Mccarthy, Afshan; Yuan, Yinyin; Marshall, Christopher J.

    2015-01-01

    Summary Polarization of cells into a protrusive front and a retracting cell body is the hallmark of mesenchymal-like cell migration. Many mRNAs are localized to protrusions, but it is unclear to what degree mRNA localization contributes toward protrusion formation. We performed global quantitative analysis of the distributions of mRNAs, proteins, and translation rates between protrusions and the cell body by RNA sequencing (RNA-seq) and quantitative proteomics. Our results reveal local translation as a key determinant of protein localization to protrusions. Accordingly, inhibition of local translation destabilizes protrusions and inhibits mesenchymal-like morphology. Interestingly, many mRNAs localized to protrusions are translationally repressed. Specific cis-regulatory elements within mRNA UTRs define whether mRNAs are locally translated or repressed. Finally, RNAi screening of RNA-binding proteins (RBPs) enriched in protrusions revealed trans-regulators of localized translation that are functionally important for protrusions. We propose that by deciphering the localized mRNA UTR code, these proteins regulate protrusion stability and mesenchymal-like morphology. PMID:26555054

  7. Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation

    PubMed Central

    Lee, Jae Ho; Lee, Gha Young; Jang, Hagoon; Choe, Sung Sik; Koo, Seung-Hoi; Kim, Jae Bum

    2014-01-01

    Sterol regulatory element binding protein1c (SREBP1c) is a key transcription factor for de novo lipogenesis during the postprandial state. During nutritional deprivation, hepatic SREBP1c is rapidly suppressed by fasting signals to prevent lipogenic pathways. However, the molecular mechanisms that control SREBP1c turnover in response to fasting status are not thoroughly understood. To elucidate which factors are involved in the inactivation of SREBP1c, we attempted to identify SREBP1c-interacting proteins by mass spectrometry analysis. Since we observed that ring finger protein20 (RNF20) ubiquitin ligase was identified as one of SREBP1c-interacting proteins, we hypothesized that fasting signaling would promote SREBP1c degradation in an RNF20-dependent manner. In this work, we demonstrate that RNF20 physically interacts with SREBP1c, leading to degradation of SREBP1c via ubiquitination. In accordance with these findings, RNF20 represses the transcriptional activity of SREBP1c and turns off the expression of lipogenic genes that are targets of SREBP1c. In contrast, knockdown of RNF20 stimulates the expression of SREBP1c and lipogenic genes and induces lipogenic activity in primary hepatocytes. Furthermore, activation of protein kinase A (PKA) with glucagon or forskolin enhances the expression of RNF20 and potentiates the ubiquitination of SREBP1c via RNF20. In wild-type and db/db mice, adenoviral overexpression of RNF20 markedly suppresses FASN promoter activity and reduces the level of hepatic triglycerides, accompanied by a decrease in the hepatic lipogenic program. Here, we reveal that RNF20-induced SREBP1c ubiquitination down-regulates hepatic lipogenic activity upon PKA activation. Conclusion: RNF20 acts as a negative regulator of hepatic fatty acid metabolism through degradation of SREBP1c upon PKA activation. Knowledge regarding this process enhances our understanding of how SREBP1c is able to turn off hepatic lipid metabolism during nutritional deprivation

  8. Global small RNA chaperone Hfq and regulatory small RNAs are important virulence regulators in Erwinia amylovora.

    PubMed

    Zeng, Quan; McNally, R Ryan; Sundin, George W

    2013-04-01

    Hfq is a global small RNA (sRNA) chaperone that interacts with Hfq-regulated sRNAs and functions in the posttranscriptional regulation of gene expression. In this work, we identified Hfq to be a virulence regulator in the Gram-negative fire blight pathogen Erwinia amylovora. Deletion of hfq in E. amylovora Ea1189 significantly reduced bacterial virulence in both immature pear fruits and apple shoots. Analysis of virulence determinants in strain Ea1189Δhfq showed that Hfq exerts pleiotropic regulation of amylovoran exopolysaccharide production, biofilm formation, motility, and the type III secretion system (T3SS). Further characterization of biofilm regulation by Hfq demonstrated that Hfq limits bacterial attachment to solid surfaces while promoting biofilm maturation. Characterization of T3SS regulation by Hfq revealed that Hfq positively regulates the translocation and secretion of the major type III effector DspE and negatively controls the secretion of the putative translocator HrpK and the type III effector Eop1. Lastly, 10 Hfq-regulated sRNAs were identified using a computational method, and two of these sRNAs, RprA and RyhA, were found to be required for the full virulence of E. amylovora. PMID:23378513

  9. Quantitative assessment of regulatory proteins in blood as markers of radiation effects in the late period after occupational exposure.

    PubMed

    Kirillova, Evgenia N; Zakharova, Maria L; Muksinova, Klara N; Drugova, Elena D; Pavlova, Olga S; Sokolova, Svetlana N

    2012-07-01

    The objective of this research was quantitative assessment of serum and membrane regulatory proteins in blood from nuclear workers as markers of radiation-induced alterations in immune homeostasis in the late period after protracted exposure of nuclear workers with different doses. The effector and regulatory lymphocytes were measured using a flow cytofluorometer in workers from the main facilities of the Mayak PA (aged ∼60 y up to 80 y) in the late period after combined exposure to external gamma-rays and internal alpha-radiation from incorporated 239Pu. The control group included non-occupationally exposed members of the Ozyorsk population matched by gender and age to the group of Mayak workers. Thirty serum proteins involved in regulation of immune homeostasis, such as growth factors, multifunctional interleukins, pro- and anti-inflammatory cytokines, and their receptors, were measured using ELISA in blood serum specimens from the Radiobiology Human Tissue Repository. The dosimetry estimates were obtained using Doses-2005. The correlation analysis revealed a statistically significant direct relationship of T-killers and plutonium body burden and a decreasing level of T-helpers with accumulated external dose in exposed individuals. There were differences in expression of membrane markers in young regulatory cells (double null T-lymphocytes, NKT-lymphocytes, regulatory T-cells, and an increase of activated forms of T-lymphocytes), which indicated an active role of regulatory cells in maintaining immune homeostasis in terms of protracted exposure. The assessment of regulatory proteins in blood indicated that growth factors (EGF, TGF-β1, PDGF), multifunctional interleukins (IL-17A, IL-18), and pro-inflammatory cytokines (IL-1β and INF-γ) could be potential markers of radiation-induced alterations in protein status. An imbalance of pro- and antiinflammatory proteins in blood and variations of protein profiles at the lower exposure levels (gamma-ray dose <1 Gy

  10. Efficient preparation and metal specificity of the regulatory protein TroR from the human pathogen Treponema pallidum.

    PubMed

    Liu, Yi; Li, Wei; Wei, Yaozhu; Jiang, Yindi; Tan, Xiangshi

    2013-10-01

    TroR is a putative metal-dependent regulatory protein that has been linked to the virulence of the human pathogen Treponema pallidum. It shares high homology with the well-known iron-dependent regulatory protein DtxR from Corynebacterium diphtheriae, as well as the manganese-dependent MntR from Bacillus subtilis. However, it has been uncertain whether manganese or zinc is the natural cofactor of TroR to date. Herein, we established an efficient method named "double-fusion tagging" to obtain soluble TroR for the first time. A series of studies, including ICP, CD, fluorescence, ITC, and electrophoresis mobility shift assay (EMSA), were performed to resolve the discrepancies in its metal-binding specificity. In addition, bioinformatic analysis as well as mutation studies were carried out to find the genetic relationships of TroR with its homology proteins. In conclusion, our findings indicate that TroR is a manganese-dependent rather than a zinc-dependent regulatory protein. PMID:23945957

  11. Effect of branched-chain amino acid supplementation during unloading on regulatory components of protein synthesis in atrophied soleus muscles.

    PubMed

    Bajotto, Gustavo; Sato, Yuzo; Kitaura, Yasuyuki; Shimomura, Yoshiharu

    2011-08-01

    Maintenance of skeletal muscle mass depends on the equilibrium between protein synthesis and protein breakdown; diminished functional demand during unloading breaks this balance and leads to muscle atrophy. The current study analyzed time-course alterations in regulatory genes and proteins in the unloaded soleus muscle and the effects of branched-chain amino acid (BCAA) supplementation on muscle atrophy and abundance of molecules that regulate protein turnover. Short-term (6 days) hindlimb suspension of rats resulted in significant losses of myofibrillar proteins, total RNA, and rRNAs and pronounced atrophy of the soleus muscle. Muscle disuse induced upregulation and increases in the abundance of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), increases in gene and protein amounts of two ubiquitin ligases (muscle RING-finger protein 1 and muscle atrophy F-box protein), and decreases in the expression of cyclin D1, the ribosomal protein S6 kinase 1, the mammalian target of rapamycin (mTOR), and ERK1/2. BCAA addition to the diet did not prevent muscle atrophy and had no apparent effect on regulators of proteasomal protein degradation. However, BCAA supplementation reduced the loss of myofibrillar proteins and RNA, attenuated the increases in 4E-BP1, and partially preserved cyclin D1, mTOR and ERK1 proteins. These results indicate that BCAA supplementation alone does not oppose protein degradation but partly preserves specific signal transduction proteins that act as regulators of protein synthesis and cell growth in the non-weight-bearing soleus muscle. PMID:21222129

  12. Global characterization of interferon regulatory factor (IRF) genes in vertebrates: Glimpse of the diversification in evolution

    PubMed Central

    2010-01-01

    Background Interferon regulatory factors (IRFs), which can be identified based on a unique helix-turn-helix DNA-binding domain (DBD) are a large family of transcription factors involved in host immune response, haemotopoietic differentiation and immunomodulation. Despite the identification of ten IRF family members in mammals, and some recent effort to identify these members in fish, relatively little is known in the composition of these members in other classes of vertebrates, and the evolution and probably the origin of the IRF family have not been investigated in vertebrates. Results Genome data mining has been performed to identify any possible IRF family members in human, mouse, dog, chicken, anole lizard, frog, and some teleost fish, mainly zebrafish and stickleback, and also in non-vertebrate deuterostomes including the hemichordate, cephalochordate, urochordate and echinoderm. In vertebrates, all ten IRF family members, i.e. IRF-1 to IRF-10 were identified, with two genes of IRF-4 and IRF-6 identified in fish and frog, respectively, except that in zebrafish exist three IRF-4 genes. Surprisingly, an additional member in the IRF family, IRF-11 was found in teleost fish. A range of two to ten IRF-like genes were detected in the non-vertebrate deuterostomes, and they had little similarity to those IRF family members in vertebrates as revealed in genomic structure and in phylogenetic analysis. However, the ten IRF family members, IRF-1 to IRF-10 showed certain degrees of conservation in terms of genomic structure and gene synteny. In particular, IRF-1, IRF-2, IRF-6, IRF-8 are quite conserved in their genomic structure in all vertebrates, and to a less degree, some IRF family members, such as IRF-5 and IRF-9 are comparable in the structure. Synteny analysis revealed that the gene loci for the ten IRF family members in vertebrates were also quite conservative, but in zebrafish conserved genes were distributed in a much longer distance in chromosomes. Furthermore

  13. Global multiple protein-protein interaction network alignment by combining pairwise network alignments

    PubMed Central

    2015-01-01

    Background A wealth of protein interaction data has become available in recent years, creating an urgent need for powerful analysis techniques. In this context, the problem of finding biologically meaningful correspondences between different protein-protein interaction networks (PPIN) is of particular interest. The PPIN of a species can be compared with that of other species through the process of PPIN alignment. Such an alignment can provide insight into basic problems like species evolution and network component function determination, as well as translational problems such as target identification and elucidation of mechanisms of disease spread. Furthermore, multiple PPINs can be aligned simultaneously, expanding the analytical implications of the result. While there are several pairwise network alignment algorithms, few methods are capable of multiple network alignment. Results We propose SMAL, a MNA algorithm based on the philosophy of scaffold-based alignment. SMAL is capable of converting results from any global pairwise alignment algorithms into a MNA in linear time. Using this method, we have built multiple network alignments based on combining pairwise alignments from a number of publicly available (pairwise) network aligners. We tested SMAL using PPINs of eight species derived from the IntAct repository and employed a number of measures to evaluate performance. Additionally, as part of our experimental investigations, we compared the effectiveness of SMAL while aligning up to eight input PPINs, and examined the effect of scaffold network choice on the alignments. Conclusions A key advantage of SMAL lies in its ability to create MNAs through the use of pairwise network aligners for which native MNA implementations do not exist. Experiments indicate that the performance of SMAL was comparable to that of the native MNA implementation of established methods such as IsoRankN and SMETANA. However, in terms of computational time, SMAL was significantly faster

  14. Developmental regulation of expression of the regulatory subunit of the cAMP-dependent protein kinase of Blastocladiella emersonii.

    PubMed

    Marques, M do V; Juliani, M H; Maia, J C; Gomes, S L

    1989-01-01

    A monospecific polyclonal antiserum to the regulatory subunit (R) of the cAMP-dependent protein kinase of Blastocladiella emersonii has been developed by immunization with purified regulatory subunit. In Western blots, the antiserum displays high affinity and specificity for the intact R monomer of Mr = 58,000, as well as for its proteolytic products of Mr = 43,000 and Mr = 36,000, even though the antiserum has been raised against the Mr = 43,000 fragment. Western blots of cell extracts prepared at different times during the life cycle of the fungus indicate that the increase in cAMP-binding activity occurring during sporulation, as well as its decrease during germination, are associated with the accumulation of the regulatory subunit during sporulation and its disappearance during germination, respectively. Pulse labeling with [35S]methionine and immunoprecipitation indicate that the accumulation of R is due to its increased synthesis during sporulation. Two-dimensional gel electrophoresis of affinity purified cell extracts obtained after [35S]methionine pulse labeling during sporulation confirms de novo synthesis of R during this stage and furthermore shows that the protein is rapidly phosphorylated after its synthesis. In vitro translation studies using RNA isolated from different stages of the life cycle followed by immunoprecipitation have shown that the time course of expression of the mRNA coding for the regulatory subunit parallels the rate of its synthesis in vivo. PMID:2912735

  15. Toward a global analysis of metabolites in regulatory mutants of yeast

    PubMed Central

    Humston, Elizabeth M.; Dombek, Kenneth M.; Tu, Benjamin P.; Young, Elton T.

    2013-01-01

    The AMP-activated protein kinase in yeast, Snf1, coordinates expression and activity of numerous intracellular signaling and developmental pathways, including those regulating cellular differentiation, response to stress, meiosis, autophagy, and the diauxic transition. Snf1 phosphorylates metabolic enzymes and transcription factors to change cellular physiology and metabolism. Adr1 and Cat8, transcription factors that activate gene expression after the diauxic transition, are regulated by Snf1; Cat8 through direct phosphorylation and Adr1 by dephosphorylation in a Snf1-dependent manner. Adr1 and Cat8 coordinately regulate numerous genes encoding enzymes of gluconeogenesis, the glyoxylate cycle, β-oxidation of fatty acids, and the utilization of alternative fermentable sugars and nonfermentable substrates. To determine the roles of Adr1, Cat8, and Snf1 in metabolism, two-dimensional gas chromatography coupled to time-of-flight mass spectrometry and liquid chromatography coupled to tandem mass spectrometry were used to identify metabolites whose levels change after the diauxic transition in wild-type-, ADR1-, CAT8-, and SNF1-deficient yeast. A discovery-based approach to data analysis utilized chemometric algorithms to identify, quantify, and compare 63 unique metabolites between wild type, adr1Δ, cat8Δ, adr1Δcat8Δ, and snf1Δ strains. The primary metabolites found to differ were those of gluconeogenesis, the glyoxylate and tricarboxylic acid cycles, and amino acid metabolism. In general, good agreement was observed between the levels of metabolites derived from these pathways and the levels of transcripts from the same strains, suggesting that transcriptional control plays a major role in regulating the levels of metabolites after the diauxic transition. PMID:21416166

  16. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    Adaptive responses associated with environmental stressors are critical to cell survival. These involve the modulation of central signaling protein functions through site-specific and enzymatically reversible oxidative modifications of methionines to coordinate cellular metabolism, energy utilization, and calcium signaling. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. Thus, depending on either the ecological niche of the organism or the cellular milieu of different organ systems, cellular metabolism can be fine-tuned to maintain optimal function in the face of variable amounts of collateral oxidative damage. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met{sup 144} or Met{sup 145} results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylationdependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in

  17. CovS/CovR of group B streptococcus: a two-component global regulatory system involved in virulence.

    PubMed

    Lamy, Marie-Cécile; Zouine, Mohammed; Fert, Juliette; Vergassola, Massimo; Couve, Elisabeth; Pellegrini, Elisabeth; Glaser, Philippe; Kunst, Frank; Msadek, Tarek; Trieu-Cuot, Patrick; Poyart, Claire

    2004-12-01

    In this study, we carried out a detailed structural and functional analysis of a Streptococcus agalactiae (GBS) two-component system which is orthologous to the CovS/CovR (CsrS/CsrR) regulatory system of Streptococcus pyogenes. In GBS, covR and covS are part of a seven gene operon transcribed from two promoters that are not regulated by CovR. A DeltacovSR mutant was found to display dramatic phenotypic changes such as increased haemolytic activity and reduced CAMP activity on blood agar. Adherence of the DeltacovSR mutant to epithelial cells was greatly increased and analysis by transmission electron microscopy revealed the presence at its surface of a fibrous extracellular matrix that might be involved in these intercellular interactions. However, the DeltacovSR mutant was unable to initiate growth in RPMI and its viability in human normal serum was greatly impaired. A major finding of this phenotypic analysis was that the CovS/CovR system is important for GBS virulence, as a 3 log increase of the LD(50) of the mutant strain was observed in the neonate rat sepsis model. The pleiotropic phenotype of the DeltacovSR mutant is in full agreement with the large number of genes controlled by CovS/CovR as seen by expression profiling analysis, many of which encode potentially secreted or cell surface-associated proteins: 76 genes are repressed whereas 63 were positively regulated. CovR was shown to bind directly to the regulatory regions of several of these genes and a consensus CovR recognition sequence was proposed using both DNase I footprinting and computational analyses. PMID:15554966

  18. Sterol regulatory element-binding proteins are regulators of the NIS gene in thyroid cells.

    PubMed

    Ringseis, Robert; Rauer, Christine; Rothe, Susanne; Gessner, Denise K; Schütz, Lisa-Marie; Luci, Sebastian; Wen, Gaiping; Eder, Klaus

    2013-05-01

    The uptake of iodide into the thyroid, an essential step in thyroid hormone synthesis, is an active process mediated by the sodium-iodide symporter (NIS). Despite its strong dependence on TSH, the master regulator of the thyroid, the NIS gene was also reported to be regulated by non-TSH signaling pathways. In the present study we provide evidence that the rat NIS gene is subject to regulation by sterol regulatory element-binding proteins (SREBPs), which were initially identified as master transcriptional regulators of lipid biosynthesis and uptake. Studies in FRTL-5 thyrocytes revealed that TSH stimulates expression and maturation of SREBPs and expression of classical SREBP target genes involved in lipid biosynthesis and uptake. Almost identical effects were observed when the cAMP agonist forskolin was used instead of TSH. In TSH receptor-deficient mice, in which TSH/cAMP-dependent gene regulation is blocked, the expression of SREBP isoforms in the thyroid was markedly reduced when compared with wild-type mice. Sterol-mediated inhibition of SREBP maturation and/or RNA interference-mediated knockdown of SREBPs reduced expression of NIS and NIS-specific iodide uptake in FRTL-5 cells. Conversely, overexpression of active SREBPs caused a strong activation of the 5'-flanking region of the rat NIS gene mediated by binding to a functional SREBP binding site located in the 5'-untranslated region of the rat NIS gene. These findings show that TSH acts as a regulator of SREBP expression and maturation in thyroid epithelial cells and that SREBPs are novel transcriptional regulators of NIS. PMID:23542164

  19. Structural Requirements for Sterol Regulatory Element-binding Protein (SREBP) Cleavage in Fission Yeast*

    PubMed Central

    Cheung, Rocky; Espenshade, Peter J.

    2013-01-01

    Sterol regulatory element-binding proteins (SREBPs) are central regulators of cellular lipid synthesis and homeostasis. Mammalian SREBPs are proteolytically activated and liberated from the membrane by Golgi Site-1 and Site-2 proteases. Fission yeast SREBPs, Sre1 and Sre2, employ a different mechanism that genetically requires the Golgi Dsc E3 ligase complex for cleavage activation. Here, we established Sre2 as a model to define structural requirements for SREBP cleavage. We showed that Sre2 cleavage does not require the N-terminal basic helix-loop-helix zipper transcription factor domain, thus separating cleavage of Sre2 from its transcription factor function. From a mutagenesis screen of 94 C-terminal residues of Sre2, we isolated 15 residues required for cleavage and further identified a glycine-leucine sequence required for Sre2 cleavage. Importantly, the glycine-leucine sequence is located at a conserved distance before the first transmembrane segment of both Sre1 and Sre2 and cleavage occurs in between this sequence and the membrane. Bioinformatic analysis revealed a broad conservation of this novel glycine-leucine motif in SREBP homologs of ascomycete fungi, including the opportunistic human pathogen Aspergillus fumigatus where SREBP is required for virulence. Consistent with this, the sequence was also required for cleavage of the oxygen-responsive transcription factor Sre1 and adaptation to hypoxia, demonstrating functional conservation of this cleavage recognition motif. These cleavage mutants will aid identification of the fungal SREBP protease and facilitate functional dissection of the Dsc E3 ligase required for SREBP activation and fungal pathogenesis. PMID:23729666

  20. A large family of anti-activators accompanying XylS/AraC family regulatory proteins.

    PubMed

    Santiago, Araceli E; Yan, Michael B; Tran, Minh; Wright, Nathan; Luzader, Deborah H; Kendall, Melissa M; Ruiz-Perez, Fernando; Nataro, James P

    2016-07-01

    AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners. PMID:27038276

  1. Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

    PubMed Central

    Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

    2014-01-01

    Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant. PMID:25279857

  2. Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

    PubMed

    Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

    2014-01-01

    Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5'- or 3'-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant. PMID:25279857

  3. Genomic insights of protein arginine methyltransferase Hmt1 binding reveals novel regulatory functions

    PubMed Central

    2012-01-01

    Background Protein arginine methylation is a post-translational modification involved in important biological processes such as transcription and RNA processing. This modification is catalyzed by both type I and II protein arginine methyltransferases (PRMTs). One of the most conserved type I PRMTs is PRMT1, the homolog of which is Hmt1 in Saccharomyces cerevisiae. Hmt1 has been shown to play a role in various gene expression steps, such as promoting the dynamics of messenger ribonucleoprotein particle (mRNP) biogenesis, pre-mRNA splicing, and silencing of chromatin. To determine the full extent of Hmt1’s involvement during gene expression, we carried out a genome-wide location analysis for Hmt1. Results A comprehensive genome-wide binding profile for Hmt1 was obtained by ChIP-chip using NimbleGen high-resolution tiling microarrays. Of the approximately 1000 Hmt1-binding sites found, the majority fall within or proximal to an ORF. Different occupancy patterns of Hmt1 across genes with different transcriptional rates were found. Interestingly, Hmt1 occupancy is found at a number of other genomic features such as tRNA and snoRNA genes, thereby implicating a regulatory role in the biogenesis of these non-coding RNAs. RNA hybridization analysis shows that Hmt1 loss-of-function mutants display higher steady-state tRNA abundance relative to the wild-type. Co-immunoprecipitation studies demonstrate that Hmt1 interacts with the TFIIIB component Bdp1, suggesting a mechanism for Hmt1 in modulating RNA Pol III transcription to regulate tRNA production. Conclusions The genome-wide binding profile of Hmt1 reveals multiple potential new roles for Hmt1 in the control of eukaryotic gene expression, especially in the realm of non-coding RNAs. The data obtained here will provide an important blueprint for future mechanistic studies on the described occupancy relationship for genomic features bound by Hmt1. PMID:23268696

  4. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC.

    PubMed Central

    Keener, J; Kustu, S

    1988-01-01

    The NTRC protein (ntrC product) of enteric bacteria activates transcription of nitrogen-regulated genes by a holoenzyme form of RNA polymerase that contains the ntrA product (sigma 54) as sigma factor. Although unmodified NTRC will bind to DNA, it must be phosphorylated to activate transcription. Both phosphorylation and dephosphorylation of NTRC occur in the presence of the NTRB protein (ntrB product). We here demonstrate rigorously that it is the NTRB protein that is a protein kinase by showing that NTRB can phosphorylate itself, whereas NTRC cannot. Phosphorylated NTRC (NTRC-P) is capable of autodephosphorylation with a first-order rate constant of 0.14-0.19 min-1 (t 1/2 of 5.0-3.6 min) at 37 degrees C. In addition, there is regulated dephosphorylation of NTRC-P. By contrast to the autophosphatase activity, regulated dephosphorylation requires three components in addition to NTRC-P: the PII regulatory protein, NTRB, and ATP. NTRC is phosphorylated within its amino-terminal domain, which is conserved in one partner of a number of two-component regulatory systems in a wide variety of eubacteria. A purified amino-terminal fragment of NTRC (approximately equal to 12.5 kDa) is sufficient for recognition by NTRB and is autodephosphorylated at the same rate as the native protein. Images PMID:2839825

  5. The role of ion-regulatory membrane proteins of excitation-contraction coupling and relaxation in inherited muscle diseases.

    PubMed

    Froemming, G R; Ohlendieck, K

    2001-01-01

    The excitation-contraction-relaxation cycle of skeletal muscle fibres depends on the finely tuned interplay between the voltage-sensing dihydropyridine receptor, the junctional ryanodine receptor Ca2+-release channel and the sarcoplasmic reticulum Ca2+-ATPase. Inherited diseases of excitation-contraction coupling and muscle relaxation such as malignant hyperthermia, central core disease, hypokalemic periodic paralysis or Brody disease are caused by mutations in these Ca2+-regulatory elements. Over twenty different mutations in the Ca2+-release channel are associated with susceptibility to the pharmacogenetic disorder malignant hyperthermia. Other mutations in the ryanodine receptor trigger central core disease. Primary abnormalities in the alpha-1 subunit of the dihydropyridine receptor underlie the molecular pathogenesis of both hypokalemic periodic paralysis and certain forms of malignant hyperthermia. Some cases of the muscle relaxation disorder named Brody disease were demonstrated to be based on primary abnormalities in the Ca2+-ATPase. Since a variety of other sarcoplasmic reticulum proteins modulate the activity of the voltage sensor, Ca2+-release channel and ion-binding proteins, mutations in these Ca2+-regulatory muscle components might be the underlying cause for novel, not yet fully characterized, genetic muscle disorders. The cell biological analysis of knock-out mice has been helpful in evaluating the biomedical consequences of defects in ion-regulatory muscle proteins. PMID:11145921

  6. Characterization of MtfA, a Novel Regulatory Output Signal Protein of the Glucose-Phosphotransferase System in Escherichia coli K-12

    PubMed Central

    Göhler, Anna-Katharina; Staab, Ariane; Gabor, Elisabeth; Homann, Karina; Klang, Elisabeth; Kosfeld, Anne; Muus, Janna-Eleni; Wulftange, Jana Selina

    2012-01-01

    The glucose-phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory and regulatory system. In addition to its central role in glucose uptake, it informs other global regulatory networks about carbohydrate availability and the physiological status of the cell. The expression of the ptsG gene encoding the glucose-PTS transporter EIICBGlc is primarily regulated via the repressor Mlc, whose inactivation is glucose dependent. During transport of glucose and dephosphorylation of EIICBGlc, Mlc binds to the B domain of the transporter, resulting in derepression of several Mlc-regulated genes. In addition, Mlc can also be inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. In this study, we identified the binding site for Mlc in the carboxy-terminal region of MtfA by measuring the effect of mutated MtfAs on ptsG expression. In addition, we demonstrated the ability of MtfA to inactivate an Mlc super-repressor, which cannot be inactivated by EIICBGlc, by using in vivo titration and gel shift assays. Finally, we characterized the proteolytic activity of purified MtfA by monitoring cleavage of amino 4-nitroanilide substrates and show Mlc's ability to enhance this activity. Based on our findings, we propose a model of MtfA as a glucose-regulated peptidase activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as they enter the stationary phase. This proteolytic activity of MtfA modulated by Mlc constitutes a newly identified PTS output signal that responds to changes in environmental conditions. PMID:22178967

  7. Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

    PubMed Central

    Teriete, Peter; Thai, Khang; Choi, Jungyuen; Marassi, Francesca M.

    2009-01-01

    FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na+/K+ ion binding site of the Na,K-ATPase α subunit. PMID:19761758

  8. Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI.

    PubMed

    Sheldon, Paul J; Busarow, Sara B; Hutchinson, C Richard

    2002-04-01

    Streptomyces antibiotic regulatory proteins (SARPs) constitute a novel family of transcriptional activators that control the expression of several diverse anti-biotic biosynthetic gene clusters. The Streptomyces peucetius DnrI protein, one of only a handful of these proteins yet discovered, controls the biosynthesis of the polyketide antitumour antibiotics daunorubicin and doxorubicin. Recently, comparative analyses have revealed significant similarities among the predicted DNA-binding domains of the SARPs and the C-terminal DNA-binding domain of the OmpR family of regulatory proteins. Using the crystal structure of the OmpR-binding domain as a template, DnrI was mapped by truncation and site-directed mutagenesis. Several highly conserved residues within the N-terminus are crucial for DNA binding and protein function. Tandemly arranged heptameric imperfect repeat sequences are found within the -35 promoter regions of target genes. Substitutions for each nucleotide within the repeats of the dnrG-dpsABCD promoter were performed by site-directed mutagenesis. The mutant promoter fragments were found to have modified binding characteristics in gel mobility shift assays. The spacing between the repeat target sequences is also critical for successful occupation by DnrI and, therefore, competent transcriptional activation of the dnrG-dpsABCD operon. PMID:11972782

  9. Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis.

    PubMed

    Wojtuszkiewicz, Anna; Schuurhuis, Gerrit J; Kessler, Floortje L; Piersma, Sander R; Knol, Jaco C; Pham, Thang V; Jansen, Gerrit; Musters, René J P; van Meerloo, Johan; Assaraf, Yehuda G; Kaspers, Gertjan J L; Zweegman, Sonja; Cloos, Jacqueline; Jimenez, Connie R

    2016-04-01

    Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividualex vivoapoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p= 0.0067 andp= 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p= 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication

  10. Staphylococcus aureus SarA is a Regulatory Protein Responsive to Redox and pH that can Support Bacteriophage Lambda Integrase-Mediated Excision/Recombination

    PubMed Central

    Fujimoto, David F.; Higginbotham, Robin H.; Maleki, Soheila J.; Segall1, Anca M.; Smeltzer, Mark S.; Hurlburt, Barry K.

    2010-01-01

    Summary Staphylococcus aureus produces a wide array of virulence factors and causes a correspondingly diverse array of infections. Production of these virulence factors is under the control of a complex network of global regulatory elements, one of which is sarA. sarA encodes a DNA-binding protein that is considered to function as a transcription factor capable of acting as either a repressor or an activator. Using competitive ELISA assays, we demonstrate that SarA is present at approximately 50,000 copies per cell, which is not characteristic of classical transcription factors. We also demonstrate that SarA is present at all stages of growth in vitro and is capable of binding DNA with high affinity but that its binding affinity and pattern of shifted complexes in electrophoretic mobility shift assays is responsive to the redox state. We also show that SarA binds to the bacteriophage lambda (λ) attachment site, attL, producing SarA-DNA complexes similar to intasomes, which consist of bacteriophage lambda integrase, E. coli integration host factor and attL DNA. In addition, SarA stimulates intramolecular excision recombination in the absence of λ excisionase, a DNA-binding accessory protein. Taken together, these data suggest that SarA may function as an architectural accessory protein. PMID:19919677

  11. Global protein synthesis in human trophoblast is resistant to inhibition by hypoxia

    PubMed Central

    Williams, S.F.; Fik, E.; Zamudio, S.; Illsley, N.P.

    2012-01-01

    Placental growth and function depend on syncytial cell processes which require the continuing synthesis of cellular proteins. The substantial energy demands of protein synthesis are met primarily from oxidative metabolism. Although the responses of individual proteins produced by the syncytiotrophoblast to oxygen deprivation have been investigated previously, there is no information available on global protein synthesis in syncytiotrophoblast under conditions of hypoxia. These studies were designed to test the hypothesis that syncytial protein synthesis is decreased in a dose-dependent manner by hypoxia. Experiments were performed to measure amino acid incorporation into proteins in primary syncytiotrophoblast cells exposed to oxygen concentrations ranging from 0 to 10%. Compared to cells exposed to normoxia (10% O2), no changes were observed following exposure to 5% or 3% O2, but after exposure to 1% O2, protein synthesis after 24 and 48 h decreased by 24% and 23% and with exposure to 0% O2, by 65% and 50%. As a consequence of these results, we hypothesized that global protein synthesis in conditions of severe hypoxia was being supported by glucose metabolism. Additional experiments were performed therefore to examine the role of glucose in supporting protein synthesis. These demonstrated that at each oxygen concentration there was a significant, decreasing linear trend in protein synthesis as glucose concentration was reduced. Under conditions of near-anoxia and in the absence of glucose, protein synthesis was reduced by >85%. Even under normoxic conditions (defined as 10% O2) and in the presence of oxidative substrates, reductions in glucose were accompanied by decreases in protein synthesis. These experiments demonstrate that syncytiotrophoblast cells are resistant to reductions in protein synthesis at O2 concentrations greater than 1%. This could be explained by our finding that a significant fraction of protein synthesis in the syncytiotrophoblast is

  12. Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae.

    PubMed Central

    van Zyl, W; Huang, W; Sneddon, A A; Stark, M; Camier, S; Werner, M; Marck, C; Sentenac, A; Broach, J R

    1992-01-01

    We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13 degrees C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDC55, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIIIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase. Images PMID:1328868

  13. Protein Structure and Evolution: Are They Constrained Globally by a Principle Derived from Information Theory?

    PubMed Central

    Hatton, Leslie; Warr, Gregory

    2015-01-01

    That the physicochemical properties of amino acids constrain the structure, function and evolution of proteins is not in doubt. However, principles derived from information theory may also set bounds on the structure (and thus also the evolution) of proteins. Here we analyze the global properties of the full set of proteins in release 13-11 of the SwissProt database, showing by experimental test of predictions from information theory that their collective structure exhibits properties that are consistent with their being guided by a conservation principle. This principle (Conservation of Information) defines the global properties of systems composed of discrete components each of which is in turn assembled from discrete smaller pieces. In the system of proteins, each protein is a component, and each protein is assembled from amino acids. Central to this principle is the inter-relationship of the unique amino acid count and total length of a protein and its implications for both average protein length and occurrence of proteins with specific unique amino acid counts. The unique amino acid count is simply the number of distinct amino acids (including those that are post-translationally modified) that occur in a protein, and is independent of the number of times that the particular amino acid occurs in the sequence. Conservation of Information does not operate at the local level (it is independent of the physicochemical properties of the amino acids) where the influences of natural selection are manifest in the variety of protein structure and function that is well understood. Rather, this analysis implies that Conservation of Information would define the global bounds within which the whole system of proteins is constrained; thus it appears to be acting to constrain evolution at a level different from natural selection, a conclusion that appears counter-intuitive but is supported by the studies described herein. PMID:25970335

  14. Evolving New Skeletal Traits by cis-Regulatory Changes in Bone Morphogenetic Proteins.

    PubMed

    Indjeian, Vahan B; Kingman, Garrett A; Jones, Felicity C; Guenther, Catherine A; Grimwood, Jane; Schmutz, Jeremy; Myers, Richard M; Kingsley, David M

    2016-01-14

    Changes in bone size and shape are defining features of many vertebrates. Here we use genetic crosses and comparative genomics to identify specific regulatory DNA alterations controlling skeletal evolution. Armor bone-size differences in sticklebacks map to a major effect locus overlapping BMP family member GDF6. Freshwater fish express more GDF6 due in part to a transposon insertion, and transgenic overexpression of GDF6 phenocopies evolutionary changes in armor-plate size. The human GDF6 locus also has undergone distinctive regulatory evolution, including complete loss of an enhancer that is otherwise highly conserved between chimps and other mammals. Functional tests show that the ancestral enhancer drives expression in hindlimbs but not forelimbs, in locations that have been specifically modified during the human transition to bipedalism. Both gain and loss of regulatory elements can localize BMP changes to specific anatomical locations, providing a flexible regulatory basis for evolving species-specific changes in skeletal form. PMID:26774823

  15. Inducing Humoral Immune Responses Against Regulatory T Cells by Foxp3-Fc(IgG) Fusion Protein.

    PubMed

    Niri, Neda Mousavi; Hadjati, Jamshid; Sadat, Mahdi; Memarnejadian, Arash; Aghasadeghi, Mohammadreza; Akbarzadeh, Abolfazl; Zarghami, Nosratollah

    2015-12-01

    The existence of a developed network of suppressory factors and cells against an immune response in different cancers has been proven; regulatory T cells are a typical issue. Therefore their depletion, elimination, or suppression has been assessed in different research studies that were not entirely successful. By applying an improved vaccine against regulatory T cells, we have evaluated the B cell response elicited by the vaccine in an experimental design. A previously described DNA vaccine and recombinant protein of Foxp3-Fc fusion were produced and used in the vaccination regimen. DNA construct and respective protein were injected into C57BL/6 mice. After 2 weeks, serum levels of IgG antibody and its subtypes against Foxp3 were investigated by ELISA. To produce recombinant Foxp3 for ELISA antigen coating, pET24a-Foxp3 vector was transformed into Escherichia coli strain BL21 as host cells. Afterward, protein was expressed and then purified using Ni-NTA agarose. SDS-PAGE and Western blot analysis were carried out to confirm protein expression. The expression analysis of Foxp3 was confirmed by SDS-PAGE followed by Western blot analysis. FOXP3-Fc DNA vaccine/fusion protein vaccination regimen could induce T helper-dependent humoral responses. Due to the effectiveness of Foxp3-Fc(IgG) in inducing humoral responses, it would be expected to be useful in developing vaccines in tumor therapies for the removal of regulatory T cells as a strategy for increasing the efficiency of other means of immunotherapy. PMID:26683176

  16. SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea

    PubMed Central

    2013-01-01

    Background Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. Results We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. Conclusions SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence

  17. Local-global alignment for finding 3D similarities in protein structures

    DOEpatents

    Zemla, Adam T.

    2011-09-20

    A method of finding 3D similarities in protein structures of a first molecule and a second molecule. The method comprises providing preselected information regarding the first molecule and the second molecule. Comparing the first molecule and the second molecule using Longest Continuous Segments (LCS) analysis. Comparing the first molecule and the second molecule using Global Distance Test (GDT) analysis. Comparing the first molecule and the second molecule using Local Global Alignment Scoring function (LGA_S) analysis. Verifying constructed alignment and repeating the steps to find the regions of 3D similarities in protein structures.

  18. Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

    PubMed

    Mitchison, A

    1997-01-01

    In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics. PMID:9148788

  19. UbiNet: an online resource for exploring the functional associations and regulatory networks of protein ubiquitylation.

    PubMed

    Nguyen, Van-Nui; Huang, Kai-Yao; Weng, Julia Tzu-Ya; Lai, K Robert; Lee, Tzong-Yi

    2016-01-01

    Protein ubiquitylation catalyzed by E3 ubiquitin ligases are crucial in the regulation of many cellular processes. Owing to the high throughput of mass spectrometry-based proteomics, a number of methods have been developed for the experimental determination of ubiquitylation sites, leading to a large collection of ubiquitylation data. However, there exist no resources for the exploration of E3-ligase-associated regulatory networks of for ubiquitylated proteins in humans. Therefore, the UbiNet database was developed to provide a full investigation of protein ubiquitylation networks by incorporating experimentally verified E3 ligases, ubiquitylated substrates and protein-protein interactions (PPIs). To date, UbiNet has accumulated 43 948 experimentally verified ubiquitylation sites from 14 692 ubiquitylated proteins of humans. Additionally, we have manually curated 499 E3 ligases as well as two E1 activating and 46 E2 conjugating enzymes. To delineate the regulatory networks among E3 ligases and ubiquitylated proteins, a total of 430 530 PPIs were integrated into UbiNet for the exploration of ubiquitylation networks with an interactive network viewer. A case study demonstrated that UbiNet was able to decipher a scheme for the ubiquitylation of tumor proteins p63 and p73 that is consistent with their functions. Although the essential role of Mdm2 in p53 regulation is well studied, UbiNet revealed that Mdm2 and additional E3 ligases might be implicated in the regulation of other tumor proteins by protein ubiquitylation. Moreover, UbiNet could identify potential substrates for a specific E3 ligase based on PPIs and substrate motifs. With limited knowledge about the mechanisms through which ubiquitylated proteins are regulated by E3 ligases, UbiNet offers users an effective means for conducting preliminary analyses of protein ubiquitylation. The UbiNet database is now freely accessible via http://csb.cse.yzu.edu.tw/UbiNet/ The content is regularly updated with the

  20. Global analysis of cellular protein flux quantifies the selectivity of basal autophagy.

    PubMed

    Zhang, Tian; Ghaemmaghami, Sina

    2016-08-01

    In eukaryotic cells, the macroautophagy pathway has been implicated in the degradation of long-lived proteins and damaged organelles. Although it has been demonstrated that macroautophagy can selectively degrade specific targets, its contribution to the basal turnover of cellular proteins had previously not been quantified on proteome-wide scales. In a recent study, we utilized dynamic proteomics to provide a global comparison of protein half-lives between wild-type and autophagy-deficient cells. Our results indicated that in quiescent fibroblasts, macroautophagy contributes to the basal turnover of a substantial fraction of the proteome. However, the contribution of macroautophagy to constitutive protein turnover is variable within the proteome. The methodology outlined in the study provides a global strategy for quantifying the selectivity of basal macroautophagy. PMID:27248575

  1. Acetyl-Phosphate Is Not a Global Regulatory Bridge between Virulence and Central Metabolism in Borrelia burgdorferi.

    PubMed

    Richards, Crystal L; Lawrence, Kevin A; Su, Hua; Yang, Youyun; Yang, X Frank; Dulebohn, Daniel P; Gherardini, Frank C

    2015-01-01

    In B. burgdorferi, the Rrp2-RpoN-RpoS signaling cascade is a distinctive system that coordinates the expression of virulence factors required for successful transition between its arthropod vector and mammalian hosts. Rrp2 (BB0763), an RpoN specific response regulator, is essential to activate this regulatory pathway. Previous investigations have attempted to identify the phosphate donor of Rrp2, including the cognate histidine kinase, Hk2 (BB0764), non-cognate histidine kinases such as Hk1, CheA1, and CheA2, and small molecular weight P-donors such as carbamoyl-phosphate and acetyl-phosphate (AcP). In a report by Xu et al., exogenous sodium acetate led to increased expression of RpoS and OspC and it was hypothesized this effect was due to increased levels of AcP via the enzyme AckA (BB0622). Genome analyses identified only one pathway that could generate AcP in B. burgdorferi: the acetate/mevalonate pathway that synthesizes the lipid, undecaprenyl phosphate (C55-P, lipid I), which is essential for cell wall biogenesis. To assess the role of AcP in Rrp2-dependent regulation of RpoS and OspC, we used a unique selection strategy to generate mutants that lacked ackA (bb0622: acetate to AcP) or pta (bb0589: AcP to acetyl-CoA). These mutants have an absolute requirement for mevalonate and demonstrate that ackA and pta are required for cell viability. When the ΔackA or Δpta mutant was exposed to conditions (i.e., increased temperature or cell density) that up-regulate the expression of RpoS and OspC, normal induction of those proteins was observed. In addition, adding 20mM acetate or 20mM benzoate to the growth media of B. burgdorferi strain B31 ΔackA induced the expression of RpoS and OspC. These data suggest that AcP (generated by AckA) is not directly involved in modulating the Rrp2-RpoN-RpoS regulatory pathway and that exogenous acetate or benzoate are triggering an acid stress response in B. burgdorferi. PMID:26681317

  2. Acetyl-Phosphate Is Not a Global Regulatory Bridge between Virulence and Central Metabolism in Borrelia burgdorferi

    PubMed Central

    Richards, Crystal L.; Lawrence, Kevin A.; Su, Hua; Yang, Youyun; Yang, X. Frank; Dulebohn, Daniel P.; Gherardini, Frank C.

    2015-01-01

    In B. burgdorferi, the Rrp2-RpoN-RpoS signaling cascade is a distinctive system that coordinates the expression of virulence factors required for successful transition between its arthropod vector and mammalian hosts. Rrp2 (BB0763), an RpoN specific response regulator, is essential to activate this regulatory pathway. Previous investigations have attempted to identify the phosphate donor of Rrp2, including the cognate histidine kinase, Hk2 (BB0764), non-cognate histidine kinases such as Hk1, CheA1, and CheA2, and small molecular weight P-donors such as carbamoyl-phosphate and acetyl-phosphate (AcP). In a report by Xu et al., exogenous sodium acetate led to increased expression of RpoS and OspC and it was hypothesized this effect was due to increased levels of AcP via the enzyme AckA (BB0622). Genome analyses identified only one pathway that could generate AcP in B. burgdorferi: the acetate/mevalonate pathway that synthesizes the lipid, undecaprenyl phosphate (C55-P, lipid I), which is essential for cell wall biogenesis. To assess the role of AcP in Rrp2–dependent regulation of RpoS and OspC, we used a unique selection strategy to generate mutants that lacked ackA (bb0622: acetate to AcP) or pta (bb0589: AcP to acetyl-CoA). These mutants have an absolute requirement for mevalonate and demonstrate that ackA and pta are required for cell viability. When the ΔackA or Δpta mutant was exposed to conditions (i.e., increased temperature or cell density) that up-regulate the expression of RpoS and OspC, normal induction of those proteins was observed. In addition, adding 20mM acetate or 20mM benzoate to the growth media of B. burgdorferi strain B31 ΔackA induced the expression of RpoS and OspC. These data suggest that AcP (generated by AckA) is not directly involved in modulating the Rrp2-RpoN-RpoS regulatory pathway and that exogenous acetate or benzoate are triggering an acid stress response in B. burgdorferi. PMID:26681317

  3. Regulatory role of the 90-kDa-heat-shock protein (Hsp90) and associated factors on gene expression.

    PubMed

    Erlejman, Alejandra G; Lagadari, Mariana; Toneatto, Judith; Piwien-Pilipuk, Graciela; Galigniana, Mario D

    2014-02-01

    The term molecular chaperone was first used to describe the ability of nucleoplasmin to prevent the aggregation of histones with DNA during the assembly of nucleosomes. Subsequently, the name was extended to proteins that mediate the post-translational assembly of oligomeric complexes protecting them from denaturation and/or aggregation. Hsp90 is a 90-kDa molecular chaperone that represents the major soluble protein of the cell. In contrast to most conventional chaperones, Hsp90 functions as a refined sensor of protein function and its principal role in the cell is to facilitate biological activity to properly folded client proteins that already have a preserved tertiary structure. Consequently, Hsp90 is related to basic cell functions such as cytoplasmic transport of soluble proteins, translocation of client proteins to organelles, and regulation of the biological activity of key signaling factors such as protein kinases, ubiquitin ligases, steroid receptors, cell cycle regulators, and transcription factors. A growing amount of evidence links the protective action of this molecular chaperone to mechanisms related to posttranslational modifications of soluble nuclear factors as well as histones. In this article, we discuss some aspects of the regulatory action of Hsp90 on transcriptional regulation and how this effect could have impacted genetic assimilation mechanism in some organisms. PMID:24389346

  4. Preservation of Gene Duplication Increases the Regulatory Spectrum of Ribosomal Protein Genes and Enhances Growth under Stress.

    PubMed

    Parenteau, Julie; Lavoie, Mathieu; Catala, Mathieu; Malik-Ghulam, Mustafa; Gagnon, Jules; Abou Elela, Sherif

    2015-12-22

    In baker's yeast, the majority of ribosomal protein genes (RPGs) are duplicated, and it was recently proposed that such duplications are preserved via the functional specialization of the duplicated genes. However, the origin and nature of duplicated RPGs' (dRPGs) functional specificity remain unclear. In this study, we show that differences in dRPG functions are generated by variations in the modality of gene expression and, to a lesser extent, by protein sequence. Analysis of the sequence and expression patterns of non-intron-containing RPGs indicates that each dRPG is controlled by specific regulatory sequences modulating its expression levels in response to changing growth conditions. Homogenization of dRPG sequences reduces cell tolerance to growth under stress without changing the number of expressed genes. Together, the data reveal a model where duplicated genes provide a means for modulating the expression of ribosomal proteins in response to stress. PMID:26686636

  5. Human C3b- and C4b-regulatory proteins: a new multi-gene family.

    PubMed

    Holers, V M; Cole, J L; Lublin, D M; Seya, T; Atkinson, J P

    1985-06-01

    The complement cascade is regulated to prevent inappropriate activation. This regulation is targeted not only at the initiation of the cascade but also at the amplification andjunctional (C3) steps. Five glycoproteins with both complement regulatory activity and binding affinity for C3b/C4b have been characterized. In plasma these molecules are factor H(H) and C4-binding protein (C4-bp) and, on cells, they are the C3b/C4b receptor (CR1), decay-accelerating factor (DAF), and gp45-70. Here Michael Holers and his colleagues review structural, functional and genetic studies of these proteins and discuss the evidence for a new multi-gene family with a common ancestral protein. PMID:25289982

  6. Structure-function analysis of the beta regulatory subunit of protein kinase CK2 by targeting embryonic stem cell.

    PubMed

    Ziercher, Léa; Filhol, Odile; Laudet, Béatrice; Prudent, Renaud; Cochet, Claude; Buchou, Thierry

    2011-10-01

    Programs that govern stem cell maintenance and pluripotency are dependent on extracellular factors and of intrinsic cell modulators. Embryonic stem (ES) cells with a specific depletion of the gene encoding the regulatory subunit of protein kinase CK2 (CK2β) revealed a viability defect. However, analysis of CK2β functions along the neural lineage established CK2β as a positive regulator for neural stem/progenitor cell (NSC) proliferation and multipotency. By using an in vitro genetic conditional approach, we demonstrate in this work that specific domains of CK2β involved in the regulatory function towards CK2 catalytic subunits are crucial structural determinants for ES cell homeostasis. PMID:21861102

  7. Genome-wide Distribution of AdpA, a Global Regulator for Secondary Metabolism and Morphological Differentiation in Streptomyces, Revealed the Extent and Complexity of the AdpA Regulatory Network

    PubMed Central

    Higo, Akiyoshi; Hara, Hirofumi; Horinouchi, Sueharu; Ohnishi, Yasuo

    2012-01-01

    AdpA is a global transcriptional activator triggering morphological differentiation and secondary metabolism in Streptomyces griseus. AdpA influences the expression of >1000 genes; however, the overall picture of the AdpA regulon remains obscure. Here, we took snapshots of the distribution of AdpA across the chromosome in living S. griseus cells using chromatin immunoprecipitation/chromatin affinity precipitation-seq analysis. In both liquid and solid cultures, AdpA bound to >1200 similar sites, which were located on not only in putative regulatory regions (65%), but also in regions (35%) that appeared not to affect transcription. Transcriptome analysis indicated that ∼40% of the AdpA-binding sites in putative regulatory regions were involved in gene regulation. AdpA was indicated to act as a transcriptional repressor as well as an activator. Expression profiles of AdpA-target genes were very different between liquid and solid cultures, despite their similar AdpA-binding profiles. We concluded that AdpA directly controls >500 genes in cooperation with other regulatory proteins. A comprehensive competitive gel mobility shift assay of AdpA with 304 selected AdpA-binding sites revealed several unique characteristics of the DNA-binding property of AdpA. This study provides the first experimental insight into the extent of the AdpA regulon, indicating that many genes are under the direct control of AdpA. PMID:22449632

  8. UbiNet: an online resource for exploring the functional associations and regulatory networks of protein ubiquitylation

    PubMed Central

    Nguyen, Van-Nui; Huang, Kai-Yao; Weng, Julia Tzu-Ya; Lai, K. Robert; Lee, Tzong-Yi

    2016-01-01

    Protein ubiquitylation catalyzed by E3 ubiquitin ligases are crucial in the regulation of many cellular processes. Owing to the high throughput of mass spectrometry-based proteomics, a number of methods have been developed for the experimental determination of ubiquitylation sites, leading to a large collection of ubiquitylation data. However, there exist no resources for the exploration of E3-ligase-associated regulatory networks of for ubiquitylated proteins in humans. Therefore, the UbiNet database was developed to provide a full investigation of protein ubiquitylation networks by incorporating experimentally verified E3 ligases, ubiquitylated substrates and protein–protein interactions (PPIs). To date, UbiNet has accumulated 43 948 experimentally verified ubiquitylation sites from 14 692 ubiquitylated proteins of humans. Additionally, we have manually curated 499 E3 ligases as well as two E1 activating and 46 E2 conjugating enzymes. To delineate the regulatory networks among E3 ligases and ubiquitylated proteins, a total of 430 530 PPIs were integrated into UbiNet for the exploration of ubiquitylation networks with an interactive network viewer. A case study demonstrated that UbiNet was able to decipher a scheme for the ubiquitylation of tumor proteins p63 and p73 that is consistent with their functions. Although the essential role of Mdm2 in p53 regulation is well studied, UbiNet revealed that Mdm2 and additional E3 ligases might be implicated in the regulation of other tumor proteins by protein ubiquitylation. Moreover, UbiNet could identify potential substrates for a specific E3 ligase based on PPIs and substrate motifs. With limited knowledge about the mechanisms through which ubiquitylated proteins are regulated by E3 ligases, UbiNet offers users an effective means for conducting preliminary analyses of protein ubiquitylation. The UbiNet database is now freely accessible via http://csb.cse.yzu.edu.tw/UbiNet/. The content is regularly updated with the

  9. The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10*

    PubMed Central

    Mou, Zhongming; Tapper, Andrew R.; Gardner, Paul D.

    2009-01-01

    Sox10 is a member of the group E Sox transcription factor family and plays key roles in neural crest development and subsequent cellular differentiation. Sox10 binds to regulatory sequences in target genes via its conserved high mobility group domain. In most cases, Sox10 exerts its transcriptional effects in concert with other DNA-binding factors, adaptor proteins, and nuclear import proteins. These interactions can lead to synergistic gene activation and can be cell type-specific. In earlier work, we demonstrated that Sox10 transactivates the nicotinic acetylcholine receptor α3 and β4 subunit genes and does so only in neuronal-like cell lines, raising the possibility that Sox10 mediates its effects via interactions with co-regulatory factors. Here we describe the identification of the armadillo repeat-containing protein, ARMCX3, as a Sox10-interacting protein. Biochemical analyses indicate that ARMCX3 is an integral membrane protein of the mitochondrial outer membrane. Others have shown that Sox10 is a nucleocytoplasmic shuttling protein. We extend this observation and demonstrate that, in the cytoplasm, Sox10 is peripherally associated with the mitochondrial outer membrane. Both Sox10 and ARMCX3 are expressed in mouse brain and spinal cord as well as several cell lines. Overexpression of ARMCX3 increased the amount of mitochondrially associated Sox10. In addition, although ARMCX3 does not possess intrinsic transcriptional activity, it does enhance transactivation of the nicotinic acetylcholine receptor α3 and β4 subunit gene promoters by Sox10. These results suggest that Sox10 is a membrane-associated factor whose transcriptional function is increased by direct interactions with ARMCX3 and raise the possibility of a signal transduction cascade between the nucleus and mitochondria through Sox10/ARMCX3 interactions. PMID:19304657

  10. P2RP: a web-based framework for the identification and analysis of regulatory proteins in prokaryotic genomes

    PubMed Central

    2013-01-01

    Background Regulatory proteins (RPs) such as transcription factors (TFs) and two-component system (TCS) proteins control how prokaryotic cells respond to changes in their external and/or internal state. Identification and annotation of TFs and TCSs is non-trivial, and between-genome comparisons are often confounded by different standards in annotation. There is a need for user-friendly, fast and convenient tools to allow researchers to overcome the inherent variability in annotation between genome sequences. Results We have developed the web-server P2RP (Predicted Prokaryotic Regulatory Proteins), which enables users to identify and annotate TFs and TCS proteins within their sequences of interest. Users can input amino acid or genomic DNA sequences, and predicted proteins therein are scanned for the possession of DNA-binding domains and/or TCS domains. RPs identified in this manner are categorised into families, unambiguously annotated, and a detailed description of their features generated, using an integrated software pipeline. P2RP results can then be outputted in user-specified formats. Conclusion Biologists have an increasing need for fast and intuitively usable tools, which is why P2RP has been developed as an interactive system. As well as assisting experimental biologists to interrogate novel sequence data, it is hoped that P2RP will be built into genome annotation pipelines and re-annotation processes, to increase the consistency of RP annotation in public genomic sequences. P2RP is the first publicly available tool for predicting and analysing RP proteins in users’ sequences. The server is freely available and can be accessed along with documentation at http://www.p2rp.org. PMID:23601859

  11. Leucine-responsive regulatory protein Lrp and PapI homologues influence phase variation of CS31A fimbriae.

    PubMed

    Graveline, Richard; Garneau, Philippe; Martin, Christine; Mourez, Michaël; Hancock, Mark A; Lavoie, Rémi; Harel, Josée

    2014-08-15

    CS31A, a K88-related surface antigen specified by the clp operon, is a member of the type P family of adhesive factors and plays a key role in the establishment of disease caused by septicemic and enterotoxigenic Escherichia coli strains. Its expression is under the control of methylation-dependent transcriptional regulation, for which the leucine-responsive regulatory protein (Lrp) is essential. CS31A is preferentially in the OFF state and exhibits distinct regulatory features compared to the regulation of other P family members. In the present study, surface plasmon resonance and DNase I protection assays showed that Lrp binds to the distal moiety of the clp regulatory region with low micromolar affinity compared to its binding to the proximal moiety, which exhibits stronger, nanomolar affinity. The complex formation was also influenced by the addition of PapI or FooI, which increased the affinity of Lrp for the clp distal and proximal regions and was required to induce phase variation. The influence of PapI or FooI, however, was predominantly associated with a more complete shutdown of clp expression, in contrast to what has previously been observed with AfaF (a PapI ortholog). Taken together, these results suggest that the preferential OFF state observed in CS31A cells is mainly due to the weak interaction of the leucine-responsive regulatory protein with the clp distal region and that the PapI homolog favors the OFF phase. Within the large repertoire of fimbrial variants in the P family, our study illustrates that having a fimbrial operon that lacks its own PapI ortholog allows it to be more flexibly regulated by other orthologs in the cell. PMID:24914179

  12. The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting

    PubMed Central

    Whitfield, Shawn T.; Burston, Helen E.; Bean, Björn D. M.; Raghuram, Nandini; Maldonado-Báez, Lymarie; Davey, Michael; Wendland, Beverly; Conibear, Elizabeth

    2016-01-01

    Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type–specific expression of alternate μ chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the μ subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the μ chain. Here we show that the variant μ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes. PMID:26658609

  13. Rethinking gene regulatory networks in light of alternative splicing, intrinsically disordered protein domains, and post-translational modifications

    PubMed Central

    Niklas, Karl J.; Bondos, Sarah E.; Dunker, A. Keith; Newman, Stuart A.

    2015-01-01

    Models for genetic regulation and cell fate specification characteristically assume that gene regulatory networks (GRNs) are essentially deterministic and exhibit multiple stable states specifying alternative, but pre-figured cell fates. Mounting evidence shows, however, that most eukaryotic precursor RNAs undergo alternative splicing (AS) and that the majority of transcription factors contain intrinsically disordered protein (IDP) domains whose functionalities are context dependent as well as subject to post-translational modification (PTM). Consequently, many transcription factors do not have fixed cis-acting regulatory targets, and developmental determination by GRNs alone is untenable. Modeling these phenomena requires a multi-scale approach to explain how GRNs operationally interact with the intra- and intercellular environments. Evidence shows that AS, IDP, and PTM complicate gene expression and act synergistically to facilitate and promote time- and cell-specific protein modifications involved in cell signaling and cell fate specification and thereby disrupt a strict deterministic GRN-phenotype mapping. The combined effects of AS, IDP, and PTM give proteomes physiological plasticity, adaptive responsiveness, and developmental versatility without inefficiently expanding genome size. They also help us understand how protein functionalities can undergo major evolutionary changes by buffering mutational consequences. PMID:25767796

  14. Stress-induced Start Codon Fidelity Regulates Arsenite-inducible Regulatory Particle-associated Protein (AIRAP) Translation*

    PubMed Central

    Zach, Lolita; Braunstein, Ilana; Stanhill, Ariel

    2014-01-01

    Initial steps in protein synthesis are highly regulated processes as they define the reading frame of the translation machinery. Eukaryotic translation initiation is a process facilitated by numerous factors (eIFs), aimed to form a “scanning” mechanism toward the initiation codon. Translation initiation of the main open reading frame (ORF) in an mRNA transcript has been reported to be regulated by upstream open reading frames (uORFs) in a manner of re-initiation. This mode of regulation is governed by the phosphorylation status of eIF2α and controlled by cellular stresses. Another mode of translational initiation regulation is leaky scanning, and this regulatory process has not been extensively studied. We have identified arsenite-inducible regulatory particle-associated protein (AIRAP) transcript to be translationally induced during arsenite stress conditions. AIRAP transcript contains a single uORF in a poor-kozak context. AIRAP translation induction is governed by means of leaky scanning and not re-initiation. This induction of AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is phosphorylated under specific conditions that induce protein misfolding and have biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning like re-initiation is responsive to stress conditions and that leaky scanning can induce ORF translation by bypassing poor kozak context of a single uORF transcript. PMID:24898249

  15. Varicella-zoster virus open reading frame 4 encodes an immediate-early protein with posttranscriptional regulatory properties.

    PubMed Central

    Defechereux, P; Debrus, S; Baudoux, L; Rentier, B; Piette, J

    1997-01-01

    Varicella-zoster virus (VZV) encodes four putative immediate-early proteins based on sequence homology with herpes simplex virus type 1: the products of ORF4, -61, -62, and -63. Until now, only two VZV proteins have been described as being truly expressed with immediate-early kinetics (IE62 and IE63). The ORF4-encoded protein can stimulate gene expression either alone or in synergy with the major regulatory protein IE62. Making use of a sequential combination of transcription and protein synthesis inhibitors (actinomycin D and cycloheximide, respectively), we demonstrated the immediate-early nature of the ORF4 gene product, which can thus be named IE4. The fact that IE4 is expressed with kinetics similar to that of IE62 further underlines the possible cooperation between these two VZV proteins in gene expression. Analysis of the IE4-mediated autologous or heterologous viral gene expression at the mRNA levels clearly indicated that IE4 may have several mechanisms of action. Activation of the two VZV genes tested could occur partly by a posttranscriptional mechanism, since increases in chloramphenicol acetyltransferase (CAT) mRNA levels do not account for the stimulation of CAT activity. On the other hand, stimulation of the human immunodeficiency virus type 1 long terminal repeat- or the cytomegalovirus promoter-associated CAT activity is correlated with an increase in the corresponding CAT mRNA. PMID:9261438

  16. Global Transcriptome Analysis of the Cold Shock Response ofShewanella oneidensis MR-1 and Mutational Analysis of Its Classical ColdShock Proteins.

    SciTech Connect

    Gao, H.; Thompson, D.K.; Zhou, J.-Z.

    2006-04-01

    This study presents a global transcriptional analysis of thecold shock response of Shewanella oneidensis MR-1 after a temperaturedownshift from 30oC to 8 or 15oC based on time series microarrayexperiments. More than 700 genes were found to be significantly affected(P<0.05) upon cold shock challenge, especially at 8oC. The temporalgene expression patterns of the classical cold shock genes varied, andonly some of them, most notably So1648 and So2787, were differentiallyregulated in response to a temperature downshift. The global response ofS. oneidensis to cold shock was also characterized by the up-regulationof genes encoding membraneproteins, DNA metabolism and translationapparatus components, metabolic proteins, regulatory proteins, andhypothetical proteins. Most of the metabolic proteins affected areinvolved in catalytic processes that generate NADH or NADPH. Mutationalanalyses confirmed that the small cold shock proteins, So1648 and So2787,are involved in the cold shock response of S. oneidensis. The analysesalso indicated that So1648 may function only at very lowtemperatures.

  17. Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection*

    PubMed Central

    Heard, William; Sklenář, Jan; Tomé, Daniel F. A.; Robatzek, Silke; Jones, Alexandra M. E.

    2015-01-01

    The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein

  18. Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

    SciTech Connect

    Selezneva, Anna I.; Cavigiolio, Giorgio; Theil, Elizabeth C.; Walden, William E.; Volz, Karl

    2006-03-01

    The iron regulatory protein IRP1 has been crystallized in a complex with ferritin IRE RNA and a complete data set has been collected to 2.8 Å resolution. Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe–4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe–4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe–S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 Å, β = 92.0°. Native data sets have been collected from several crystals with resolution extending to 2.8 Å and the structure has been solved by molecular replacement.

  19. Mutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complex

    PubMed Central

    Casey, Mairead; Vaughan, Carl J.; He, Jie; Hatcher, Cathy J.; Winter, Jordan M.; Weremowicz, Stanislawa; Montgomery, Kate; Kucherlapati, Raju; Morton, Cynthia C.; Basson, Craig T.

    2000-01-01

    Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1α gene encoding the R1α regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1α frameshift mutations in three unrelated families result in haploinsufficiency of R1α and cause Carney complex. We did not detect any truncated R1α protein encoded by mutant PRKAR1α. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1α deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1α alleles and that wild-type R1α protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1α to R2β regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1α haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org. J. Clin. Invest. 106:R31–R38 (2000). PMID:10974026

  20. The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits.

    PubMed Central

    Zolnierowicz, S; Van Hoof, C; Andjelković, N; Cron, P; Stevens, I; Merlevede, W; Goris, J; Hemmings, B A

    1996-01-01

    Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein micro-sequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription-PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either alpha or beta isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the epsilon isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates. PMID:8694763

  1. Global stability of protein folding from an empirical free energy function.

    PubMed

    Ruiz-Blanco, Yasser B; Marrero-Ponce, Yovani; Paz, Waldo; García, Yamila; Salgado, Jesús

    2013-03-21

    The principles governing protein folding stand as one of the biggest challenges of Biophysics. Modeling the global stability of proteins and predicting their tertiary structure are hard tasks, due in part to the variety and large number of forces involved and the difficulties to describe them with sufficient accuracy. We have developed a fast, physics-based empirical potential, intended to be used in global structure prediction methods. This model considers four main contributions: Two entropic factors, the hydrophobic effect and configurational entropy, and two terms resulting from a decomposition of close-packing interactions, namely the balance of the dispersive interactions of folded and unfolded states and electrostatic interactions between residues. The parameters of the model were fixed from a protein data set whose unfolding free energy has been measured at the "standard" experimental conditions proposed by Maxwell et al. (2005) and a large data set of 1151 monomeric proteins obtained from the PDB. A blind test with proteins taken from ProTherm database, at similar experimental conditions, was carried out. We found a good correlation with the test data set, proving the effectiveness of our model for predicting protein folding free energies in considered standard conditions. Such a prediction compares favorably against estimations made with FoldX's function and the force field GROMOS96. This model constitutes a valuable tool for the fast evaluation of protein structure stability in 3D structure prediction methods. PMID:23313334

  2. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    SciTech Connect

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

    2013-07-12

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  3. Increased expression of the maize immunoglobulin binding protein homolog b-70 in three zein regulatory mutants.

    PubMed Central

    Boston, R S; Fontes, E B; Shank, B B; Wrobel, R L

    1991-01-01

    Plants carrying floury-2, Defective endosperm-B30, or Mucronate mutations overproduce b-70, a maize homolog of the mammalian immunoglobulin binding protein. During endosperm development in these mutants, levels of both b-70 protein and RNA increase dramatically between 14 days and 20 days after pollination. At later stages, b-70 RNA levels decline while protein levels remain high. The increase in b-70 RNA levels is endosperm specific and dependent on gene dosage in the floury-2 mutant. In all three mutants, the increases in b-70 RNA and protein levels are inversely proportional to changes in zein synthesis. Although b-70 polypeptides can be extracted from purified protein bodies, they carry a carboxy-terminal endoplasmic reticulum retention signal, HDEL. We propose that induction of b-70 in these mutants is a cellular response to abnormally folded or improperly assembled storage proteins and probably reflects its role as a polypeptide chain binding protein. PMID:1840924

  4. Signal Regulatory Protein alpha (SIRPalpha)+ Cells in the Adaptive Response to ESAT-6/CFP-10 Protein of Tuberculous Mycobacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10(CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the eff...

  5. Androgen-dependent protein interactions within an intron 1 regulatory region of the 20-kDa protein gene.

    PubMed

    Avellar, M C; Gregory, C W; Power, S G; French, F S

    1997-07-11

    The 20-kDa protein gene is androgen regulated in rat ventral prostate. Intron 1 contains a 130-base pair complex response element (D2) that binds androgen (AR) and glucocorticoid receptor (GR) but transactivates only with AR in transient cotransfection assays in CV1 cells using the reporter vector D2-tkCAT. To better understand the function of this androgen-responsive unit, nuclear protein interactions with D2 were analyzed by DNase I footprinting in ventral prostate nuclei of intact or castrated rats and in vitro with ventral prostate nuclear protein extracts from intact, castrated, and testosterone-treated castrated rats. Multiple androgen-dependent protected regions and hypersensitive sites were identified in the D2 region with both methods. Mobility shift assays with 32P-labeled oligonucleotides spanning D2 revealed specific interactions with ventral prostate nuclear proteins. Four of the D2-protein complexes decreased in intensity within 24 h of castration. UV cross-linking of the androgen-dependent DNA binding proteins identified protein complexes of approximately 140 and 55 kDa. The results demonstrate androgen-dependent nuclear protein-DNA interactions within the complex androgen response element D2. PMID:9211911

  6. Differential expression of complement proteins and regulatory decay accelerating factor in relation to differentiation of cultured human colon adenocarcinoma cell lines.

    PubMed Central

    Bernet-Camard, M F; Coconnier, M H; Hudault, S; Servin, A L

    1996-01-01

    Self protection of host cells against inadvertent injury resulting from attack by autologous complement proteins is well reported for vascular epithelium. In intestinal epithelium, the expression of C complement proteins and regulatory proteins remains currently poorly reported. This study looked at the distribution of C complement proteins and regulatory decay accelerating factor (DAF) in four cultured human intestinal cell lines of embryogenic or colon cancer origins. C3 and C4 proteins and DAF were widely present in human colon adenocarcinoma T84, HT-29 glc-/+ cells compared with human embryonic INT407 cells. In contrast, no expression of C5, C5b-9, and CR1 was seen for any of the cell lines. Taking advantage of the Caco-2 cells, which spontaneously differentiate in culture, it was seen that the C3, C4, and DAF were present in undifferentiated cells and that their expression increased as a function of the cell differentiation. These results, taken together with other reports on the presence of C complement proteins and DAF in the intestinal cells infer that the expression of regulatory C complement proteins develops in parallel with the expression of C proteins to protect these cells against the potential injury resulting from the activation of these local C proteins. Moreover, the finding that the pathogenic C1845 Escherichia coli binds to the membrane bound DAF in the cultured human intestinal cells synthetising locally C proteins and regulatory C proteins supports the hypothesis that E coli could promote inflammatory disorders by blocking local regulatory protein function. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8801206

  7. Unique N-terminal Arm of Mycobacterium tuberculosis PhoP Protein Plays an Unusual Role in Its Regulatory Function*

    PubMed Central

    Das, Arijit Kumar; Kumar, Vijjamarri Anil; Sevalkar, Ritesh Rajesh; Bansal, Roohi; Sarkar, Dibyendu

    2013-01-01

    Mycobacterium tuberculosis PhoP, a master regulator involved in complex lipid biosynthesis and expression of unknown virulence determinants, is composed of an N-terminal receiver domain and a C-terminal effector domain. The two experimentally characterized PhoP orthologs, from Escherichia coli and Salmonella enterica, display vastly different regulatory capabilities. Here, we demonstrate that the 20-residue-long N-terminal arm unique to M. tuberculosis PhoP plays an essential role in the expanded regulatory capabilities of this important regulator. Although the arm is not required for overall structural stability and/or phosphorylation of the PhoP N-domain, strikingly it is essential for phosphorylation-coupled transcription regulation of target genes. Consistent with this view, arm truncation of PhoP is accompanied by a conformational change of the effector domain, presenting a block in activation subsequent to phosphorylation. These results suggest that presence of the arm, unique to this regulator that shares an otherwise highly conserved domain structure with members of the protein family, contributes to the mechanism of inter-domain interactions. Thus, we propose that the N-terminal arm is an adaptable structural feature of M. tuberculosis PhoP, which evolved to fine-tune regulatory capabilities of the transcription factor in response to the changing physiology of the bacilli within its host. PMID:23963455

  8. Expression of protein kinase A regulatory subunits in benign and malignant human thyroid tissues: A systematic review.

    PubMed

    Del Gobbo, Alessandro; Peverelli, Erika; Treppiedi, Donatella; Lania, Andrea; Mantovani, Giovanna; Ferrero, Stefano

    2016-08-01

    In this review, we discuss the molecular mechanisms and prognostic implications of the protein kinase A (PKA) signaling pathway in human tumors, with special emphasis on the malignant thyroid. The PKA signaling pathway is differentially activated by the expression of regulatory subunits 1 (R1) and 2 (R2), whose levels change during development, differentiation, and neoplastic transformation. Following the identification of gene mutations within the PKA regulatory subunit R1A (PRKAR1A) that cause Carney complex-associated neoplasms, several investigators have studied PRKAR1A expression in sporadic thyroid tumors. The PKA regulatory subunit R2B (PRKAR2B) is highly expressed in benign, as well as in malignant differentiated and undifferentiated lesions. PRKAR1A is highly expressed in follicular adenomas and malignant lesions with a statistically significant gradient between benign and malignant tumors; however, it is not expressed in hyperplastic nodules. Although the importance of PKA in human malignancy outcomes is not completely understood, PRKAR1A expression correlates with tumor dimension in malignant lesions. Additional studies are needed to determine whether a relationship exists between PKA subunit expression and clinical outcomes, particularly in undifferentiated tumors. In conclusion, the R1A subunit might be a good molecular candidate for the targeted treatment of malignant thyroid tumors. PMID:27321957

  9. Construction of hormonally responsive intact cell hybrids by cell fusion: transfer of. beta. -adrenergic receptor and nucleotide regulatory protein(s) in normal and desensitized cells

    SciTech Connect

    Schulster, D.; Salmon, D.M.

    1985-01-01

    Fusion of normal, untreated human erythrocytes with desensitized turkey erythrocytes increases isoproterenol stimulation of cyclic (/sup 3/H)AMP accumulation over basal rates. Moreover, pretreatment of the human erythrocytes with cholera toxin before they are fused with desensitized turkey erthythrocytes leads to a large stimulation with isoproterenol. This is even greater and far more rapid than the response obtained if turkey erythrocytes are treated directly with cholera toxin. It is concluded that the stimulation in the fused system is due to the transfer of an ADP-ribosylated subunit of nucleotide regulatory protein.

  10. The Sorcerer II Global Ocean Sampling Expedition: Expanding theUniverse of Protein Families

    SciTech Connect

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B.; Halpern,Aaron L.; Williamson, Shannon J.; Remington, Karin; Eisen, Jonathan A.; Heidelberg, Karla B.; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S.; Li, Huiying; Mashiyama, Susan T.; Joachimiak, Marcin P.; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A.; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael,Benjamin J.; Bafna, Vineet; Friedman, Robert; Brenner, Steven E.; Godzik,Adam; Eisenberg, David; Dixon, Jack E.; Taylor, Susan S.; Strausberg,Robert L.; Frazier, Marvin; Venter, J.Craig

    2006-03-23

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

  11. The Sorcerer II Global Ocean Sampling Expedition: Expanding the Universe of Protein Families

    PubMed Central

    Yooseph, Shibu; Sutton, Granger; Rusch, Douglas B; Halpern, Aaron L; Williamson, Shannon J; Remington, Karin; Eisen, Jonathan A; Heidelberg, Karla B; Manning, Gerard; Li, Weizhong; Jaroszewski, Lukasz; Cieplak, Piotr; Miller, Christopher S; Li, Huiying; Mashiyama, Susan T; Joachimiak, Marcin P; van Belle, Christopher; Chandonia, John-Marc; Soergel, David A; Zhai, Yufeng; Natarajan, Kannan; Lee, Shaun; Raphael, Benjamin J; Bafna, Vineet; Friedman, Robert; Brenner, Steven E; Godzik, Adam; Eisenberg, David; Dixon, Jack E; Taylor, Susan S; Strausberg, Robert L; Frazier, Marvin; Venter, J. Craig

    2007-01-01

    Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature. PMID:17355171

  12. Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins

    NASA Technical Reports Server (NTRS)

    Toroser, D.; Athwal, G. S.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    We report an Mg2+-dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.

  13. Fasting-Induced Protein Phosphatase 1 Regulatory Subunit Contributes to Postprandial Blood Glucose Homeostasis via Regulation of Hepatic Glycogenesis

    PubMed Central

    Luo, Xiaolin; Zhang, Yongxian; Ruan, Xiangbo; Jiang, Xiaomeng; Zhu, Lu; Wang, Xiao; Ding, Qiurong; Liu, Weizhong; Pan, Yi; Wang, Zhenzhen; Chen, Yan

    2011-01-01

    OBJECTIVE Most animals experience fasting–feeding cycles throughout their lives. It is well known that the liver plays a central role in regulating glycogen metabolism. However, how hepatic glycogenesis is coordinated with the fasting–feeding cycle to control postprandial glucose homeostasis remains largely unknown. This study determines the molecular mechanism underlying the coupling of hepatic glycogenesis with the fasting–feeding cycle. RESEARCH DESIGN AND METHODS Through a series of molecular, cellular, and animal studies, we investigated how PPP1R3G, a glycogen-targeting regulatory subunit of protein phosphatase 1 (PP1), is implicated in regulating hepatic glycogenesis and glucose homeostasis in a manner tightly orchestrated with the fasting–feeding cycle. RESULTS PPP1R3G in the liver is upregulated during fasting and downregulated after feeding. PPP1R3G associates with glycogen pellet, interacts with the catalytic subunit of PP1, and regulates glycogen synthase (GS) activity. Fasting glucose level is reduced when PPP1R3G is overexpressed in the liver. Hepatic knockdown of PPP1R3G reduces postprandial elevation of GS activity, decreases postprandial accumulation of liver glycogen, and decelerates postprandial clearance of blood glucose. Other glycogen-targeting regulatory subunits of PP1, such as PPP1R3B, PPP1R3C, and PPP1R3D, are downregulated by fasting and increased by feeding in the liver. CONCLUSIONS We propose that the opposite expression pattern of PPP1R3G versus other PP1 regulatory subunits comprise an intricate regulatory machinery to control hepatic glycogenesis during the fasting–feeding cycle. Because of its unique expression pattern, PPP1R3G plays a major role to control postprandial glucose homeostasis during the fasting–feeding transition via its regulation on liver glycogenesis. PMID:21471512

  14. Evidence for the existence of an Ns-type regulatory protein in Trypanosoma cruzi membranes.

    PubMed Central

    Eisenschlos, C D; Paladini, A A; Molina y Vedia, L; Torres, H N; Flawiá, M M

    1986-01-01

    The existence of a GTP-binding protein of the Ns type in Trypanosoma cruzi was explored. Epimastigote membranes were labelled by cholera toxin in the presence of [adenine-14C]NAD+. After SDS/polyacrylamide-gel electrophoresis of extracted membrane proteins, a single labelled polypeptide band of apparent Mr approx. 45,000 was detected. Epimastigote cells were treated with N-ethylmaleimide and electrofused to lymphoma S49 cells lacking the Ns protein. Evidence indicates that in such electrofusion-generated cell hybrids a heterologous adenylate cyclase system was reconstituted with the Ns protein provided by T. cruzi epimastigotes. Images Fig. 2. PMID:3099761

  15. Regulatory protein phosphorylation in Mycoplasma pneumoniae. A PP2C-type phosphatase serves to dephosphorylate HPr(Ser-P).

    PubMed

    Halbedel, Sven; Busse, Julia; Schmidl, Sebastian R; Stülke, Jörg

    2006-09-01

    Among the few regulatory events in the minimal bacterium Mycoplasma pneumoniae is the phosphorylation of the HPr phosphocarrier protein of the phosphotransferase system. In the presence of glycerol, HPr is phosphorylated in an ATP-dependent manner by the HPr kinase/phosphorylase. The role of the latter enzyme was studied by constructing a M. pneumoniae hprK mutant defective in HPr kinase/phosphorylase. This mutant strain no longer exhibited HPr kinase activity but, surprisingly, still had phosphatase activity toward serine-phosphorylated HPr (HPr(Ser-P)). An inspection of the genome sequence revealed the presence of a gene (prpC) encoding a presumptive protein serine/threonine phosphatase of the PP2C family. The phosphatase PrpC was purified and its biochemical activity in HPr(Ser-P) dephosphorylation demonstrated. Moreover, a prpC mutant strain was isolated and found to be impaired in HPr(Ser-P) dephosphorylation. Homologues of PrpC are present in many bacteria possessing HPr(Ser-P), suggesting that PrpC may play an important role in adjusting the cellular HPr phosphorylation state and thus controlling the diverse regulatory functions exerted by the different forms of HPr. PMID:16857667

  16. Epitopes of human immunodeficiency virus regulatory proteins tat, nef, and rev are expressed in normal human tissue.

    PubMed Central

    Parmentier, H. K.; van Wichen, D. F.; Meyling, F. H.; Goudsmit, J.; Schuurman, H. J.

    1992-01-01

    The expression of regulatory proteins tat, rev, and nef of human immunodeficiency virus type-1 (HIV-1) and tat of HIV-2 was studied in frozen sections of lymph nodes from HIV-1-infected individuals, and various tissues from uninfected persons. In HIV-1-positive lymph nodes, monoclonal antibodies to HIV-1-tat stained solitary cells in the germinal centers and interfollicular zones, and vascular endothelium. Staining by an anti-nef monoclonal antibody was restricted to follicular dendritic cells, whereas anti-rev antibody bound to fibriohistiocytes and high endothelial venules. The antibodies used labeled several cell types in tissues from uninfected individuals. Anti-HIV-1-tat antibodies labeled blood vessels and Hassall's corpuscles in skin and thymus; goblet cells in intestinal tissue and trachea; neural cells in brain and spinal cord; and zymogen-producing cells in pancreas. Anti-rev antibody stained high endothelial venules, Hassall's corpuscles and histiocytes. One anti-nef antibody solely stained follicular dendritic cells in spleen, tonsil, lymph node and Peyer's patches, whereas two other anti-nef antibodies bound to astrocytes, solitary cells in the interfollicular zones of lymph nodes, and skin cells. The current results hamper the immunohistochemical study for pathogenetic and diagnostic use of HIV regulatory protein expression in infected tissue specimens or cells. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1279980

  17. Global approaches to study protein-protein interactions among viruses and hosts.

    PubMed

    Mendez-Rios, Jorge; Uetz, Peter

    2010-02-01

    While high-throughput protein-protein interaction screens were first published approximately 10 years ago, systematic attempts to map interactions among viruses and hosts started only a few years ago. HIV-human interactions dominate host-pathogen interaction databases (with approximately 2000 interactions) despite the fact that probably none of these interactions have been identified in systematic interaction screens. Recently, combinations of protein interaction data with RNAi and other functional genomics data allowed researchers to model more complex interaction networks. The rapid progress in this area promises a flood of new data in the near future, with clinical applications as soon as structural and functional genomics catches up with next-generation sequencing of human variation and structure-based drug design. PMID:20143950

  18. Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence.

    PubMed

    Kirwan, Angie F; Bibby, Ashley C; Mvilongo, Thierry; Riedel, Heimo; Burke, Thomas; Millis, Sherri Z; Parissenti, Amadeo M

    2003-07-15

    The N-terminal pseudosubstrate site within the protein kinase Calpha (PKCalpha)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCalpha-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940-8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCalpha-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Ralpha(39-177): this protein contained the full regulatory domain of human PKCalpha fused to glutathione S-transferase (GST), but lacked amino acids 1-38 (including the pseudosubstrate sequence) and amino acids 178-270 (including the C2 region)]. GST-Ralpha(39-177) significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCalpha holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33-86 within GST-Ralpha(39-177) dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159-242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of

  19. Control of human papillomavirus type 11 origin of replication by the E2 family of transcription regulatory proteins.

    PubMed

    Chiang, C M; Dong, G; Broker, T R; Chow, L T

    1992-09-01

    Replication of human papillomavirus type 11 (HPV-11) DNA requires the full-length viral E1 and E2 proteins (C.-M. Chiang, M. Ustav, A. Stenlund, T. F. Ho, T. R. Broker, and L. T. Chow, Proc. Natl. Acad. Sci. USA 89:5799-5803, 1992). Using transient transfection of subgenomic HPV DNA into hamster CHO and human 293 cells, we have localized an origin of replication (ori) to an 80-bp segment in the upstream regulatory region spanning nucleotide 1. It overlaps the E6 promoter region and contains a short A + T-rich segment and a sequence which is homologous to the binding site of the bovine papillomavirus type 1 (BPV-1) E1 protein in the BPV-1 ori. However, unlike the BPV-1 ori, for which half an E2-responsive sequence (E2-RS) or binding site suffices, an intact binding site is essential for the HPV-11 ori. Replication was more efficient when additional E2-RSs were present. The intact HPV-11 genome also replicated in both cell lines when supplied with E1 and E2 proteins. Expression vectors of transcription repressor proteins that lack the N-terminal domain essential for E2 transcriptional trans activation did not support replication in collaboration with the E1 expression vector. Rather, cotransfection with the repressor expression vectors inhibited ori replication by the E1 and E2 proteins. These results demonstrate the importance of the N-terminal domain of the E2 protein in DNA replication and indicate that the family of E2 proteins positively and negatively regulates both viral DNA replication and E6 promoter transcription. PMID:1323690

  20. Drug Transporters and Na+/H+ Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

    PubMed Central

    Walsh, Dustin R.; Nolin, Thomas D.

    2015-01-01

    Drug transporters govern the absorption, distribution, and elimination of pharmacologically active compounds. Members of the solute carrier and ATP binding-cassette drug transporter family mediate cellular drug uptake and efflux processes, thereby coordinating the vectorial movement of drugs across epithelial barriers. To exert their physiologic and pharmacological function in polarized epithelia, drug transporters must be targeted and stabilized to appropriate regions of the cell membrane (i.e., apical versus basolateral). Despite the critical importance of drug transporter membrane targeting, the mechanisms that underlie these processes are largely unknown. Several clinically significant drug transporters possess a recognition sequence that binds to PSD-95/Drosophila discs large/ZO-1 (PDZ) proteins. PDZ proteins, such as the Na+/H+ exchanger regulatory factor (NHERF) family, act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is particularly high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia, tissues important to drug disposition. Several recent studies have highlighted NHERF proteins as determinants of drug transporter function secondary to their role in controlling membrane abundance and localization. Mounting evidence strongly suggests that NHERF proteins may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development. PMID:26092975

  1. Evaluation of Affinity-Tagged Protein Expression Strategies using Local and Global Isotope Ratio Measurements

    SciTech Connect

    Hervey, IV, William Judson; Khalsa-Moyers, Gurusahai K; Lankford, Patricia K; Owens, Elizabeth T; McKeown, Catherine K; Lu, Tse-Yuan S; Foote, Linda J; Morrell-Falvey, Jennifer L; McDonald, W Hayes; Pelletier, Dale A; Hurst, Gregory {Greg} B

    2009-01-01

    Protein enrichments of engineered, affinity-tagged (or bait ) fusion proteins with interaction partners are often laden with background, non-specific proteins, due to interactions that occur in vitro as an artifact of the technique. Furthermore, the in vivo expression of the bait protein may itself affect physiology or metabolism. In this study, intrinsic affinity purification challenges were investigated in a model protein complex, DNA-dependent RNA polymerase (RNAP), encompassing chromosome- and plasmid-encoding strategies for bait proteins in two different microbial species: Escherichia coli and Rhodopseudomonas palustris. Isotope ratio measurements of bait protein expression strains relative to native, wild-type strains were performed by liquid chromatography tandem mass spectrometry (LC-MS-MS) to assess bait protein expression strategies in each species. Authentic interacting proteins of RNAP were successfully discerned from artifactual co-isolating proteins by the isotopic differentiation of interactions as random or targeted (I-DIRT) method (A. J. Tackett et al. J. Proteome Res. 2005, 4 (5), 1752-1756). To investigate broader effects of bait protein production in the bacteria, we compared proteomes from strains harboring a plasmid that encodes an affinity-tagged subunit (RpoA) of the RNAP complex with the corresponding wild-type strains using stable isotope metabolic labeling. The ratio of RpoA abundance in plasmid strains versus wild type was 0.8 for R. palustris and 1.7 for E. coli. While most other proteins showed no appreciable difference, proteins significantly increased in abundance in plasmid-encoded bait-expressing strains of both species included the plasmid encoded antibiotic resistance protein, GenR and proteins involved in amino acid biosynthesis. Together, these local, complex-specific and more global, whole proteome isotopic abundance ratio measurements provided a tool for evaluating both in vivo and in vitro effects of plasmid

  2. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles.

    PubMed

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  3. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

    PubMed Central

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  4. The Role of Metal Regulatory Proteins in Brain Oxidative Stress: A Tutorial

    PubMed Central

    2012-01-01

    The proteins that regulate the metabolism of a metal must also play a role in regulating the redox activity of the metal. Metals are intrinsic to a substantial number of biological processes and the proteins that regulate those activities are also considerable in number. The role these proteins play in a wide range of physiological processes involves them directly and indirectly in a variety of disease processes. Similarly, it may be therapeutically advantageous to pharmacologically alter the activity of these metal containing proteins to influence disease processes. This paper will introduce the reader to a number of important proteins in both metal metabolism and oxidative stress, with an emphasis on the brain. Potential pharmacological targets will be considered. PMID:23304261

  5. From local structure to a global framework: recognition of protein folds

    PubMed Central

    Joseph, Agnel Praveen; de Brevern, Alexandre G.

    2014-01-01

    Protein folding has been a major area of research for many years. Nonetheless, the mechanisms leading to the formation of an active biological fold are still not fully apprehended. The huge amount of available sequence and structural information provides hints to identify the putative fold for a given sequence. Indeed, protein structures prefer a limited number of local backbone conformations, some being characterized by preferences for certain amino acids. These preferences largely depend on the local structural environment. The prediction of local backbone conformations has become an important factor to correctly identifying the global protein fold. Here, we review the developments in the field of local structure prediction and especially their implication in protein fold recognition. PMID:24740960

  6. Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

    SciTech Connect

    Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg . E-mail: jkobarg@lnls.br

    2006-03-17

    Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro.

  7. CCAAT displacement protein (CDP/cut) binds a negative regulatory element in the human tryptophan hydroxylase gene.

    PubMed

    Teerawatanasuk, N; Skalnik, D G; Carr, L G

    1999-01-01

    Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of serotonin, a neurotransmitter that has been implicated in many psychiatric illnesses. The mechanism of transcriptional regulation of the human TPH gene is largely unknown. We have identified a negative regulatory element located between nucleotides -310 and -220 in the human TPH (hTPH) gene. Electromobility shift analyses performed with the -310/-220 hTPH probe and nuclear extract from P815-HTR (a TPH-expressing cell line) revealed two slow migrating protein-DNA complexes, designated I and II. CCAAT displacement protein (CDP/Cut) is involved in complex I formation as shown in electromobility shift analysis, using consensus oligonucleotide competitor and antibody. Mutations in the CDP/Cut binding site not only disrupted the CDP-DNA complex but also disrupted the second complex, suggesting that the core binding sequences of the two proteins are overlapping. The functional importance of these protein-DNA interactions was assessed by transiently transfecting wild-type and mutant pTPH/luciferase reporter constructs into P815-HTR cells. Mutations in the core CDP/Cut site resulted in an approximately fourfold increase in relative luciferase activities. Because CDP/Cut has been shown to repress transcription of many target genes, we speculate that disruption of the CDP/Cut binding was responsible, at least in part, for the activation of hTPH gene. PMID:9886051

  8. Structural Dynamics and Conformational Equilibria of SERCA Regulatory Proteins in Membranes by Solid-State NMR Restrained Simulations

    PubMed Central

    De Simone, Alfonso; Mote, Kaustubh R.; Veglia, Gianluigi

    2014-01-01

    Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes. PMID:24940774

  9. Epithelial Splicing Regulatory Protein 1 (ESRP1) is a new regulator of stomach smooth muscle development and plasticity.

    PubMed

    Sagnol, Sébastien; Marchal, Stéphane; Yang, Yinshan; Allemand, Frédéric; de Santa Barbara, Pascal

    2016-06-15

    In vertebrates, stomach smooth muscle development is a complex process that involves the tight transcriptional or post-transcriptional regulation of different signalling pathways. Here, we identified the RNA-binding protein Epithelial Splicing Regulatory Protein 1 (ESRP1) as an early marker of developing and undifferentiated stomach mesenchyme. Using a gain-of-function approach, we found that in chicken embryos, sustained expression of ESRP1 impairs stomach smooth muscle cell (SMC) differentiation and FGFR2 splicing profile. ESRP1 overexpression in primary differentiated stomach SMCs induced their dedifferentiation, promoted specific-FGFR2b splicing and decreased FGFR2c-dependent activity. Moreover, co-expression of ESRP1 and RBPMS2, another RNA-binding protein that regulates SMC plasticity and Bone Morphogenetic Protein (BMP) pathway inhibition, synergistically promoted SMC dedifferentiation. Finally, we also demonstrated that ESRP1 interacts with RBPMS2 and that RBPMS2-mediated SMC dedifferentiation requires ESRP1. Altogether, these results show that ESRP1 is expressed also in undifferentiated stomach mesenchyme and demonstrate its role in SMC development and plasticity. PMID:27108394

  10. Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism

    PubMed Central

    Rumpf, Sebastian; Bagley, Joshua A.; Thompson-Peer, Katherine L.; Zhu, Sijun; Gorczyca, David; Beckstead, Robert B.; Jan, Lily Yeh; Jan, Yuh Nung

    2014-01-01

    The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process—also known as dendrite pruning—depends on the ubiquitin–proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. Here, we show that mutation or inhibition of Valosin-Containing Protein (VCP), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, we find that VCP inhibition causes an altered splicing pattern of the large pruning gene molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR–DNA-binding protein of 43 kilo-Dalton (TDP-43). Our data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, our results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism. PMID:24799714

  11. The Escherichia coli regulatory protein OxyR discriminates between methylated and unmethylated states of the phage Mu mom promoter.

    PubMed Central

    Bölker, M; Kahmann, R

    1989-01-01

    Expression of the phage Mu mom gene is transcriptionally regulated by DNA methylation. Three GATC sites upstream of the mom promoter have to be methylated by the Escherichia coli deoxyadenosine methylase (Dam) to allow initiation of transcription. An E. coli dam strain was mutagenized with Tn5 in an attempt to isolate mutants which allow mom gene expression. Three independent Tn5 mutants were isolated, each mapped to a gene at 89.6 min which we designate momR. The wildtype gene was cloned and sequenced, it encodes a protein of 305 amino acids. The protein belongs to a group of related bacterial activators recently identified as the LysR family (Henikoff et al., 1988). MomR protein was overproduced and purified. Expression of momR is autoregulated; MomR binds to a 43 bp region upstream of its coding sequence. In the mom promoter MomR protects a 43 bp region containing the three GATC sites. Specific binding to these sequences was observed only with unmethylated DNA. Fortuitously, we learned that MomR is identical to OxyR, a regulatory protein responding to oxidative stress. We discuss the implications of this control for Mu development. Images PMID:2551682

  12. Accessible surface area of proteins from purely sequence information and the importance of global features

    NASA Astrophysics Data System (ADS)

    Faraggi, Eshel; Zhou, Yaoqi; Kloczkowski, Andrzej

    2014-03-01

    We present a new approach for predicting the accessible surface area of proteins. The novelty of this approach lies in not using residue mutation profiles generated by multiple sequence alignments as descriptive inputs. Rather, sequential window information and the global monomer and dimer compositions of the chain are used. We find that much of the lost accuracy due to the elimination of evolutionary information is recouped by the use of global features. Furthermore, this new predictor produces similar results for proteins with or without sequence homologs deposited in the Protein Data Bank, and hence shows generalizability. Finally, these predictions are obtained in a small fraction (1/1000) of the time required to run mutation profile based prediction. All these factors indicate the possible usability of this work in de-novo protein structure prediction and in de-novo protein design using iterative searches. Funded in part by the financial support of the National Institutes of Health through Grants R01GM072014 and R01GM073095, and the National Science Foundation through Grant NSF MCB 1071785.

  13. Simultaneous coexpression of Borrelia burgdorferi Erp proteins occurs through a specific, erp locus-directed regulatory mechanism.

    PubMed

    El-Hage, Nazira; Stevenson, Brian

    2002-08-01

    An individual Borrelia burgdorferi bacterium can encode as many as 13 different Erp (OspE/F-related) proteins from mono-and bicistronic loci that are carried on up to 10 separate plasmids. We demonstrate through multilabel immunofluorescence analyses that individual bacteria simultaneously coexpress their entire Erp protein repertoire. While it has been proposed that B. burgdorferi controls expression of Erp and other plasmid-encoded proteins through changes in DNA topology, we observed regulated Erp expression in the absence of detectable differences in DNA supercoiling. Likewise, inhibition of DNA gyrase had no detectable effect on Erp expression. Furthermore, expression of loci physically adjacent to erp loci was observed to be independently regulated. It is concluded that Erp expression is regulated by a mechanism(s) directed at erp loci and not by a global, plasmid-wide mechanism. PMID:12142424

  14. Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A.

    PubMed

    Shu, Y; Yang, H; Hallberg, E; Hallberg, R

    1997-06-01

    The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation. PMID:9154823

  15. Over-expression of GTP-cyclohydrolase 1 feedback regulatory protein attenuates LPS and cytokine-stimulated nitric oxide production.

    PubMed

    Nandi, Manasi; Kelly, Peter; Vallance, Patrick; Leiper, James

    2008-02-01

    GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate-limiting step for the de novo production of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS). The GTP-CH1-BH(4) pathway is emerging as an important regulator in a number of pathologies associated with over-production of nitric oxide (NO) and hence a more detailed understanding of this pathway may lead to novel therapeutic targets for the treatment of certain vascular diseases. GTP-CH1 activity can be inhibited by BH(4) through its protein-protein interactions with GTP-CH1 regulatory protein (GFRP), and transcriptional and post-translational modification of both GTP-CH1 and GFRP have been reported in response to proinflammatory stimuli. However, the functional significance of GFRP/GTP-CH1 interactions on NO pathways has not yet been demonstrated. We aimed to investigate whether over-expression of GFRP could affect NO production in living cells. Over-expression of N-terminally Myc-tagged recombinant human GFRP in the murine endothelial cell line sEnd 1 resulted in no significant effect on basal BH(4) nor NO levels but significantly attenuated the rise in BH(4) and NO observed following lipopolysaccharide and cytokine stimulation of cells. This study demonstrates that GFRP can play a direct regulatory role in iNOS-mediated NO synthesis and suggests that the allosteric regulation of GTP-CH1 activity by GFRP may be an important mechanism regulating BH(4) and NO levels in vivo. PMID:18372436

  16. Temperature inducible β-sheet structure in the transactivation domains of retroviral regulatory proteins of the Rev family

    NASA Astrophysics Data System (ADS)

    Thumb, Werner; Graf, Christine; Parslow, Tristram; Schneider, Rainer; Auer, Manfred

    1999-11-01

    The interaction of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev with cellular cofactors is crucial for the viral life cycle. The HIV-1 Rev transactivation domain is functionally interchangeable with analog regions of Rev proteins of other retroviruses suggesting common folding patterns. In order to obtain experimental evidence for similar structural features mediating protein-protein contacts we investigated activation domain peptides from HIV-1, HIV-2, VISNA virus, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) by CD spectroscopy, secondary structure prediction and sequence analysis. Although different in polarity and hydrophobicity, all peptides showed a similar behavior with respect to solution conformation, concentration dependence and variations in ionic strength and pH. Temperature studies revealed an unusual induction of β-structure with rising temperatures in all activation domain peptides. The high stability of β-structure in this region was demonstrated in three different peptides of the activation domain of HIV-1 Rev in solutions containing 40% hexafluoropropanol, a reagent usually known to induce α-helix into amino acid sequences. Sequence alignments revealed similarities between the polar effector domains from FIV and EIAV and the leucine rich (hydrophobic) effector domains found in HIV-1, HIV-2 and VISNA. Studies on activation domain peptides of two dominant negative HIV-1 Rev mutants, M10 and M32, pointed towards different reasons for the biological behavior. Whereas the peptide containing the M10 mutation (L 78E 79→D 78L 79) showed wild-type structure, the M32 mutant peptide (L 78L 81L 83→A 78A 81A 83) revealed a different protein fold to be the reason for the disturbed binding to cellular cofactors. From our data, we conclude, that the activation domain of Rev proteins from different viral origins adopt a similar fold and that a β-structural element is involved in binding to a

  17. Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes.

    PubMed

    Yoneyama, T; Hatakeyama, K

    2001-04-01

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively. PMID:11274478

  18. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice

    PubMed Central

    Sheehan, Michael J.; Lee, Victoria; Corbett-Detig, Russell; Bi, Ke; Beynon, Robert J.; Hurst, Jane L.; Nachman, Michael W.

    2016-01-01

    Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation) and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup) gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP) scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones. PMID:26938775

  19. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice.

    PubMed

    Sheehan, Michael J; Lee, Victoria; Corbett-Detig, Russell; Bi, Ke; Beynon, Robert J; Hurst, Jane L; Nachman, Michael W

    2016-03-01

    Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation) and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup) gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP) scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones. PMID:26938775

  20. Kinetic oscillations in the expression of messenger RNA, regulatory protein, and nonprotein coding RNA

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2008-06-01

    The interplay of messenger RNA (mRNA), protein, produced via translation of this RNA, and nonprotein coding RNA (ncRNA) may include regulation of the ncRNA production by protein and (i) ncRNA-mRNA association or (ii) ncRNA-protein association resulting in degradation of the corresponding complex. The kinetic models, describing these two scenarios and taking into account that the association of ncRNA with a target occurs after ncRNA conversion from the initial form to the final form (e.g., from a long RNA to microRNA), are found to predict oscillations provided that the rate of ncRNA formation increases with increasing protein population.

  1. Local and global structural drivers for the photoactivation of the orange carotenoid protein

    PubMed Central

    Gupta, Sayan; Guttman, Miklos; Leverenz, Ryan L.; Zhumadilova, Kulyash; Pawlowski, Emily G.; Petzold, Christopher J.; Lee, Kelly K.; Ralston, Corie Y.; Kerfeld, Cheryl A.

    2015-01-01

    Photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of small angle X-ray scattering (SAXS), X-ray hydroxyl radical footprinting, circular dichroism, and H/D exchange mass spectrometry, we identified both the local and global structural changes in the OCP upon photoactivation. SAXS and H/D exchange data showed that global tertiary structural changes, including complete domain dissociation, occur upon photoactivation, but with alteration of secondary structure confined to only the N terminus of the OCP. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection. PMID:26385969

  2. Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

    SciTech Connect

    Senogles, S.E.; Caron, M.G.

    1986-05-01

    The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..S binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.

  3. Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins.

    PubMed

    Verrastro, Ivan; Tveen-Jensen, Karina; Woscholski, Rudiger; Spickett, Corinne M; Pitt, Andrew R

    2016-01-01

    Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured. PMID:26561776

  4. Global Analysis of Protein Damage by the Lipid Electrophile 4-Hydroxy-2-nonenal*S⃞

    PubMed Central

    Codreanu, Simona G.; Zhang, Bing; Sobecki, Scott M.; Billheimer, Dean D.; Liebler, Daniel C.

    2009-01-01

    Lipid peroxidation yields a variety of electrophiles, which are thought to contribute to the molecular pathogenesis of diseases involving oxidative stress, yet little is known of the scope of protein damage caused by lipid electrophiles. We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 μm HNE. HNE Michael adducts were biotinylated by reaction with biotinamidohexanoic acid hydrazide, captured with streptavidin, and the captured proteins were resolved by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry. Of the 1500+ proteins identified, 417 displayed a statistically significant increase in adduction with increasing HNE exposure concentration. We further identified 18 biotin hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high resolution liquid chromatography-tandem mass spectrometry. A subset of the identified HNE targets were validated with a streptavidin capture and immunoblotting approach, which enabled detection of adducts at HNE exposures as low as 1 μm. Protein interaction network analysis indicated several subsystems impacted by endogenous electrophiles in oxidative stress, including the 26 S proteasomal and chaperonin containing TCP-1 (CCT) systems involved in protein-folding and degradation, as well as the COP9 signalosome, translation initiation complex, and a large network of ribonucleoproteins. Global analyses of protein lipid electrophile adducts provide a systems-level perspective on the mechanisms of diseases involving oxidative stress. PMID:19054759

  5. Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis.

    PubMed

    Scholz, Roland; Imami, Koshi; Scott, Nichollas E; Trimble, William S; Foster, Leonard J; Finlay, B Brett

    2015-07-01

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation

  6. Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

    PubMed Central

    Zayas, Margarita; Long, Gang; Madan, Vanesa; Bartenschlager, Ralf

    2016-01-01

    Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles. PMID:26727512

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

    PubMed

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

    2016-01-01

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

  8. Quercetin and vitamin E attenuate Bonny Light crude oil-induced alterations in testicular apoptosis, stress proteins and steroidogenic acute regulatory protein in Wistar rats.

    PubMed

    Ebokaiwe, Azubuike P; Mathur, Premendu P; Farombi, Ebenezer O

    2016-10-01

    Studies have shown the reproductive effects of Bonny Light crude oil (BLCO) via the mechanism of oxidative stress and testicular apoptosis. We investigated the protective role of quercetin and vitamin E on BLCO-induced testicular apoptosis. Experimental rats were divided into four groups of four each. Animals were orally administered 2 ml/kg corn oil (control: group 1), BLCO-800 mg/kg body weight + 10 mg/kg quercetin (group 2), BLCO-800 mg/kg body weight + 50 mg/kg vitamin E (group 3) and BLCO-800 mg/kg body weight only (group 4) for 7 d. Protein levels of caspase 3, FasL, NF-kB, steroidogenic acute regulatory protein and stress response proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunofluorescence staining was used to quantify the expression of caspase 3, FasL and NF-kB. Apoptosis was quantified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Administration of BLCO resulted in a significant increase in the levels of stress response proteins and apoptosis-related proteins by 50% and above after 7 d following BLCO exposure and a concomitant increase in expression of caspase 3, FasL and NF-kB expression by immunofluorescence staining. Apoptosis showed a significant increase in TUNEL positive cells. Co-administration with quercetin or vitamin E reversed BLCO-induced apoptosis and levels of stress protein, relative to control. These findings suggest that quercetin and vitamin E may confer protection against BLCO-induced testicular oxidative stress-related apoptosis. PMID:26821606

  9. Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA-protein complexes with iron boxes.

    PubMed Central

    Flores, Francisco J; Martín, Juan F

    2004-01-01

    In high G+C Gram-positive bacteria, the control of expression of genes involved in iron metabolism is exerted by a DmdR [divalent (bivalent) metal-dependent regulatory protein] in the presence of Fe2+ or other bivalent ions. The dmdR1 and dmdR2 genes of Streptomyces coelicolor were overexpressed in Escherichia coli and the DmdR1 and DmdR2 proteins were purified to homogeneity. Electrophoretic mobility-shift assays showed that both DmdR1 and DmdR2 bind to the 19-nt tox and desA iron boxes forming two different complexes in each case. Increasing the concentrations of DmdR1 or DmdR2 protein shifted these complexes from their low-molecular-mass form to the high-molecular-mass complexes. Formation of the DNA-protein complexes was prevented by the bivalent metal chelating agent 2,2'-dipyridyl and by antibodies specific against the DmdR proteins. Cross-linking with glutaraldehyde of pure DmdR1 or DmdR2 proteins showed that DmdR1 forms dimers, whereas DmdR2 is capable of forming dimers and probably tetramers. Ten different iron boxes were found in a search for iron boxes in the genome of S. coelicolor. Most of them correspond to putative genes involved in siderophore biosynthesis. Since the nucleotide sequence of these ten boxes is identical (or slightly different) with the synthetic DNA fragment containing the desA box used in the present study, it is proposed that DmdR1 and DmdR2 bind to the iron boxes upstream of at least ten different genes in S. coelicolor. PMID:14960152

  10. High-yield soluble expression, purification and characterization of human steroidogenic acute regulatory protein (StAR) fused to a cleavable Maltose-Binding Protein (MBP).

    PubMed

    Sluchanko, Nikolai N; Tugaeva, Kristina V; Faletrov, Yaroslav V; Levitsky, Dmitrii I

    2016-03-01

    Steroidogenic acute regulatory protein (StAR) is responsible for the rapid delivery of cholesterol to mitochondria where the lipid serves as a source for steroid hormones biosynthesis in adrenals and gonads. Despite many successful investigations, current understanding of the mechanism of StAR action is far from being completely clear. StAR was mostly obtained using denaturation/renaturation or in minor quantities in a soluble form at decreased temperatures that, presumably, limited the possibilities for its consequent detailed exploration. In our hands, existing StAR expression constructs could be bacterially expressed almost exclusively as insoluble forms, even upon decreased expression temperatures and in specific strains of Escherichia coli, and isolated protein tended to aggregate and was difficult to handle. To maximize the yield of soluble protein, optimized StAR sequence encompassing functional domain STARD1 (residues 66-285) was fused to the C-terminus of His-tagged Maltose-Binding Protein (MBP) with the possibility to cleave off the whole tag by 3C protease. The developed protocol of expression and purification comprising of a combination of subtractive immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography allowed us to obtain up to 25 mg/1 L culture of completely soluble StAR protein, which was (i) homogenous according to SDS-PAGE, (ii) gave a single symmetrical peak on a gel-filtration, (iii) showed the characteristic CD spectrum and (iv) pH-dependent ability to bind a fluorescently-labeled cholesterol analogue. We conclude that our strategy provides fully soluble and native StAR protein which in future could be efficiently used for biotechnology and drug discovery aimed at modulation of steroids production. PMID:26555181

  11. Interleukin-1 family cytokines and their regulatory proteins in normal pregnancy and pre-eclampsia.

    PubMed

    Southcombe, J H; Redman, C W G; Sargent, I L; Granne, I

    2015-09-01

    Maternal systemic inflammation is a feature of pre-eclampsia, a condition in pregnancy characterized by hypertension and proteinuria. Pre-eclampsia is caused by the placenta; many placental factors contribute to the syndrome's progression, and proinflammatory cytokines have been identified previously as one such mediator. The interleukin (IL)-1 family of cytokines are key regulators of the inflammatory network, and two naturally occurring regulatory molecules for IL-1 family cytokines, IL-1RA and sST2, have been found previously to be elevated in maternal blood from women with pre-eclampsia. Here we investigate more recently identified IL-1 family cytokines and regulatory molecules, IL-1RAcP, IL-37, IL-18BP, IL-36α/β/γ/Ra and IL-38 in pre-eclampsia. Pregnant women have more circulating IL-18BP and IL-36Ra than non-pregnant women, and sIL-1RAcP is elevated from women with pre-eclampsia compared to normal pregnancies. The placenta expresses all the molecules, and IL-37 and IL-18BP are up-regulated significantly in pre-eclampsia placentas compared to those from normal pregnancies. Together, these changes contribute to the required inhibition of maternal systemic cytotoxic immunity in normal pregnancy; however, in pre-eclampsia the same profile is not seen. Interestingly, the increased circulating levels of sIL-1RAcP and increased placental IL-18BP and IL-37, the latter of which we show to be induced by hypoxic damage to the placenta, are all factors which are anti-inflammatory. While the placenta is often held responsible for the damage and clinical symptoms of pre-eclampsia by the research community, here we show that the pre-eclampsia placenta is also trying to prevent inflammatory damage to the mother. PMID:25693732

  12. Hook Proteins: Association with Alzheimer Pathology and Regulatory Role of Hook3 in Amyloid Beta Generation

    PubMed Central

    Arsalan-Werner, Annika; Hilbrich, Isabel; Jäger, Carsten; Flach, Katharina; Suttkus, Anne; Lachmann, Ingolf; Arendt, Thomas; Holzer, Max

    2015-01-01

    Defects in intracellular transport are implicated in the pathogenesis of Alzheimer’s disease (AD). Hook proteins are a family of cytoplasmic linker proteins that participate in endosomal transport. In this study we show that Hook1 and Hook3 are expressed in neurons while Hook2 is predominantly expressed in astrocytes. Furthermore, Hook proteins are associated with pathological hallmarks in AD; Hook1 and Hook3 are localized to tau aggregates and Hook2 to glial components within amyloid plaques. Additionally, the expression of Hook3 is reduced in AD. Modelling of Hook3 deficiency in cultured cells leads to slowing of endosomal transport and increases β-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD. PMID:25799409

  13. Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

    SciTech Connect

    Saraswat, L.D.; Filutowicz, M.

    1986-05-01

    The cDNA for the bovine type I regulatory subunit of cAMP-dependent protein kinase has been inserted into the expression vector pUC7. When E. coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 2-4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-N/sub 3/-(/sup 32/P)-cAMP following transfer from SDS polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of IPTG. R-subunit accumulated in large amounts only in the stationary phase of growth. The addition of IPTG during the log phase of growth actually blocked the accumulation of R-subunit. The soluble, dimeric R-subunit was purifided to homogeneity by affinity chromatography. This R-subunit bound 2 mol cAMP/mol R monomer, reassociated with C-subunit to form cAMP-dependent holoenzyme, and migrated as a dimer on SDS polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties the expressed protein was indistinguishable from R/sup I/ purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z gene of the vector. The NH/sub 2/-terminal sequence was confirmed by amino acid sequencing.

  14. The GTPase regulatory proteins Pix and Git control tissue growth via the Hippo pathway.

    PubMed

    Dent, Lucas G; Poon, Carole L C; Zhang, Xiaomeng; Degoutin, Joffrey L; Tipping, Marla; Veraksa, Alexey; Harvey, Kieran F

    2015-01-01

    The Salvador-Warts-Hippo (Hippo) pathway is a conserved regulator of organ size and is deregulated in human cancers. In epithelial tissues, the Hippo pathway is regulated by fundamental cell biological properties, such as polarity and adhesion, and coordinates these with tissue growth. Despite its importance in disease, development, and regeneration, the complete set of proteins that regulate Hippo signaling remain undefined. To address this, we used proteomics to identify proteins that bind to the Hippo (Hpo) kinase. Prominent among these were PAK-interacting exchange factor (known as Pix or RtGEF) and G-protein-coupled receptor kinase-interacting protein (Git). Pix is a conserved Rho-type guanine nucleotide exchange factor (Rho-GEF) homologous to Beta-PIX and Alpha-PIX in mammals. Git is the single Drosophila melanogaster homolog of the mammalian GIT1 and GIT2 proteins, which were originally identified in the search for molecules that interact with G-protein-coupled receptor kinases. Pix and Git form an oligomeric scaffold to facilitate sterile 20-like kinase activation and have also been linked to GTPase regulation. We show that Pix and Git regulate Hippo-pathway-dependent tissue growth in D. melanogaster and that they do this in parallel to the known upstream regulator Fat cadherin. Pix and Git influence activity of the Hpo kinase by acting as a scaffold complex, rather than enzymes, and promote Hpo dimerization and autophosphorylation of Hpo's activation loop. Therefore, we provide important new insights into an ancient signaling network that controls the growth of metazoan tissues. PMID:25484297

  15. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    SciTech Connect

    Lee, A L

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the {delta}-Al-{var_epsilon} activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a {beta}{alpha}{beta}-{beta}{alpha}{beta} pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel {beta}-sheet. In addition {sup 15}N T{sub 1}, T{sub 2}, and {sup 15}N/{sup 1}H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone {sup 1}H, {sup 13}C, and {sup 15}N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and {sup 15}N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

  16. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation*

    PubMed Central

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2015-01-01

    Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome. PMID:26140926

  17. GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits. Purification, cDNA cloning, and bacterial expression.

    PubMed

    Yoneyama, T; Brewer, J M; Hatakeyama, K

    1997-04-11

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated Mr of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinity-purified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average Mr of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes. PMID:9092499

  18. Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

    PubMed Central

    Castillo, Ana Fernanda; Fan, Jinjiang; Papadopoulos, Vassilios; Podestá, Ernesto J.

    2011-01-01

    Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis. PMID:21829656

  19. Insulin counter-regulatory factors, fibrinogen and C-reactive protein during olanzapine administration: effects of the antidiabetic metformin.

    PubMed

    Baptista, Trino; Sandia, Ignacio; Lacruz, Anny; Rangel, Nairy; de Mendoza, Soaira; Beaulieu, Serge; Contreras, Quilianio; Galeazzi, Tatiana; Vargas, Doritza

    2007-03-01

    In this study, the Authors assessed some insulin counter-regulatory factors, fibrinogen and C-reactive protein after olanzapine administration, and the effect of metformin on these variables, 37 patients with chronic schizophrenia were given olanzapine (10 mg/day for 14 weeks). Nineteen patients received metformin (850-2550 mg/day) and 18 received placebo in a randomized, double-blind protocol. The following variables were quantified before and after olanzapine: cortisol, leptin, tumor necrosis factor-alpha, glucagon, growth hormone, fibrinogen and C-reactive protein. Results were correlated with the changes in body weight and the insulin resistance index. We have reported elsewhere that metformin did not prevent olanzapine-induced weight gain, and the insulin resistance index significantly decreased after metformin and placebo; Baptista T, et al. Can J Psychiatry 2006; 51: 192-196. Cortisol, tumor necrosis factor-alpha and fibrinogen levels significantly decreased in both groups. Glucagon significantly increased after metformin (P=0.03). Leptin tended to increase after placebo (P=0.1) and displayed a small nonsignificant reduction after metformin. The C-reactive protein did not change significantly in any group. Contrarily to most published studies, olanzapine was associated with decreased insulin resistance. Decrements in cortisol, fibrinogen and tumor necrosis factor-alpha levels point to an improvement in the metabolic profile. The trend for leptin to increase after placebo, but not after metformin in spite of similar weight gain suggests a beneficial effect of this antidiabetic agent. PMID:17293706

  20. Inducer effect on the complex formation between rat liver nuclear proteins and cytochrome P450 2B gene regulatory elements.

    PubMed

    Duzhak, T G; Schwartz, E I; Gulyaeva, L F; Lyakhovich, V V

    2002-09-01

    DNA gel retardation assay has been applied to the investigation of complexes between rat liver nuclear proteins and Barbie box positive regulatory element of cytochrome P450 2B (CYP2B) genes. The intensities of B1 and B2 bands detected in the absence of an inducer increased after 30 min protein incubation with phenobarbital (PB) or triphenyldioxane (TPD), but not with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOPOB). In addition, a new complex (B3 band) was for the first time detected under induction by PB, TPD, and TCPOPOB. Increase in the incubation time up to 2 h facilitated the formation of other new complexes (B4 and B5 bands), which were detected only in the presence of TPD. The use of [3H]TPD in hybridization experiments revealed that this inducer, capable of binding to Barbie box DNA, is also present in B4 and B5 complexes. It is probable that the investigated compounds activate the same proteins at the initial induction steps, which correlates with the formation of B1, B2, and B3 complexes. The further induction step might be inducer-specific, as indicated by the formation of B4 and B5 complexes in the presence of TPD only. Thus, the present data suggest the possibility of specific gene activation signaling pathways that are dependent on a particular inducer. PMID:12387719

  1. Crystallization and preliminary X-ray diffraction data for the aconitase form of human iron-regulatory protein 1

    SciTech Connect

    Dupuy, J.; Darnault, C.; Moulis, J. M.

    2005-05-01

    Two crystal forms of the aconitase version of recombinant human IRP1 are reported. Iron-regulatory proteins (IRPs) 1 and 2 are closely related molecules involved in animal iron metabolism. Both proteins can bind to specific mRNA regions called iron-responsive elements and thereby control the expression of proteins involved in the uptake, storage and utilization of iron. In iron-replete cells, IRP1, but not IRP2, binds a [4Fe–4S] cluster and functions as a cytoplasmic aconitase, with simultaneous loss of its RNA-binding ability. Whereas IRP2 is known to be involved in Fe homeostasis, the role of IRP1 is less clear; it may provide a link between citrate and iron metabolisms and be involved in oxidative stress response. Here, two crystal forms of the aconitase version of recombinant human IRP1 are reported. An X-ray fluorescence measurement performed on a gold-derivative crystal showed the unexpected presence of zinc, in addition to gold and iron. Both native and MAD X-ray data at the Au, Fe and Zn absorption edges have been collected from these crystals.

  2. Crystal structures of GCN2 protein kinase C-terminal domains suggest regulatory differences in yeast and mammals.

    PubMed

    He, Hongzhen; Singh, Isha; Wek, Sheree A; Dey, Souvik; Baird, Thomas D; Wek, Ronald C; Georgiadis, Millie M

    2014-05-23

    In response to amino acid starvation, GCN2 phosphorylation of eIF2 leads to repression of general translation and initiation of gene reprogramming that facilitates adaptation to nutrient stress. GCN2 is a multidomain protein with key regulatory domains that directly monitor uncharged tRNAs which accumulate during nutrient limitation, leading to activation of this eIF2 kinase and translational control. A critical feature of regulation of this stress response kinase is its C-terminal domain (CTD). Here, we present high resolution crystal structures of murine and yeast CTDs, which guide a functional analysis of the mammalian GCN2. Despite low sequence identity, both yeast and mammalian CTDs share a core subunit structure and an unusual interdigitated dimeric form, albeit with significant differences. Disruption of the dimeric form of murine CTD led to loss of translational control by GCN2, suggesting that dimerization is critical for function as is true for yeast GCN2. However, although both CTDs bind single- and double-stranded RNA, murine GCN2 does not appear to stably associate with the ribosome, whereas yeast GCN2 does. This finding suggests that there are key regulatory differences between yeast and mammalian CTDs, which is consistent with structural differences. PMID:24719324

  3. In vivo promoter analysis on refeeding response of hepatic sterol regulatory element-binding protein-1c expression

    SciTech Connect

    Takeuchi, Yoshinori; Yahagi, Naoya; Nakagawa, Yoshimi; Matsuzaka, Takashi; Shimizu, Ritsuko; Sekiya, Motohiro; Iizuka, Yoko; Ohashi, Ken; Gotoda, Takanari; Yamamoto, Masayuki; Nagai, Ryozo; Kadowaki, Takashi; Yamada, Nobuhiro; Osuga, Jun-ichi; Shimano, Hitoshi

    2007-11-16

    Sterol regulatory element-binding protein (SREBP)-1c is the master regulator of lipogenic gene expression in liver. The mRNA abundance of SREBP-1c is markedly induced when animals are refed after starvation, although the regulatory mechanism is so far unknown. To investigate the mechanism of refeeding response of SREBP-1c gene expression in vivo, we generated a transgenic mouse model that carries 2.2 kb promoter region fused to the luciferase reporter gene. These transgenic mice exhibited refeeding responses of the reporter in liver and adipose tissues with extents essentially identical to those of endogenous SREBP-1c mRNA. The same results were obtained from experiments using adenovirus-mediated SREBP-1c-promoter-luciferase fusion gene transduction to liver. These data demonstrate that the regulation of SREBP-1c gene expression is at the transcription level, and that the 2.2 kb 5'-flanking region is sufficient for this regulation. Moreover, when these transgenic or adenovirus-infected mice were placed on insulin-depleted state by streptozotocin treatment, the reporter expression was upregulated as strongly as in control mice, demonstrating that this regulation is not dominated by serum insulin level. These mice are the first models to provide the mechanistic insight into the transcriptional regulation of SREBP-1c gene in vivo.

  4. Increased protein kinase A type Iα regulatory subunit expression in parathyroid gland adenomas of patients with primary hyperparathyroidism.

    PubMed

    Hibi, Yatsuka; Kambe, Fukushi; Imai, Tsuneo; Ogawa, Kimio; Shimizu, Yoshimi; Shibata, Masahiro; Kagawa, Chikara; Mizuno, Yutaka; Ito, Asako; Iwase, Katsumi

    2013-01-01

    Protein kinase A (PKA) regulatory subunit type Iα (RIα) is a major regulatory subunit that functions as an inhibitor of PKA kinase activity. We have previously demonstrated that elevated RIα expression is associated with diffuse-to-nodular transformation of hyperplasia in parathyroid glands of renal hyperparathyroidism. The aim of the current study was to determine whether or not RIα expression is increased in adenomas of primary hyperparathyroidism (PHPT), because monoclonal proliferation has been demonstrated in both adenomas and nodular hyperplasia. Surgical specimens comprising 22 adenomas and 11 normal glands, obtained from 22 patients with PHPT, were analyzed. Western blot and immunohistochemical analyses were employed to evaluate RIα expression. PKA activities were determined in several adenomas highly expressing RIα. RIα expression was also separately evaluated in chief and oxyphilic cells using the "Allred score" system. Expression of proliferating cell nuclear antigen (PCNA), a proliferation marker, was also immunohistochemically examined. Western blot analysis revealed that 5 out of 8 adenomas highly expressed RIα, compared with normal glands. PKA activity in adenomas was significantly less than in normal glands. Immunohistochemical analysis further demonstrated high expression of RIα in 20 out of 22 adenomas. In adenomas, the greater RIα expression and more PCNA positive cells were observed in both chief and oxyphilic cells. The present study suggested that high RIα expression could contribute to monoclonal proliferation of parathyroid cells by impairing the cAMP/PKA signaling pathway. PMID:23197043

  5. Molecular characterization of global regulatory RNA species that control pathogenicity factors in Erwinia amylovora and Erwinia herbicola pv. gypsophilae.

    PubMed

    Ma, W; Cui, Y; Liu, Y; Dumenyo, C K; Mukherjee, A; Chatterjee, A K

    2001-03-01

    rsmB(Ecc) specifies a nontranslatable RNA regulator that controls exoprotein production and pathogenicity in soft rot-causing Erwinia carotovora subsp. carotovora. This effect of rsmB(Ecc) RNA is mediated mostly by neutralizing the function of RsmA(Ecc), an RNA-binding protein of E. carotovora subsp. carotovora, which acts as a global negative regulator. To determine the occurrence of functional homologs of rsmB(Ecc) in non-soft-rot-causing Erwinia species, we cloned the rsmB genes of E. amylovora (rsmB(Ea)) and E. herbicola pv. gypsophilae (rsmB(Ehg)). We show that rsmB(Ea) in E. amylovora positively regulates extracellular polysaccharide (EPS) production, motility, and pathogenicity. In E. herbicola pv. gypsophilae, rsmB(Ehg) elevates the levels of transcripts of a cytokinin (etz) gene and stimulates the production of EPS and yellow pigment as well as motility. RsmA(Ea) and RsmA(Ehg) have more than 93% identity to RsmA(Ecc) and, like the latter, function as negative regulators by affecting the transcript stability of the target gene. The rsmB genes reverse the negative effects of RsmA(Ea), RsmA(Ehg), and RsmA(Ecc), but the extent of reversal is highest with homologous combinations of rsm genes. These observations and findings that rsmB(Ea) and rsmB(Ehg) RNA bind RsmA(Ecc) indicate that the rsmB effect is channeled via RsmA. Additional support for this conclusion comes from the observation that the rsmB genes are much more effective as positive regulators in a RsmA(+) strain of E. carotovora subsp. carotovora than in its RsmA(-) derivative. E. herbicola pv. gypsophilae produces a 290-base rsmB transcript that is not subject to processing. By contrast, E. amylovora produces 430- and 300-base rsmB transcripts, the latter presumably derived by processing of the primary transcript as previously noted with the transcripts of rsmB(Ecc). Southern blot hybridizations revealed the presence of rsmB homologs in E. carotovora, E. chrysanthemi, E. amylovora, E. herbicola, E

  6. Preparation and crystallization of the stimulatory and inhibitory complexes of GTP cyclohydrolase I and its feedback regulatory protein GFRP.

    PubMed

    Maita, N; Okada, K; Hirotsu, S; Hatakeyama, K; Hakoshima, T

    2001-08-01

    Mammalian GTP cyclohydrolase I is a decameric enzyme in the first and rate-limiting step in the biosynthesis of tetrahydrobiopterin, which is an essential cofactor for enzymes producing neurotransmitters such as catecholamines and for nitric oxide synthases. The enzyme is dually regulated by its feedback regulatory protein GFRP in the presence of its stimulatory effector phenylalanine and its inhibitory effector biopterin. Here, both the stimulatory and inhibitory complexes of rat GTP cyclohydrolase I bound to GFRP were crystallized by vapour diffusion. Diffraction data sets at resolutions of 3.0 and 2.64 A were collected for the stimulatory and inhibitory complexes, respectively. Each complex consists of two GTPCHI pentamer rings and two GFRP pentamer rings, with pseudo-52 point-group symmetry. PMID:11468403

  7. Modulation of cell cycle regulatory protein expression and suppression of tumor growth by mimosine in nude mice.

    PubMed

    Chang, H C; Weng, C F; Yen, M H; Chuang, L Y; Hung, W C

    2000-10-01

    Our previous results demonstrated that the plant amino acid mimosine blocked cell cycle progression and suppressed proliferation of human lung cancer cells in vitro by multiple mechanisms. Inhibition of cyclin D1 expression or induction of cyclin-dependent kinase inhibitor p21WAF1 expression was found in mimosine-treated lung cancer cells. However, whether mimosine may modulate the expression of these cell cycle regulatory proteins and suppress tumor growth in vivo is unknown. In this study, we examined the anti-cancer effect of mimosine on human H226 lung cancer cells grown in nude mice. Our results demonstrated that mimosine inhibits cyclin D1 and induces p21WAF1 expression in vivo. Furthermore, results of TUNEL analysis indicated that mimosine may induce apoptosis to suppress tumor growth in nude mice. Collectively, these results suggest that mimosine exerts anti-cancer effect in vivo and might be useful in the therapy of lung cancer. PMID:10995875

  8. Surveying the lipogenesis landscape in Yarrowia lipolytica through understanding the function of a Mga2p regulatory protein mutant.

    PubMed

    Liu, Leqian; Markham, Kelly; Blazeck, John; Zhou, Nijia; Leon, Dacia; Otoupal, Peter; Alper, Hal S

    2015-09-01

    Lipogenic organisms represent great starting points for metabolic engineering of oleochemical production. While previous engineering efforts were able to significantly improve lipid production in Yarrowia lipolytica, the lipogenesis landscape, especially with respect to regulatory elements, has not been fully explored. Through a comparative genomics and transcriptomics approach, we identified and validated a mutant mga2 protein that serves as a regulator of desaturase gene expression and potent lipogenesis factor. The resulting strain is enriched in unsaturated fatty acids. Comparing the underlying mechanism of this mutant to other previously engineered strains suggests that creating an imbalance between glycolysis and the TCA cycle can serve as a driving force for lipogenesis when combined with fatty acid catabolism overexpressions. Further comparative transcriptomics analysis revealed both distinct and convergent rewiring associated with these different genotypes. Finally, by combining metabolic engineering targets, it is possible to further engineer a strain containing the mutant mga2 gene to a lipid production titer of 25g/L. PMID:26219673

  9. The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity

    PubMed Central

    Kidani, Yoko; Elsaesser, Heidi; Hock, M Benjamin; Vergnes, Laurent; Williams, Kevin J; Argus, Joseph P; Marbois, Beth N; Komisopoulou, Evangelia; Wilson, Elizabeth B; Osborne, Timothy F; Graeber, Thomas G; Reue, Karen; Brooks, David G; Bensinger, Steven J

    2013-01-01

    Newly activated CD8+ T cells reprogram their metabolism to meet the extraordinary biosynthetic demands of clonal expansion; however, the signals mediating metabolic reprogramming remain poorly defined. Herein, we demonstrate an essential role for sterol regulatory element binding proteins (SREBPs) in the acquisition of effector cell metabolism. Without SREBP signaling, CD8+ T cells are unable to blast, resulting in markedly attenuated clonal expansion during viral infection. Mechanistic studies indicate that SREBPs are essential to meet the heightened lipid requirements of membrane synthesis during blastogenesis. SREBPs are dispensable for homeostatic proliferation, indicating a context-specific requirement for SREBPs in effector responses. These studies provide insights into the molecular signals underlying metabolic reprogramming of CD8+ T cells during the transition from quiescence to activation. PMID:23563690

  10. A predicted protein interactome identifies conserved global networks and disease resistance subnetworks in maize

    PubMed Central

    Musungu, Bryan; Bhatnagar, Deepak; Brown, Robert L.; Fakhoury, Ahmad M.; Geisler, Matt

    2015-01-01

    Interactomes are genome-wide roadmaps of protein-protein interactions. They have been produced for humans, yeast, the fruit fly, and Arabidopsis thaliana and have become invaluable tools for generating and testing hypotheses. A predicted interactome for Zea mays (PiZeaM) is presented here as an aid to the research community for this valuable crop species. PiZeaM was built using a proven method of interologs (interacting orthologs) that were identified using both one-to-one and many-to-many orthology between genomes of maize and reference species. Where both maize orthologs occurred for an experimentally determined interaction in the reference species, we predicted a likely interaction in maize. A total of 49,026 unique interactions for 6004 maize proteins were predicted. These interactions are enriched for processes that are evolutionarily conserved, but include many otherwise poorly annotated proteins in maize. The predicted maize interactions were further analyzed by comparing annotation of interacting proteins, including different layers of ontology. A map of pairwise gene co-expression was also generated and compared to predicted interactions. Two global subnetworks were constructed for highly conserved interactions. These subnetworks showed clear clustering of proteins by function. Another subnetwork was created for disease response using a bait and prey strategy to capture interacting partners for proteins that respond to other organisms. Closer examination of this subnetwork revealed the connectivity between biotic and abiotic hormone stress pathways. We believe PiZeaM will provide a useful tool for the prediction of protein function and analysis of pathways for Z. mays researchers and is presented in this paper as a reference tool for the exploration of protein interactions in maize. PMID:26089837

  11. Protein structure prediction and potential energy landscape analysis using continuous global minimization

    SciTech Connect

    Dill, K.A.; Phillips, A.T.; Rosen, J.B.

    1997-12-01

    Proteins require specific three-dimensional conformations to function properly. These {open_quotes}native{close_quotes} conformations result primarily from intramolecular interactions between the atoms in the macromolecule, and also intermolecular interactions between the macromolecule and the surrounding solvent. Although the folding process can be quite complex, the instructions guiding this process are specified by the one-dimensional primary sequence of the protein or nucleic acid: external factors, such as helper (chaperone) proteins, present at the time of folding have no effect on the final state of the protein. Many denatured proteins spontaneously refold into functional conformations once denaturing conditions are removed. Indeed, the existence of a unique native conformation, in which residues distant in sequence but close in proximity exhibit a densely packed hydrophobic core, suggests that this three-dimensional structure is largely encoded within the sequential arrangement of these specific amino acids. In any case, the native structure is often the conformation at the global minimum energy. In addition to the unique native (minimum energy) structure, other less stable structures exist as well, each with a corresponding potential energy. These structures, in conjunction with the native structure, make up an energy landscape that can be used to characterize various aspects of the protein structure. 22 refs., 10 figs., 2 tabs.

  12. Trm9-Catalyzed tRNA Modifications Regulate Global Protein Expression by Codon-Biased Translation

    PubMed Central

    Deng, Wenjun; Babu, I. Ramesh; Su, Dan; Yin, Shanye; Begley, Thomas J.; Dedon, Peter C.

    2015-01-01

    Post-transcriptional modifications of transfer RNAs (tRNAs) have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5) and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modifications catalyzed by tRNA methyltransferase 9 (Trm9) in tRNAArg(UCU) and tRNAGlu(UUC) and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell. PMID:26670883

  13. Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates.

    PubMed Central

    Norbury, C; Blow, J; Nurse, P

    1991-01-01

    The p34cdc2 protein kinase is a conserved regulator of the eukaryotic cell cycle. Here we show that residues Thr14 and Tyr15 of mouse p34cdc2 become phosphorylated as mouse fibroblasts proceed through the cell cycle. We have mutated these residues and measured protein kinase activity of the p34cdc2 variants in a Xenopus egg extract. Phosphorylation of residues 14 and 15, which lie within the presumptive ATP-binding region of p34cdc2, normally restrains the protein kinase until it is specifically dephosphorylated and activated at the G2/M transition. Regulation by dephosphorylation of Tyr15 is conserved from fission yeast to mammals, while an extra level of regulation of mammalian p34cdc2 involves Thr14 dephosphorylation. In the absence of phosphorylation on these two residues, the kinase still requires cyclin B protein for its activation. Inhibition of DNA synthesis inhibits activation of wild-type p34cdc2 in the Xenopus system, but a mutant which cannot be phosphorylated at residues 14 and 15 escapes this inhibition, suggesting that these phosphorylation events form part of the pathway linking completion of DNA replication to initiation of mitosis. Images PMID:1655417

  14. Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: Relationship with cell proliferation

    SciTech Connect

    Mantovani, G.; Lania, A.G.; Bondioni, S.; Peverelli, E.; Pedroni, C.; Ferrero, S.; Pellegrini, C.; Vicentini, L.; Arnaldi, G.; Bosari, S.; Beck-Peccoz, P.; Spada, A.

    2008-01-01

    The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing's syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas (n = 16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas (n = 5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-cAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.

  15. Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase

    PubMed Central

    Bacot-Davis, Valjean R.; Ciomperlik, Jessica J.; Basta, Holly A.; Cornilescu, Claudia C.; Palmenberg, Ann C.

    2014-01-01

    Cardiovirus Leader (L) proteins induce potent antihost inhibition of active cellular nucleocytoplasmic trafficking by triggering aberrant hyperphosphorylation of nuclear pore proteins (Nup). To achieve this, L binds protein RanGTPase (Ran), a key trafficking regulator, and diverts it into tertiary or quaternary complexes with required kinases. The activity of L is regulated by two phosphorylation events not required for Ran binding. Matched NMR studies on the unphosphorylated, singly, and doubly phosphorylated variants of Mengovirus L (LM) show both modifications act together to partially stabilize a short internal α-helix comprising LM residues 43–46. This motif implies that ionic and Van der Waals forces contributed by phosphorylation help organize downstream residues 48–67 into a new interface. The full structure of LM as bound to Ran (unlabeled) and Ran (216 aa) as bound by LM (unlabeled) places LM into the BP1 binding site of Ran, wrapped by the conformational flexible COOH tail. The arrangement explains the tight KD for this complex and places the LM zinc finger and phosphorylation interface as surface exposed and available for subsequent reactions. The core structure of Ran, outside the COOH tail, is not altered by LM binding and remains accessible for canonical RanGTP partner interactions. Pull-down assays identify at least one putative Ran:LM partner as an exportin, Crm1, or CAS. A model of Ran:LM:Crm1, based on the new structures suggests LM phosphorylation status may mediate Ran’s selection of exportin(s) and cargo(s), perverting these native trafficking elements into the lethal antihost Nup phosphorylation pathways. PMID:25331866

  16. Complement regulatory protein expression by a human oligodendrocyte cell line: cytokine regulation and comparison with astrocytes.

    PubMed Central

    Gasque, P; Morgan, B P

    1996-01-01

    Rat oligodendrocytes spontaneously activate complement (C) and lack the C inhibitor CD59. As a consequence, rat oligodendrocytes are susceptible to lysis by autologous C in vitro. Expression of C inhibitors on human oligodendrocytes in vitro and other human glia has yet to be well characterized. We have previously shown expression at the mRNA level of the membrane inhibitors CD59, decay-accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46) in human astrocytes. We here examine the expression of membrane and secreted C inhibitors by the oligodendrocyte cell line, HOG. HOG cells abundantly expressed CD59, assessed at protein and mRNA level, and expressed DAF and MCP, albeit at a lower level. Expression of all three inhibitors was enhanced by incubation with interferon-gamma or with phorbol ester (PMA). Complement receptor type 1 (CR1; CD35) was neither expressed constitutively nor induced by cytokines. HOG also constitutively secreted C1-inhibitor, S-protein and clusterin. Factor H was secreted only after stimulation with cytokines. C4b binding protein was expressed at a very low level and was detected only at the mRNA level by reverse transcriptase-polymerase chain reaction (RT-PCR). For comparison, astrocyte expression of CD59, DAF, MCP and CR1 was confirmed at the mRNA and protein levels. HOG did not activate C spontaneously, as judged by the lack of deposition of C fragments, and were not lysed by C even after inhibition of CD59 and DAF using specific monoclonal antibodies. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8958045

  17. Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

    PubMed Central

    2010-01-01

    Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified. Conclusions

  18. Improving understanding of chromatin regulatory proteins and potential implications for drug discovery.

    PubMed

    Rafehi, Haloom; Khan, Abdul Waheed; El-Osta, Assam

    2016-04-01

    Many epigenetic-based therapeutics, including drugs such as histone deacetylase inhibitors, are now used in the clinic or are undergoing advanced clinical trials. The study of chromatin-modifying proteins has benefited from the rapid advances in high-throughput sequencing methods, the organized efforts of major consortiums and by individual groups to profile human epigenomes in diverse tissues and cell types. However, while such initiatives have carefully characterized healthy human tissue, disease epigenomes and drug-epigenome interactions remain very poorly understood. Reviewed here is how high-throughput sequencing improves our understanding of chromatin regulator proteins and the potential implications for the study of human disease and drug development and discovery. PMID:26923902

  19. Immune Protection of Retroviral Vectors Upon Molecular Painting with the Complement Regulatory Protein CD59.

    PubMed

    Heider, Susanne; Kleinberger, Sandra; Kochan, Feliks; Dangerfield, John A; Metzner, Christoph

    2016-07-01

    Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications. PMID:27170144

  20. Domains with transcriptional regulatory activity within the ALL1 and AF4 proteins involved in acute leukemia.

    PubMed Central

    Prasad, R; Yano, T; Sorio, C; Nakamura, T; Rallapalli, R; Gu, Y; Leshkowitz, D; Croce, C M; Canaani, E

    1995-01-01

    The ALLI gene, located at chromosome band 11q23, is involved in acute leukemia through a series of chromosome translocations and fusion to a variety of genes, most frequently to A4 and AF9. The fused genes encode chimeric proteins proteins. Because the Drosophila homologue of ALL1, trithorax, is a positive regulator of homeotic genes and acts at the level of transcription, it is conceivable that alterations in ALL1 transcriptional activity may underlie its action in malignant transformation. To begin studying this, we examined the All1, AF4, AF9, and AF17 proteins for the presence of potential transcriptional regulatory domains. This was done by fusing regions of the proteins to the yeast GAL4 DNA binding domain and assaying their effect on transcription of a reporter gene. A domain of 55 residues positioned at amino acids 2829-2883 of ALL1 was identified as a very strong activator. Further analysis of this domain by in vitro mutagenesis pointed to a core of hydrophobic and acidic residues as critical for the activity. An ALL1 domain that repressed transcription of the reporter gene coincided with the sequence homologous to a segment of DNA methyltransferase. An AF4 polypeptide containing residues 480-560 showed strong activation potential. The C-terminal segment of AF9 spanning amino acids 478-568 transactivated transcription of the reporter gene in HeLa but not in NIH 3T3 cells. These results suggest that ALL1, AF4, and probably AF9 interact with the transcriptional machinery of the cell. Images Fig. 1 Fig. 4 Fig. 5 PMID:8618864

  1. Anxiety-like behavior of mice produced by conditional central expression of the HIV-1 regulatory protein, Tat

    PubMed Central

    Paris, Jason J.; Singh, Harminder D.; Ganno, Michelle L.; Jackson, Pauline; McLaughlin, Jay P.

    2014-01-01

    Rationale Human immunodeficiency virus (HIV) infection is associated with substantial increases in generalized anxiety. The HIV regulatory protein, transactivator of transcription (Tat), has been implicated in the neuropathogenesis related to HIV-1 infection. However, direct examination of the effect of Tat on behavioral measures of anxiety has not been demonstrated. Objective To identify whether expression of the Tat1-86 protein exerts dose-dependent and persistent anxiety-like effects in a whole animal model, the GT-tg bigenic mouse. Methods GT-tg mice and C57BL/6J controls were administered doxycycline in a dose- (0, 50, 100, or 125 mg/kg, i.p., for 7 days) or duration- (100 mg/kg, i.p., for 0, 1, 3, 5, or 14 days) dependent manner to induce Tat1-86 in brain. Mice were assessed for anxiety-like behavior in an open field, social interaction, or marble burying task 0, 7, and/or 14 days later. Central expression of Tat1-86 protein was verified with Western blot analyses. Results Doxycycline produced no effects on C57BL/6J controls that lacked the Tat1-86 transgene. Among GT-tg mice, doxycycline (100 mg/kg for 3, 5, or 7 days) significantly increased anxiety-like behavior in all tasks, commensurate with enhanced Western blot labeling of Tat1-86 protein in brain, displaying optimal effects with the 7-day regimen. Greater exposure to doxycycline (either 125 mg/kg for 7 days or 100 mg/kg for 14 days) impaired locomotor behavior; whereas, lower dosing (below 100 mg/kg) produced only transient increases in anxiety-like behavior. Conclusions Expression of HIV-1-Tat1-86 in GT-tg mouse brain produces exposure-dependent, persistent increases in anxiety-like behavior. PMID:24352568

  2. Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

    PubMed

    Liu, Xia; Ji, Baoju; Sun, Mengyi; Wu, Weijiang; Huang, Lili; Sun, Aihua; Zong, Yangyong; Xia, Sheng; Shi, Liyun; Qian, Hui; Xu, Wenrong; Shao, Qixiang

    2015-07-01

    Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy. PMID:25809415

  3. Recycling of a regulatory protein by degradation of the RNA to which it binds.

    PubMed

    Deikus, Gintaras; Babitzke, Paul; Bechhofer, David H

    2004-03-01

    When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3'-to-5' exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase(-) strain showed accumulation of a stable, TRAP-protected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase(-) strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription. PMID:14976255

  4. Expression of regulatory proteins in choroid plexus changes in early stages of Alzheimer disease.

    PubMed

    Krzyzanowska, Agnieszka; García-Consuegra, Inés; Pascual, Consuelo; Antequera, Desiree; Ferrer, Isidro; Carro, Eva

    2015-04-01

    Recent studies indicate that the choroid plexus has important physiologic and pathologic roles in Alzheimer disease (AD). To obtain additional insight on choroid plexus function, we performed a proteomic analysis of choroid plexus samples from patients with AD stages I to II (n = 16), III to IV (n = 16), and V to VI (n = 11) and 7 age-matched control subjects. We used 2-dimensional differential gel electrophoresis coupled with mass spectrometry to generate a complete picture of changes in choroid plexus protein expression occurring in AD patients. We identified 6 proteins: 14-3-3 β/α, 14-3-3 ε, moesin, proteasome activator complex subunit 1, annexin V, and aldehyde dehydrogenase, which were significantly regulated in AD patient samples (p < 0.05, >1.5-fold variation in expression vs control samples). These proteins are implicated in major physiologic functions including mitochondrial dysfunction and apoptosis regulation. These findings contribute additional significance to the emerging importance of molecular and functional changes of choroid plexus function in the pathophysiology of AD. PMID:25756589

  5. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

    PubMed Central

    Di Roberto, Raphaël B.; Chang, Belinda; Trusina, Ala; Peisajovich, Sergio G.

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While some mutants show enhanced binding affinity to the foreign pheromone, others only display weakened interactions with the network's negative regulators. Importantly, the latter changes have a limited impact on overall pathway regulation, despite their considerable effect on sensitivity. Our results demonstrate that a new receptor–ligand pair can evolve through network-altering mutations independently of receptor–ligand binding, and suggest a potential role for such mutations in disease. PMID:27487915

  6. Direct control of type IIA topoisomerase activity by a chromosomally encoded regulatory protein

    PubMed Central

    Vos, Seychelle M.; Lyubimov, Artem Y.; Hershey, David M.; Schoeffler, Allyn J.; Sengupta, Sugopa; Nagaraja, Valakunja; Berger, James M.

    2014-01-01

    Precise control of supercoiling homeostasis is critical to DNA-dependent processes such as gene expression, replication, and damage response. Topoisomerases are central regulators of DNA supercoiling commonly thought to act independently in the recognition and modulation of chromosome superstructure; however, recent evidence has indicated that cells tightly regulate topoisomerase activity to support chromosome dynamics, transcriptional response, and replicative events. How topoisomerase control is executed and linked to the internal status of a cell is poorly understood. To investigate these connections, we determined the structure of Escherichia coli gyrase, a type IIA topoisomerase bound to YacG, a recently identified chromosomally encoded inhibitor protein. Phylogenetic analyses indicate that YacG is frequently associated with coenzyme A (CoA) production enzymes, linking the protein to metabolism and stress. The structure, along with supporting solution studies, shows that YacG represses gyrase by sterically occluding the principal DNA-binding site of the enzyme. Unexpectedly, YacG acts by both engaging two spatially segregated regions associated with small-molecule inhibitor interactions (fluoroquinolone antibiotics and the newly reported antagonist GSK299423) and remodeling the gyrase holoenzyme into an inactive, ATP-trapped configuration. This study establishes a new mechanism for the protein-based control of topoisomerases, an approach that may be used to alter supercoiling levels for responding to changes in cellular state. PMID:24990966

  7. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions.

    PubMed

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala; Peisajovich, Sergio G

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While some mutants show enhanced binding affinity to the foreign pheromone, others only display weakened interactions with the network's negative regulators. Importantly, the latter changes have a limited impact on overall pathway regulation, despite their considerable effect on sensitivity. Our results demonstrate that a new receptor-ligand pair can evolve through network-altering mutations independently of receptor-ligand binding, and suggest a potential role for such mutations in disease. PMID:27487915

  8. Regulatory Implications of Structural Changes in Tyr201 of the Oxygen Sensor Protein FixL.

    PubMed

    Yamawaki, Takeo; Ishikawa, Haruto; Mizuno, Misao; Nakamura, Hiro; Shiro, Yoshitsugu; Mizutani, Yasuhisa

    2016-07-26

    FixL is a heme-based oxygen-sensing histidine kinase that induces the expression of nitrogen fixation genes under hypoxic conditions. Oxygen dissociation from heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, activating autophosphorylation activity. Conversely, oxygen binding inhibits FixL kinase activity. It is essential to elucidate the changes that occur in the protein structure upon this oxygen dissociation for understanding of the allosteric transduction mechanism. We measured ultraviolet resonance Raman spectra of FixL and its mutants for deoxy, oxy, and carbonmonoxy forms to examine the changes in protein structure upon oxygen dissociation. The observed spectral changes indicated that Tyr201 and its neighboring residues undergo structural changes upon oxygen dissociation. Kinase assays showed that substitution of Tyr201 significantly decreased the inhibition of kinase activity upon oxygen binding. These data mean that weakening of the hydrogen bond of Tyr201 that is induced by oxygen dissociation is essential for inhibition of kinase activity. We also observed spectral changes in Tyr residues in the kinase domain upon oxygen dissociation from FixL, which is the first observation of oxygen-dependent structural changes in the kinase domain of FixL. The observed structural changes support the allosteric transduction pathway of FixL which we proposed previously [ Yano, S., Ishikawa, H., Mizuno, M., Nakamura, H., Shiro, Y., and Mizutani, Y. ( 2013 ) J. Phys. Chem. B 117 , 15786 - 15791 ]. PMID:27367650

  9. Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways.

    PubMed

    Hatori, Megumi; Hirota, Tsuyoshi; Iitsuka, Michiko; Kurabayashi, Nobuhiro; Haraguchi, Shogo; Kokame, Koichi; Sato, Ryuichiro; Nakai, Akira; Miyata, Toshiyuki; Tsutsui, Kazuyoshi; Fukada, Yoshitaka

    2011-03-22

    The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by >2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF)1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7α-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7α-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-day-dependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland. PMID:21383147

  10. The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila

    PubMed Central

    2013-01-01

    Background The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription. Results and discussion Here we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters. Conclusions Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes. PMID:23442797

  11. Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome

    PubMed Central

    Hebert, Alexander S.; Dittenhafer-Reed, Kristin E.; Yu, Wei; Bailey, Derek J.; Selen, Ebru Selin; Boersma, Melissa D.; Carson, Joshua J.; Tonelli, Marco; Balloon, Allison; Higbee, Alan J.; Westphall, Michael S.; Pagliarini, David J.; Prolla, Tomas A.; Assadi-Porter, Fariba; Roy, Sushmita; Denu, John M.; Coon, Joshua J.

    2013-01-01

    Summary Calorie restriction (CR) extends lifespan in diverse species. Mitochondria play a key role in CR adaptation, however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR vs. control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites −2,193 from mitochondrial proteins rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance. PMID:23201123

  12. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

    PubMed

    Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

    2016-06-01

    Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria. PMID:27183143

  13. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

    2014-01-01

    STUDY QUESTION Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood–testis barrier (BTB)? SUMMARY ANSWER Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. WHAT IS KNOWN ALREADY Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. STUDY DESIGN, SIZE AND DURATION We examined the effects of two environmental toxicants: cadmium chloride (0.5–20 µM) and bisphenol A (0.4–200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. PARTICIPANTS/MATERIALS, SETTING, METHODS Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). MAIN RESULTS AND THE ROLE OF CHANCE Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by

  14. Loa loa Microfilariae evade complement attack in vivo by acquiring regulatory proteins from host plasma.

    PubMed

    Haapasalo, Karita; Meri, Taru; Jokiranta, T Sakari

    2009-09-01

    Loa loa is a filarial nematode that infects humans. The adults live in subcutaneous tissues and produce microfilariae that live for several weeks in the blood circulation in order to be transmitted to another person via blood meals of a dipterian vector. As microfilariae live in continuous contact with plasma, it is obvious that they evade the complement system. We studied markers of complement activation and signs of complement regulation on Loa loa microfilariae in vivo. The microfilariae were isolated from anticoagulated blood samples of a Loa loa-infected Caucasian patient. C1q and some mannose-binding lectin but only a limited amount of C3b or C4b fragments and practically no C5 or C5b-9 were present on the microfilariae. The covalently microfilaria-bound C3 and C4 depositions were mainly inactive iC3b, C3c, and iC4b fragments indicating that microfilariae had regulated complement activation in vivo. Also, in vitro deposition of C3b onto the microfilariae upon serum exposure was limited. The patient-isolated microfilariae were found to carry the host complement regulators factor H and C4b-binding protein on the outermost layer, so called sheath. The microfilaria-bound factor H was functionally active. Binding of the complement regulators to the microfilariae was confirmed in vitro using (125)I-labeled factor H and C4b-binding protein. In conclusion, our study shows that Loa loa microfilariae block complement activation and acquire the host complement regulators factor H and C4b-binding protein in blood circulation. This is the first time that binding of complement regulators onto nonviral pathogens has been demonstrated to occur in humans in vivo. PMID:19528206

  15. Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2

    PubMed Central

    Cooperman, Sharon S.; Meyron-Holtz, Esther G.; Olivierre-Wilson, Hayden; Ghosh, Manik C.; McConnell, Joseph P.; Rouault, Tracey A.

    2005-01-01

    Iron-regulatory proteins (IRPs) 1 and 2 posttranscriptionally regulate expression of transferrin receptor (TfR), ferritin, and other iron metabolism proteins. Mice with targeted deletion of IRP2 overexpress ferritin and express abnormally low TfR levels in multiple tissues. Despite this misregulation, there are no apparent pathologic consequences in tissues such as the liver and kidney. However, in the central nervous system, evidence of abnormal iron metabolism in IRP2-/- mice precedes the development of adult-onset progressive neurodegeneration, characterized by widespread axonal degeneration and neuronal loss. Here, we report that ablation of IRP2 results in iron-limited erythropoiesis. TfR expression in erythroid precursors of IRP2-/- mice is reduced, and bone marrow iron stores are absent, even though transferrin saturation levels are normal. Marked overexpression of 5-aminolevulinic acid synthase 2 (Alas2) results from loss of IRP-dependent translational repression, and markedly increased levels of free protoporphyrin IX and zinc protoporphyrin are generated in IRP2-/- erythroid cells. IRP2-/- mice represent a new paradigm of genetic microcytic anemia. We postulate that IRP2 mutations or deletions may be a cause of refractory microcytic anemia and bone marrow iron depletion in patients with normal transferrin saturations, elevated serum ferritins, elevated red cell protoporphyrin IX levels, and adult-onset neurodegeneration. PMID:15831703

  16. Regulation of steroid 5-{alpha} reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    SciTech Connect

    Seo, Young-Kyo; Zhu, Bing; Jeon, Tae-Il; Osborne, Timothy F.

    2009-11-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  17. Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

    PubMed Central

    Chantalat, L; Leroy, D; Filhol, O; Nueda, A; Benitez, M J; Chambaz, E M; Cochet, C; Dideberg, O

    1999-01-01

    Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation. PMID:10357806

  18. Post-Translational Modifications of Kaposi’s Sarcoma-Associated Herpesvirus Regulatory Proteins – SUMO and KSHV

    PubMed Central

    Campbell, Mel; Izumiya, Yoshihiro

    2011-01-01

    KSHV latency can be envisioned as an outcome that is balanced between factors that promote viral gene expression and lytic replication against those that facilitate gene silencing and establish or maintain latency. A large body of work has focused on the activities of the key viral regulatory proteins involved in KSHV latent or lytic states. Moreover, recent studies have also begun to document the importance of epigenetic landscape evolution of the KSHV viral genome during latency and reactivation. However, one area of KSHV molecular virology that remains largely unanswered is the precise role of post-translational modifications on the activities of viral factors that function during latency and reactivation. In this review, we will summarize the post-translational modifications associated with three viral factors whose activities contribute to the viral state. The viral proteins discussed are the two major KSHV encoded transcription factors, K-Rta (KSHV replication and transcriptional activator) and K-bZIP (KSHV basic leucine zipper) and the viral latency-associated nuclear antigen (LANA). A special emphasis will be placed on the role of the sumoylation pathway in the modulation of the KSHV lifecycle. Newly uncovered small ubiquitin-like modifier (SUMO)-associated properties of LANA and K-Rta will also be presented, namely LANA histone targeting SUMO E3 ligase activity and K-Rta SUMO-targeted ubiquitin ligase function. PMID:22347876

  19. Cd47-Signal Regulatory Protein α (Sirpα) Regulates Fcγ and Complement Receptor–Mediated Phagocytosis

    PubMed Central

    Oldenborg, Per-Arne; Gresham, Hattie D.; Lindberg, Frederik P.

    2001-01-01

    In autoimmune hemolytic anemia (AIHA), circulating red blood cells (RBCs) opsonized with autoantibody are recognized by macrophage Fcγ and complement receptors. This triggers phagocytosis and elimination of RBCs from the circulation by splenic macrophages. We recently found that CD47 on unopsonized RBCs binds macrophage signal regulatory protein α (SIRPα), generating a negative signal that prevents phagocytosis of the unopsonized RBCs. We show here that clearance and phagocytosis of opsonized RBCs is also regulated by CD47-SIRPα. The inhibition generated by CD47-SIRPα interaction is strongly attenuated but not absent in mice with only residual activity of the phosphatase Src homology 2 domain–containing protein tyrosine phosphatase (SHP)-1, suggesting that most SIRPα signaling in this system is mediated by SHP-1 phosphatase activity. The macrophage phagocytic response is controlled by an integration of the inhibitory SIRPα signal with prophagocytic signals such as from Fcγ and complement receptor activation. Thus, augmentation of inhibitory CD47-SIRPα signaling may prevent or attenuate RBC clearance in AIHA. PMID:11283158

  20. LPS induces KH-type splicing regulatory protein-dependent processing of microRNA-155 precursors in macrophages.

    PubMed

    Ruggiero, Tina; Trabucchi, Michele; De Santa, Francesca; Zupo, Simona; Harfe, Brian D; McManus, Michael T; Rosenfeld, M Geoff; Briata, Paola; Gherzi, Roberto

    2009-09-01

    The importance of post-transcriptional mechanisms for the regulation of the homoeostasis of the immune system and the response to challenge by microorganisms is becoming increasingly appreciated. We investigated the contribution of microRNAs (miRNAs) to macrophage activation induced by lipopolysaccharide (LPS). We first observed that Dicer knockout in bone marrow-derived macrophages (BMDMs) increases the LPS-induced expression of some inflammation mediators. miRNA microarray analysis in BMDMs revealed that LPS significantly induces the expression of a single miRNA, miR-155, and this induction depends on enhanced miR-155 maturation from its precursors. The single-strand RNA-binding protein KH-type splicing regulatory protein (KSRP) binds to the terminal loop of miR-155 precursors and promotes their maturation. Both inhibition of miR-155 and KSRP knockdown enhance the LPS-induced expression of select inflammation mediators, and the effect of KSRP knockdown is reverted by mature miR-155. Our studies unveil the existence of an LPS-dependent post-transcriptional regulation of miR-155 biogenesis. Once induced, miR-155 finely tunes the expression of select inflammation mediators in response to LPS. PMID:19423639

  1. Genome-wide targeting of the epigenetic regulatory protein CTCF to gene promoters by the transcription factor TFII-I

    PubMed Central

    Peña-Hernández, Rodrigo; Marques, Maud; Hilmi, Khalid; Zhao, Teijun; Saad, Amine; Alaoui-Jamali, Moulay A.; del Rincon, Sonia V.; Ashworth, Todd; Roy, Ananda L.; Emerson, Beverly M.; Witcher, Michael

    2015-01-01

    CCCTC-binding factor (CTCF) is a key regulator of nuclear chromatin structure and gene regulation. The impact of CTCF on transcriptional output is highly varied, ranging from repression to transcriptional pausing and transactivation. The multifunctional nature of CTCF may be directed solely through remodeling chromatin architecture. However, another hypothesis is that the multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique functions. Consistent with this hypothesis, our mass spectrometry analyses of CTCF interacting partners reveal a previously undefined association with the transcription factor general transcription factor II-I (TFII-I). Biochemical fractionation of CTCF indicates that a distinct CTCF complex incorporating TFII-I is assembled on DNA. Unexpectedly, we found that the interaction between CTCF and TFII-I is essential for directing CTCF to the promoter proximal regulatory regions of target genes across the genome, particularly at genes involved in metabolism. At genes coregulated by CTCF and TFII-I, we find knockdown of TFII-I results in diminished CTCF binding, lack of cyclin-dependent kinase 8 (CDK8) recruitment, and an attenuation of RNA polymerase II phosphorylation at serine 5. Phenotypically, knockdown of TFII-I alters the cellular response to metabolic stress. Our data indicate that TFII-I directs CTCF binding to target genes, and in turn the two proteins cooperate to recruit CDK8 and enhance transcription initiation. PMID:25646466

  2. The C-terminus of DSX(F5) protein acts as a novel regulatory domain in Bombyx mori.

    PubMed

    Duan, Jianping; Meng, Xianxin; Ma, Sanyuan; Wang, Feng; Guo, Huozhen; Zhang, Liying; Zhao, Ping; Kan, Yunchao; Yao, Lunguang; Xia, Qingyou

    2016-08-01

    The doublesex gene regulates the somatic sexual development of Bombyx mori by alternatively splicing into sex-specific splice forms. In our previous study, the splice form Bmdsx (F7) , which encodes the BmDSX(F5) protein, was found to be expressed in a female-specific manner and to contain a novel C-terminus. In this study, we aimed to investigate the role of this C-terminus. Two transgenic lines, L1 and L2, were constructed to ectopically express Bmdsx (F7) in males. Phenotype and W chromosome-specific polymerase chain reaction (PCR) analysis showed that developmental abnormalities and sex reversal did not occur. Moreover, the sex ratio was also normal. Quantitative PCR revealed that the expression levels of SP1 and Vg were upregulated in the fat body of transgenic males. Additionally, the expression level of PBP was downregulated in the antenna of transgenic males. The results suggested that the C-terminus of BmDSX(F5) functioned as a regulatory domain during regulation of downstream target gene expression and that BmDSX(F5) participated in the sexual development of somatic cells together with other DSX proteins in B. mori. PMID:26975733

  3. Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins

    PubMed Central

    Ochiai, Ayasa; Miyata, Shingo; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2015-01-01

    Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity. PMID:26431033

  4. Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.

    PubMed

    Graham, Morag R; Smoot, Laura M; Migliaccio, Cristi A Lux; Virtaneva, Kimmo; Sturdevant, Daniel E; Porcella, Stephen F; Federle, Michael J; Adams, Gerald J; Scott, June R; Musser, James M

    2002-10-15

    Two-component gene regulatory systems composed of a membrane-bound sensor and cytoplasmic response regulator are important mechanisms used by bacteria to sense and respond to environmental stimuli. Group A Streptococcus, the causative agent of mild infections and life-threatening invasive diseases, produces many virulence factors that promote survival in humans. A two-component regulatory system, designated covRS (cov, control of virulence; csrRS), negatively controls expression of five proven or putative virulence factors (capsule, cysteine protease, streptokinase, streptolysin S, and streptodornase). Inactivation of covRS results in enhanced virulence in mouse models of invasive disease. Using DNA microarrays and quantitative RT-PCR, we found that CovR influences transcription of 15% (n = 271) of all chromosomal genes, including many that encode surface and secreted proteins mediating host-pathogen interactions. CovR also plays a central role in gene regulatory networks by influencing expression of genes encoding transcriptional regulators, including other two-component systems. Differential transcription of genes influenced by covR also was identified in mouse soft-tissue infection. This analysis provides a genome-scale overview of a virulence gene network in an important human pathogen and adds insight into the molecular mechanisms used by group A Streptococcus to interact with the host, promote survival, and cause disease. PMID:12370433

  5. High affinity binding proteins for the regulatory subunit of cAMP-dependent protein kinase: Cloning, characterization and expression of P150 and P75

    SciTech Connect

    Bregman, D.B.

    1989-01-01

    cAMP-dependent protein kinase II-B appears to be adapted for function in the mammalian central nervous system (CNS) via the properties of its regulatory subunit (RII-B). RII-B is selectively expressed in brain, tightly associated with cerebral cortex membranes and avidly complexed by the bovine brain calmodulin-binding protein designated P75. Complexes of RII-B and P75 polypeptides can be purified to near-homogeneity from either membrane or cytosolic fractions of brain homogenates, suggested that the binding protein plays a role in determining the subcellular localization and/or other CNS-specific properties of protein kinase II-B. In general, a single high-affinity RII-B binding protein is expressed in the brains of mammals, but the size of the protein varies (e.g., cow: 75 kDa; rat: 150 kDa). To investigate these non-abundant and CNS-enriched RII-B binding proteins, cDNAs for bovine brain P75 and rat brain P150, the homologue of P75, have been cloned and characterized. The cDNAs were retrieved from bovine and rat lambda gt11 expression libraries using {sup 32}P-RII-B as a functional probe. cDNA inserts subcloned into pIN-IA2 or pET-3b expression plasmids directed the production of partial P75 and P150 polypeptides in E. coli that exhibited RII-B binding activity. P150 cDNAs hybridize with 3 rat mRNAs (7.3, 5.0, 4.2 kb) that are present in brain and lung, but not other tissues. These mRNAs are not detected in fetal brain, but are expressed during the period of post-natal synaptogenesis and in adult rats. The P75 cDNA hybridizes to a 6 kb bovine brain mRNA. Finally, 3'-deletion analysis demonstrated that the C-terminal 15-25 amino acids of P150 or P75 are essential for binding with RII-B.

  6. Iron Regulatory Protein 1 Suppresses Hypoxia-Induced Iron Uptake Proteins Expression and Decreases Iron Levels in HepG2 Cells.

    PubMed

    Cheng, Chun-Ming; Wang, Dan; Cao, Xian; Luo, Qian-Qian; Lu, Ya-Peng; Zhu, Li

    2015-09-01

    Transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1) are important proteins for cellular iron uptake, and both are regulated transcriptionally through the binding of hypoxia-inducible factor 1 (HIF-1) to hypoxia-responsive elements (HREs) under hypoxic conditions. These proteins are also regulated post-transcriptionally through the binding of iron regulatory protein 1 (IRP1) to iron-responsive elements (IREs) located in the mRNA untranslated region (UTR) to control cellular iron homeostasis. In iron-deficient cells, IRP1-IRE interactions stabilize TfR1 and DMT1 mRNAs, enhancing iron uptake. However, little is known about the impact of IRP1 on the regulation of cellular iron homeostasis under hypoxia. Thus, to investigate the role of IRP1 in hypoxic condition, overexpression and knockdown assays were performed using HepG2 cells. The overexpression of IRP1 suppressed the hypoxia-induced increase in TfR1 and DMT1 (+IRE) expression and reduced the stability of TfR1 and DMT1 (+IRE) mRNAs under hypoxia, whereas IRP1 knockdown further increased the hypoxia-induced expression of both proteins, preventing the decrease in IRE-dependent luciferase activity induced by hypoxia. Under hypoxic conditions, ferrous iron uptake, the labile iron pool (LIP), and total intracellular iron reduced when IRP1 was overexpressed and further increased when IRP1 was knocked down. IRP1 expression declined and TfR1/DMT1 (+IRE) expression increased with the time of hypoxia prolonged, whereas the binding of IRP1 to the IRE of TfR1/DMT1 mRNA maintained. In summary, IRP1 suppressed TfR1/DMT1 (+IRE) expression, limited the cellular iron content and decreased lactate dehydrogenase (LDH) release induced by hypoxia. PMID:25727755

  7. Regulatory T cells in the humoral response of protein deficient mice.

    PubMed

    Price, P; Turner, K J

    1979-01-01

    Cell suspensions from the spleen or thymus of mice fed normally or mice that were protein deficient were injected into mice from each dietary group and also syngeneic nudes. Antigen, polyvinyl pyrrolidone (PVP), was injected at the stage of cell transfer and the antibody titres of the recipient animals were compared with those of control animals given only antigen. The regime was repeated using cell suspensions from donor animals which had been primed with antigen. These experiments showed that spleen cells were suppressive only when transferred from deficient to normal mice. Thymocytes generally lacked suppressive effects, except when given to irradiated mice also injected with "normal" spleen cells. However, thymocytes from deficient mice were marginally enhancing in nude mice, deficient mice and older "normals". To explain these results, it is suggested that responses to PVP are determined by distinct "suppressor-inducing" and "suppressor" T cells which act via helper T cells. The latter probably affect B cells directly and largely influence IgG production. It also appears likely that the ratio of helper to suppressor (inducer and effector) T cells is increased by protein deficiency. PMID:311719

  8. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    NASA Astrophysics Data System (ADS)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian

    2016-07-01

    The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of peptide conformations. These findings are consistent with experimental results, and give a molecular level interpretation for the reduced cytotoxicity of Vpr13-33 to some extent upon graphene exposure. Meanwhile, this study provides some significant insights into the detailed mechanism of graphene-induced protein conformation transition.

  9. FOXP3+ regulatory T cell development and function require histone/protein deacetylase 3

    PubMed Central

    Wang, Liqing; Liu, Yujie; Han, Rongxiang; Beier, Ulf H.; Bhatti, Tricia R.; Akimova, Tatiana; Greene, Mark I.; Hiebert, Scott W.; Hancock, Wayne W.

    2015-01-01

    Treg dysfunction is associated with a variety of inflammatory diseases. Treg populations are defined by expression of the oligomeric transcription factor FOXP3 and inability to produce IL-2, a cytokine required for T cell maintenance and survival. FOXP3 activity is regulated post-translationally by histone/protein acetyltransferases and histone/protein deacetylases (HDACs). Here, we determined that HDAC3 mediates both the development and function of the two main Treg subsets, thymus-derived Tregs and induced Tregs (iTregs). We determined that HDAC3 and FOXP3 physically interact and that HDAC3 expression markedly reduces Il2 promoter activity. In murine models, conditional deletion of Hdac3 during thymic Treg development restored Treg production of IL-2 and blocked the suppressive function of Tregs. HDAC3-deficient mice died from autoimmunity by 4–6 weeks of age; however, injection of WT FOXP3+ Tregs prolonged survival. Adoptive transfer of Hdac3-deficient Tregs, unlike WT Tregs, did not control T cell proliferation in naive mice and did not prevent allograft rejection or colitis. HDAC3 also regulated the development of iTregs, as HDAC3-deficient conventional T cells were not converted into iTregs under polarizing conditions and produced large amounts of IL-2, IL-6, and IL-17. We conclude that HDAC3 is essential for the normal development and suppressive functions of thymic and peripheral FOXP3+ Tregs. PMID:25642770

  10. Novel Functions for the Endocytic Regulatory Proteins MICAL-L1 AND EHD1 in Mitosis

    PubMed Central

    Reinecke, James B.; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

    2014-01-01

    During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle. PMID:25287187

  11. Global Regulation of Gene Expression by the MafR Protein of Enterococcus faecalis.

    PubMed

    Ruiz-Cruz, Sofía; Espinosa, Manuel; Goldmann, Oliver; Bravo, Alicia

    2015-01-01

    Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. However, as an opportunistic pathogen, it is able to colonize other host niches and cause life-threatening infections. Its adaptation to new environments involves global changes in gene expression. The EF3013 gene (here named mafR) of E. faecalis strain V583 encodes a protein (MafR, 482 residues) that has sequence similarity to global response regulators of the Mga/AtxA family. The enterococcal OG1RF genome also encodes the MafR protein (gene OG1RF_12293). In this work, we have identified the promoter of the mafR gene using several in vivo approaches. Moreover, we show that MafR influences positively the transcription of many genes on a genome-wide scale. The most significant target genes encode components of PTS-type membrane transporters, components of ABC-type membrane transporters, and proteins involved in the metabolism of carbon sources. Some of these genes were previously reported to be up-regulated during the growth of E. faecalis in blood and/or in human urine. Furthermore, we show that a mafR deletion mutant strain induces a significant lower degree of inflammation in the peritoneal cavity of mice, suggesting that enterococcal cells deficient in MafR are less virulent. Our work indicates that MafR is a global transcriptional regulator. It might facilitate the adaptation of E. faecalis to particular host niches and, therefore, contribute to its potential virulence. PMID:26793169

  12. Global Regulation of Gene Expression by the MafR Protein of Enterococcus faecalis

    PubMed Central

    Ruiz-Cruz, Sofía; Espinosa, Manuel; Goldmann, Oliver; Bravo, Alicia

    2016-01-01

    Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. However, as an opportunistic pathogen, it is able to colonize other host niches and cause life-threatening infections. Its adaptation to new environments involves global changes in gene expression. The EF3013 gene (here named mafR) of E. faecalis strain V583 encodes a protein (MafR, 482 residues) that has sequence similarity to global response regulators of the Mga/AtxA family. The enterococcal OG1RF genome also encodes the MafR protein (gene OG1RF_12293). In this work, we have identified the promoter of the mafR gene using several in vivo approaches. Moreover, we show that MafR influences positively the transcription of many genes on a genome-wide scale. The most significant target genes encode components of PTS-type membrane transporters, components of ABC-type membrane transporters, and proteins involved in the metabolism of carbon sources. Some of these genes were previously reported to be up-regulated during the growth of E. faecalis in blood and/or in human urine. Furthermore, we show that a mafR deletion mutant strain induces a significant lower degree of inflammation in the peritoneal cavity of mice, suggesting that enterococcal cells deficient in MafR are less virulent. Our work indicates that MafR is a global transcriptional regulator. It might facilitate the adaptation of E. faecalis to particular host niches and, therefore, contribute to its potential virulence. PMID:26793169

  13. Association between glucokinase regulatory protein (GCKR) and apolipoprotein A5 (APOA5) gene polymorphisms and triacylglycerol concentrations in fasting, postprandial, and fenofibrate-treated states

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Hypertriglyceridemia is a risk factor for cardiovascular disease. Variation in the apolipoprotein A5 (APOA5) and glucokinase regulatory protein (GCKR) genes has been associated with fasting plasma triacylglycerol. Objective: We investigated the combined effects of the GCKR rs780094C-->T,...

  14. Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene.

    PubMed Central

    Rincón-Limas, D E; Amaya-Manzanares, F; Niño-Rosales, M L; Yu, Y; Yang, T P; Patel, P I

    1995-01-01

    The hypoxanthine phosphoribosyltransferase (HPRT) gene is constitutively expressed at low levels in all tissues but at higher levels in the brain; the significance and mechanism of this differential expression are unknown. We previously identified a 182-bp element (hHPRT-NE) within the 5'-flanking region of the human HPRT (hHPRT) gene, which is involved not only in conferring neuronal specificity but also in repressing gene expression in nonneuronal tissues. Here we report that this element interacts with different nuclear proteins, some of which are present specifically in neuronal cells (complex I) and others of which are present in cells showing constitutive expression of the gene (complex II). In addition, we found that complex I factors are expressed in human NT2/D1 cells following induction of neuronal differentiation by retinoic acid. This finding correlates with an increase of HPRT gene transcription following neuronal differentiation. We also mapped the binding sites for both complexes to a 60-bp region (Ff; positions -510 to -451) which, when analyzed in transfection assays, functioned as a repressor element analogous to the full-length hHPRT-NE sequence. Methylation interference footprintings revealed a minimal unique DNA motif, 5'-GGAAGCC-3', as the binding site for nuclear proteins from both neuronal and nonneuronal sources. However, site-directed mutagenesis of the footprinted region indicated that different nucleotides are essential for the associations of these two complexes. Moreover, UV cross-linking experiments showed that both complexes are formed by the association of several different proteins. Taken together, these data suggest that differential interaction of DNA-binding factors with this regulatory element plays a crucial role in the brain-preferential expression of the gene, and they should lead to the isolation of transcriptional regulators important in neuronal expression of the HPRT gene. PMID:8524221

  15. Infantile 4-tert-octylphenol exposure transiently inhibits rat ovarian steroidogenesis and steroidogenic acute regulatory protein (StAR) expression

    SciTech Connect

    Myllymaeki, S.A. . E-mail: saanmy@utu.fi; Karjalainen, M.; Haavisto, T.E.; Toppari, J.; Paranko, J.

    2005-08-22

    Phenolic compounds, such as 4-tert-octylphenol (OP), have been shown to interfere with rat ovarian steroidogenesis. However, little is known about steroidogenic effects of infantile OP exposure on immature ovary. The aim of the present study was to investigate the effects of infantile OP exposure on plasma FSH, LH, estradiol, and progesterone levels in 14-day-old female rats. The effect on ovarian steroidogenic acute regulatory protein (StAR) and FSH receptor (FSHr) expression was analyzed by Western blotting. Ex vivo analysis was carried out for follicular estradiol, progesterone, testosterone, and cAMP production. Sprague-Dawley rats were given OP (0, 10, 50, or 100 mg/kg) subcutaneously on postnatal days 6, 8, 10, and 12. On postnatal day 14, plasma FSH was decreased and progesterone increased significantly at a dose of 100 mg OP/kg. In addition, the highest OP dose advanced the time of vaginal opening in puberty. OP had no effect on infantile LH and estradiol levels or ovarian FSHr content. Ovarian StAR protein content and ex vivo hormone and cAMP production were decreased at all OP doses compared to controls. However, hormone levels recovered independent on FSH and even increased above the control level during a prolonged culture. On postnatal day 35, no statistically significant differences were seen between control and OP-exposed animals in plasma FSH, LH, estradiol, and progesterone levels, or in ovarian StAR protein content. The results indicate that the effect of OP on the infantile ovary is reversible, while more permanent effects in the hypothalamus and pituitary, as described earlier, are involved in the reduction of circulating FSH levels and premature vaginal opening.

  16. The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

    PubMed

    Vivancos, Julien; Spinner, Lara; Mazubert, Christelle; Charlot, Florence; Paquet, Nicolas; Thareau, Vincent; Dron, Michel; Nogué, Fabien; Charon, Céline

    2012-03-01

    The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM. PMID:22170036

  17. Oxytocin receptor gene sequences in owl monkeys and other primates show remarkable interspecific regulatory and protein coding variation.

    PubMed

    Babb, Paul L; Fernandez-Duque, Eduardo; Schurr, Theodore G

    2015-10-01

    The oxytocin (OT) hormone pathway is involved in numerous physiological processes, and one of its receptor genes (OXTR) has been implicated in pair bonding behavior in mammalian lineages. This observation is important for understanding social monogamy in primates, which occurs in only a small subset of taxa, including Azara's owl monkey (Aotus azarae). To examine the potential relationship between social monogamy and OXTR variation, we sequenced its 5' regulatory (4936bp) and coding (1167bp) regions in 25 owl monkeys from the Argentinean Gran Chaco, and examined OXTR sequences from 1092 humans from the 1000 Genomes Project. We also assessed interspecific variation of OXTR in 25 primate and rodent species that represent a set of phylogenetically and behaviorally disparate taxa. Our analysis revealed substantial variation in the putative 5' regulatory region of OXTR, with marked structural differences across primate taxa, particularly for humans and chimpanzees, which exhibited unique patterns of large motifs of dinucleotide A+T repeats upstream of the OXTR 5' UTR. In addition, we observed a large number of amino acid substitutions in the OXTR CDS region among New World primate taxa that distinguish them from Old World primates. Furthermore, primate taxa traditionally defined as socially monogamous (e.g., gibbons, owl monkeys, titi monkeys, and saki monkeys) all exhibited different amino acid motifs for their respective OXTR protein coding sequences. These findings support the notion that monogamy has evolved independently in Old World and New World primates, and that it has done so through different molecular mechanisms, not exclusively through the oxytocin pathway. PMID:26025428

  18. Homolog detection using global sequence properties suggests an alternate view of structural encoding in protein sequences

    PubMed Central

    Scheraga, Harold A.; Rackovsky, S.

    2014-01-01

    We show that a Fourier-based sequence distance function is able to identify structural homologs of target sequences with high accuracy. It is shown that Fourier distances correlate very strongly with independently determined structural distances between molecules, a property of the method that is not attainable using conventional representations. It is further shown that the ability of the Fourier approach to identify protein folds is statistically far in excess of random expectation. It is then shown that, in actual searches for structural homologs of selected target sequences, the Fourier approach gives excellent results. On the basis of these results, we suggest that the global information detected by the Fourier representation is an essential feature of structure encoding in protein sequences and a key to structural homology detection. PMID:24706836

  19. Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB

    PubMed Central

    Coelho, Miguel B; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher WJ

    2015-01-01

    Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3. PMID:25599992

  20. Regulatory roles of tumor necrosis factor alpha-induced proteins (TNFAIPs) 3 and 9 in arthritis.

    PubMed

    Matsumoto, Isao; Inoue, Asuka; Takai, Chinatsu; Umeda, Naoto; Tanaka, Yuki; Kurashima, Yuko; Sumida, Takayuki

    2014-07-01

    Tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) have proved to be important in rheumatoid arthritis (RA) because the outcome of RA has greatly improved with the recent availability of biologics targeting them. It is well accepted that these cytokines are involved in the activation of the nuclear factor-κB (NF-κB) signaling pathway, but our understanding of the dependency of these pro-inflammatory cytokines and the link between them in RA is currently limited. Recently, we and others proved the importance of TNFα-induced protein (TNFAIP), due to the spontaneous development of arthritis in deficient animals that are dependent on IL-6. To date, nine TNFAIPs have been identified, and TNFAIP3 and TNFAIP9 were found to be clearly associated with mouse and human arthritis. In this review, we compare and discuss recent TNFAIP topics, especially focusing on TNFAIP3 and TNFAIP9 in autoimmune arthritis in mice and humans. PMID:24704577

  1. Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein

    PubMed Central

    Suzuki, Takashi; Brown, Judy J.; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5’-UTR for MTP-A. We generated reporter constructs in which the 5’-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5’-UTR, but not by the MTP-A 5’-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5’-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity. PMID:26771188

  2. Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein.

    PubMed

    Suzuki, Takashi; Brown, Judy J; Swift, Larry L

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5'-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5'-UTR for MTP-A. We generated reporter constructs in which the 5'-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5'-UTR, but not by the MTP-A 5'-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity. PMID:26771188

  3. Residue-level global and local ensemble-ensemble comparisons of protein domains.

    PubMed

    Clark, Sarah A; Tronrud, Dale E; Karplus, P Andrew

    2015-09-01

    Many methods of protein structure generation such as NMR-based solution structure determination and template-based modeling do not produce a single model, but an ensemble of models consistent with the available information. Current strategies for comparing ensembles lose information because they use only a single representative structure. Here, we describe the ENSEMBLATOR and its novel strategy to directly compare two ensembles containing the same atoms to identify significant global and local backbone differences between them on per-atom and per-residue levels, respectively. The ENSEMBLATOR has four components: eePREP (ee for ensemble-ensemble), which selects atoms common to all models; eeCORE, which identifies atoms belonging to a cutoff-distance dependent common core; eeGLOBAL, which globally superimposes all models using the defined core atoms and calculates for each atom the two intraensemble variations, the interensemble variation, and the closest approach of members of the two ensembles; and eeLOCAL, which performs a local overlay of each dipeptide and, using a novel measure of local backbone similarity, reports the same four variations as eeGLOBAL. The combination of eeGLOBAL and eeLOCAL analyses identifies the most significant differences between ensembles. We illustrate the ENSEMBLATOR's capabilities by showing how using it to analyze NMR ensembles and to compare NMR ensembles with crystal structures provides novel insights compared to published studies. One of these studies leads us to suggest that a "consistency check" of NMR-derived ensembles may be a useful analysis step for NMR-based structure determinations in general. The ENSEMBLATOR 1.0 is available as a first generation tool to carry out ensemble-ensemble comparisons. PMID:26032515

  4. Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth

    PubMed Central

    2013-01-01

    Background Regulation of lipid metabolism via activation of sterol regulatory element binding proteins (SREBPs) has emerged as an important function of the Akt/mTORC1 signaling axis. Although the contribution of dysregulated Akt/mTORC1 signaling to cancer has been investigated extensively and altered lipid metabolism is observed in many tumors, the exact role of SREBPs in the control of biosynthetic processes required for Akt-dependent cell growth and their contribution to tumorigenesis remains unclear. Results We first investigated the effects of loss of SREBP function in non-transformed cells. Combined ablation of SREBP1 and SREBP2 by siRNA-mediated gene silencing or chemical inhibition of SREBP activation induced endoplasmic reticulum (ER)-stress and engaged the unfolded protein response (UPR) pathway, specifically under lipoprotein-deplete conditions in human retinal pigment epithelial cells. Induction of ER-stress led to inhibition of protein synthesis through increased phosphorylation of eIF2α. This demonstrates for the first time the importance of SREBP in the coordination of lipid and protein biosynthesis, two processes that are essential for cell growth and proliferation. SREBP ablation caused major changes in lipid composition characterized by a loss of mono- and poly-unsaturated lipids and induced accumulation of reactive oxygen species (ROS) and apoptosis. Alterations in lipid composition and increased ROS levels, rather than overall changes to lipid synthesis rate, were required for ER-stress induction. Next, we analyzed the effect of SREBP ablation in a panel of cancer cell lines. Importantly, induction of apoptosis following SREBP depletion was restricted to lipoprotein-deplete conditions. U87 glioblastoma cells were highly susceptible to silencing of either SREBP isoform, and apoptosis induced by SREBP1 depletion in these cells was rescued by antioxidants or by restoring the levels of mono-unsaturated fatty acids. Moreover, silencing of SREBP1

  5. Global proteomic analysis of protein acetylation affecting metabolic regulation in Daphnia pulex.

    PubMed

    Kwon, Oh Kwang; Sim, Juhee; Kim, Sun Ju; Oh, Hye Ryeung; Nam, Doo Hyun; Lee, Sangkyu

    2016-02-01

    Daphnia (Daphnia pulex) is a small planktonic crustacean and a key constituent of aquatic ecosystems. It is generally used as a model organism to study environmental toxic problems. In the past decade, genomic and proteomic datasets of Daphnia have been developed. The proteomic dataset allows for the investigation of toxicological effects in the context of "Daphnia proteomics," resulting in greater insights for toxicological research. To exploit Daphnia for ecotoxicological research, information on the post-translational modification (PTM) of proteins is necessary, as this is a critical regulator of biological processes. Acetylation of lysine (Kac) is a reversible and highly regulated PTM that is associated with diverse biological functions. However, a comprehensive description of Kac in Daphnia is not yet available. To understand the cellular distribution of lysine acetylation in Daphnia, we identified 98 acetylation sites in 65 proteins by immunoprecipitation using an anti-acetyllysine antibody and a liquid chromatography system supported by mass spectroscopy. We identified 28 acetylated sites related to metabolic proteins and six acetylated enzymes associated with the TCA cycle in Daphnia. From GO and KEGG enrichment analyses, we showed that Kac in D. pulex is highly enriched in proteins associated with metabolic processes. Our data provide the first global analysis of Kac in D. pulex and is an important resource for the functional analysis of Kac in this organism. PMID:26700148

  6. Loss of N-terminal Acetylation Suppresses A Prion Phenotype By Modulating Global Protein Folding

    PubMed Central

    Holmes, William M.; Mannakee, Brian K.; Gutenkunst, Ryan N.; Serio, Tricia R.

    2014-01-01

    N-terminal acetylation is among the most ubiquitous of protein modifications in eukaryotes. While loss of N-terminal acetylation is associated with many abnormalities, the molecular basis of these effects is known for only a few cases, where acetylation of single factors has been linked to binding avidity or metabolic stability. In contrast, the impact of N-terminal acetylation for the majority of the proteome, and its combinatorial contributions to phenotypes, are unknown. Here, by studying the yeast prion [PSI+], an amyloid of the Sup35 protein, we show that loss of N-terminal acetylation promotes general protein misfolding, a redeployment of chaperones to these substrates, and a corresponding stress response. These proteostasis changes, combined with the decreased stability of unacetylated Sup35 amyloid, reduce the size of prion aggregates and reverse their phenotypic consequences. Thus, loss of N-terminal acetylation, and its previously unanticipated role in protein biogenesis, globally resculpts the proteome to create a unique phenotype. PMID:25023910

  7. Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery

    PubMed Central

    Freibaum, Brian D.; Chitta, Raghu; High, Anthony A.; Taylor, J. Paul

    2010-01-01

    TDP-43 is a highly conserved and ubiquitously expressed member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins. Recently, TDP-43 was shown to be a major disease protein in the ubiquitinated inclusions characteristic of most cases of amyotrophic lateral sclerosis (ALS), tau-negative frontotemporal lobar degeneration (FTLD), and inclusion body myopathy. In these diseases, TDP-43 is redistributed from its predominantly nuclear location to ubiquitin-positive, cytoplasmic foci. The extent to which TDP-43 drives pathophysiology is unknown, but the identification of mutations in TDP-43 in familial forms of ALS and FTLD-U suggests an important role for this protein in pathogenesis. Little is known about TDP-43 function and only a few TDP-43 interacting proteins have been previously identified, which makes further insight into both the normal and pathological functions of TDP-43 difficult. Here we show, via a global proteomic approach, that TDP-43 has extensive interaction with proteins that regulate RNA metabolism. Some interactions with TDP-43 were found to be dependent on RNA-binding, whereas other interactions are RNA-independent. Disease-causing mutations in TDP-43 (A315T and M337V) do not alter its interaction profile. TDP-43 interacting proteins largely cluster into two distinct interaction networks, a nuclear/splicing cluster and a cytoplasmic/translation cluster, strongly suggesting that TDP-43 has multiple roles in RNA metabolism and functions in both the nucleus and the cytoplasm. Finally, we found numerous TDP-43 interactors that are known components of stress granules and, indeed, we find that TDP-43 is also recruited to stress granules. PMID:20020773

  8. Proteomics analyses for the global proteins in the brain tissues of different human prion diseases.

    PubMed

    Shi, Qi; Chen, Li-Na; Zhang, Bao-Yun; Xiao, Kang; Zhou, Wei; Chen, Cao; Zhang, Xiao-Mei; Tian, Chan; Gao, Chen; Wang, Jing; Han, Jun; Dong, Xiao-Ping

    2015-04-01

    Proteomics changes of brain tissues have been described in different neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. However, the brain proteomics of human prion disease remains less understood. In the study, the proteomics patterns of cortex and cerebellum of brain tissues of sporadic Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD were analyzed with isobaric tags for relative and absolute quantitation combined with multidimensional liquid chromatography and MS analysis, with the brains from three normal individuals as controls. Global protein profiling, significant pathway, and functional categories were analyzed. In total, 2287 proteins were identified with quantitative information both in cortex and cerebellum regions. Cerebellum tissues appeared to contain more up- and down-regulated proteins (727 proteins) than cortex regions (312 proteins) of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD. Viral myocarditis, Parkinson's disease, Alzheimer's disease, lysosome, oxidative phosphorylation, protein export, and drug metabolism-cytochrome P450 were the most commonly affected pathways of the three kinds of diseases. Almost coincident biological functions were identified in the brain tissues of the three diseases. In all, data here demonstrate that the brain tissues of Creutzfeldt-Jakob disease, fatal familial insomnia, and G114V genetic CJD have obvious proteomics changes at their terminal stages, which show the similarities not only among human prion diseases but also with other neurodegeneration diseases. This is the first study to provide a reference proteome map for human prion diseases and will be helpful for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases. PMID:25616867