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Sample records for acetylation targets protein

  1. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian; Nielsen, Michael L; Rehman, Michael; Walther, Tobias C; Olsen, Jesper V; Mann, Matthias

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600...... lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling......, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other...

  2. Targeted amino-terminal acetylation of recombinant proteins in E. coli.

    Matthew Johnson

    Full Text Available One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co-expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.

  3. N-terminal acetylation inhibits protein targeting to the endoplasmic reticulum.

    Gabriella M A Forte

    2011-05-01

    Full Text Available Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%-80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species.

  4. Protein Acetylation in Archaea, Bacteria, and Eukaryotes

    Jörg Soppa

    2010-01-01

    Full Text Available Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which—Alba—was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.

  5. Regulation of intermediary metabolism by protein acetylation

    Guan, Kun-Liang; Xiong, Yue

    2010-01-01

    Extensive studies during the past four decades have identified important roles for lysine acetylation in the regulation of nuclear transcription. Recent proteomic analyses on protein acetylation uncovered a large number of acetylated proteins in the cytoplasm and mitochondria, including most enzymes involved in intermediate metabolism. Acetylation regulates metabolic enzymes by multiple mechanisms, including via enzymatic activation or inhibition, and by influencing protein stability. Convers...

  6. Dynamic Protein Acetylation in Plant–Pathogen Interactions

    Song, Gaoyuan; Walley, Justin W.

    2016-01-01

    Pathogen infection triggers complex molecular perturbations within host cells that results in either resistance or susceptibility. Protein acetylation is an emerging biochemical modification that appears to play central roles during host–pathogen interactions. To date, research in this area has focused on two main themes linking protein acetylation to plant immune signaling. Firstly, it has been established that proper gene expression during defense responses requires modulation of histone acetylation within target gene promoter regions. Second, some pathogens can deliver effector molecules that encode acetyltransferases directly within the host cell to modify acetylation of specific host proteins. Collectively these findings suggest that the acetylation level for a range of host proteins may be modulated to alter the outcome of pathogen infection. This review will focus on summarizing our current understanding of the roles of protein acetylation in plant defense and highlight the utility of proteomics approaches to uncover the complete repertoire of acetylation changes triggered by pathogen infection. PMID:27066055

  7. Search for novel targets of the PII signal transduction protein in Bacteria identifies the BCCP component of acetyl-CoA carboxylase as a PII binding partner.

    Rodrigues, Thiago E; Gerhardt, Edileusa C M; Oliveira, Marco A; Chubatsu, Leda S; Pedrosa, Fabio O; Souza, Emanuel M; Souza, Gustavo A; Müller-Santos, Marcelo; Huergo, Luciano F

    2014-02-01

    The PII family comprises a group of widely distributed signal transduction proteins. The archetypal function of PII is to regulate nitrogen metabolism in bacteria. As PII can sense a range of metabolic signals, it has been suggested that the number of metabolic pathways regulated by PII may be much greater than described in the literature. In order to provide experimental evidence for this hypothesis a PII protein affinity column was used to identify PII targets in Azospirillum brasilense. One of the PII partners identified was the biotin carboxyl carrier protein (BCCP), a component of the acetyl-CoA carboxylase which catalyses the committed step in fatty acid biosynthesis. As BCCP had been previously identified as a PII target in Arabidopsis thaliana we hypothesized that the PII -BCCP interaction would be conserved throughout Bacteria. In vitro experiments using purified proteins confirmed that the PII -BCCP interaction is conserved in Escherichia coli. The BCCP-PII interaction required MgATP and was dissociated by increasing 2-oxoglutarate. The interaction was modestly affected by the post-translational uridylylation status of PII ; however, it was completely dependent on the post-translational biotinylation of BCCP. PMID:24329683

  8. Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

    Ouidir, Tassadit; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2015-07-01

    Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology. PMID:25900529

  9. Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells

    Reversible lysine acetylation is a highly regulated post-translational protein modification that is known to regulate several signaling pathways. However, little is known about the radiation-induced changes in the acetylome. In this study, we analyzed the acute post-translational acetylation changes in primary human cardiac microvascular endothelial cells 4 h after a gamma radiation dose of 2 Gy. The acetylated peptides were enriched using anti-acetyl conjugated agarose beads. A total of 54 proteins were found to be altered in their acetylation status, 23 of which were deacetylated and 31 acetylated. Pathway analyses showed three protein categories particularly affected by radiation-induced changes in the acetylation status: the proteins involved in the translation process, the proteins of stress response, and mitochondrial proteins. The activation of the canonical and non-canonical Wnt signaling pathways affecting actin cytoskeleton signaling and cell cycle progression was predicted. The protein expression levels of two nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 1 and sirtuin 3, were significantly but transiently upregulated 4 but not 24 h after irradiation. The status of the p53 protein, a target of sirtuin 1, was found to be rapidly stabilized by acetylation after radiation exposure. These findings indicate that post-translational modification of proteins by acetylation and deacetylation is essentially affecting the radiation response of the endothelium. (author)

  10. Emerging Functions for N-Terminal Protein Acetylation in Plants

    Gibbs, Daniel J.

    2015-01-01

    N-terminal (Nt-) acetylation is a widespread but poorly understood co-translational protein modification. Two reports now shed light onto the proteome-wide dynamics and protein-specific consequences of Nt-acetylation in relation to plant development, stress-response, and protein stability, identifying this modification as a key regulator of diverse aspects of plant growth and behaviour.

  11. Protein lysine acetylation in bacteria: Current state of the art.

    Ouidir, Tassadit; Kentache, Takfarinas; Hardouin, Julie

    2016-01-01

    Post-translational modifications of proteins are key events in cellular metabolism and physiology regulation. Lysine acetylation is one of the best studied protein modifications in eukaryotes, but, until recently, ignored in bacteria. However, proteomic advances have highlighted the diversity of bacterial lysine-acetylated proteins. The current data support the implication of lysine acetylation in various metabolic pathways, adaptation and virulence. In this review, we present a broad overview of the current knowledge of lysine acetylation in bacteria. We emphasize particularly the significant contribution of proteomics in this field. PMID:26390373

  12. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle. PMID:26920270

  13. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins

    Michael N. Davies

    2016-01-01

    Full Text Available Lysine acetylation (AcK, a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT, an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.

  14. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins.

    Davies, Michael N; Kjalarsdottir, Lilja; Thompson, J Will; Dubois, Laura G; Stevens, Robert D; Ilkayeva, Olga R; Brosnan, M Julia; Rolph, Timothy P; Grimsrud, Paul A; Muoio, Deborah M

    2016-01-12

    Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK. PMID:26748706

  15. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha;

    2013-01-01

    The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double...... quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco) mannan, and xyloglucan as well as overall cell wall acetylation is affected differently...... in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell...

  16. Protein Acetylation Is Involved in Salmonella enterica Serovar Typhimurium Virulence.

    Sang, Yu; Ren, Jie; Ni, Jinjing; Tao, Jing; Lu, Jie; Yao, Yu-Feng

    2016-06-01

    Salmonella causes a range of diseases in different hosts, including enterocolitis and systemic infection. Lysine acetylation regulates many eukaryotic cellular processes, but its function in bacteria is largely unexplored. The acetyltransferase Pat and NAD(+)-dependent deacetylase CobB are involved in the reversible protein acetylation in Salmonella Typhimurium. Here, we used cell and animal models to evaluate the virulence of pat and cobB deletion mutants in S. Typhimurium and found that pat is critical for bacterial intestinal colonization and systemic infection. Next, to understand the underlying mechanism, genome-wide transcriptome was analyzed. RNA sequencing data showed that the expression of Salmonella pathogenicity island 1 (SPI-1) is partially dependent on pat In addition, we found that HilD, a key transcriptional regulator of SPI-1, is a substrate of Pat. The acetylation of HilD by Pat maintained HilD stability and was essential for the transcriptional activation of HilA. Taken together, these results suggest that a protein acetylation system regulates SPI-1 expression by controlling HilD in a posttranslational manner to mediate S. Typhimurium virulence. PMID:26810370

  17. New lysine-acetylated proteins screened by immunoaffinity and liquid chromatography-mass spectrometry

    2010-01-01

    The lack of selective extraction specific for lysine-acetylated proteins has been a major problem in the field of acetylation biology,though acetylation plays a key role in many biological processes.In this paper,we report for the first time the proteomic screening of lysine-acetylated proteins from a mouse liver tissue,by a new approach of immunoaffinity purification of lysine-acetylated peptides combined with nano-HPLC/MS/MS analysis.We have found 20 lysine-acetylated proteins with 21 lysine-acetylated sites,among which 12 lysine-acetylated proteins and 16 lysine-acetylated sites have never been reported before.Notably,three acetyltransferases harboring in mitochondrion are newly discovered acetyltransferases responsible for the acetylation of nonhistone proteins.We have explored the significant patterns of residue preference by the hierarchical clustering analysis of amino acid residues surrounding acetylation sites,which could be helpful to the prediction of new sites of lysine acetylation.Our findings provide more candidates for studying the important roles played by acetylation in diverse cellular pathways and related human diseases.

  18. p53 targets simian virus 40 large T antigen for acetylation by CBP.

    Poulin, Danielle L; Kung, Andrew L; DeCaprio, James A

    2004-08-01

    Simian virus 40 (SV40) large T antigen (T Ag) interacts with the tumor suppressor p53 and the transcriptional coactivators CBP and p300. Binding of these cellular proteins in a ternary complex has been implicated in T Ag-mediated transformation. It has been suggested that the ability of CBP/p300 to modulate p53 function underlies p53's regulation of cell proliferation and tumorigenesis. In this study, we provide further evidence that CBP activity may be mediated through its synergistic action with p53. We demonstrate that SV40 T Ag is acetylated in vivo in a p53-dependent manner and T Ag acetylation is largely mediated by CBP. The acetylation of T Ag is dependent on its interaction with p53 and on p53's interaction with CBP. We have mapped the site of acetylation on T Ag to the C-terminal lysine residue 697. This acetylation site is conserved between the T antigens of the human polyomaviruses JC and BK, which are also known to interact with p53. We show that both JC and BK T antigens are also acetylated at corresponding sites in vivo. While other proteins are known to be acetylated by CBP/p300, none are known to depend on p53 for acetylation. T Ag acetylation may provide a regulatory mechanism for T Ag binding to a cellular factor or play a role in another aspect of T Ag function. PMID:15254196

  19. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    Crosby, Heidi A [University of Wisconsin, Madison; Pelletier, Dale A [ORNL; Hurst, Gregory {Greg} B [ORNL; Escalante-Semerena, Jorge C [University of Wisconsin, Madison

    2012-01-01

    Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

  20. Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response

    Bennetzen, Martin; Andersen, J.S.; Lasen, D.H.;

    2013-01-01

    -dependent posttranslational modifications (PT Ms). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry......-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP 300 and CREBBP, are dynamically acetylated; (2) that nuclear...... to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure...

  1. The chromosomal protein HMGBI inhibits DNA replication in vitro. The role of post-synthetic acetylation

    The effect of HMGB1 protein on the replication of closed circular plasmid DNA in cell free extract have been studied using parental form of the protein, post-synthetically acetylated HMGB1 and HMGB1 lacking its acidic C-terminal tail. We have shown that HMGB1 protein inhibits DNA replication and that this effect is eliminated upon either acetylation of the protein or removal of the acidic C-terminal domain. An explanation of these findings suggests interactions of HMGB1 with a protein(s) of the replication complex resulting in reduction of its functional efficiency. Acetylation of HMGB1 affects these interactions in a way that restores the initial replication capacity of the system. The eventual protein-protein interactions are supposed to proceed via the C-terminal domain of HMGB1. (authors)

  2. In vitro phosphorylation and acetylation of the murine pocket protein Rb2/p130.

    Muhammad Saeed

    Full Text Available The retinoblastoma protein (pRb and the related proteins Rb2/p130 and 107 represent the "pocket protein" family of cell cycle regulators. A key function of these proteins is the cell cycle dependent modulation of E2F-regulated genes. The biological activity of these proteins is controlled by acetylation and phosphorylation in a cell cycle dependent manner. In this study we attempted to investigate the interdependence of acetylation and phosphorylation of Rb2/p130 in vitro. After having identified the acetyltransferase p300 among several acetyltransferases to be associated with Rb2/p130 during S-phase in NIH3T3 cells in vivo, we used this enzyme and the CDK4 protein kinase for in vitro modification of a variety of full length Rb2/p130 and truncated versions with mutations in the acetylatable lysine residues 1079, 128 and 130. Mutation of these residues results in the complete loss of Rb2/p130 acetylation. Replacement of lysines by arginines strongly inhibits phosphorylation of Rb2/p130 by CDK4; the inhibitory effect of replacement by glutamines is less pronounced. Preacetylation of Rb2/p130 strongly enhances CDK4-catalyzed phosphorylation, whereas deacetylation completely abolishes in vitro phosphorylation. In contrast, phosphorylation completely inhibits acetylation of Rb2/p130 by p300. These results suggest a mutual interdependence of modifications in a way that acetylation primes Rb2/p130 for phosphorylation and only dephosphorylated Rb2/p130 can be subject to acetylation. Human papillomavirus 16-E7 protein, which increases acetylation of Rb2/p130 by p300 strongly reduces phosphorylation of this protein by CDK4. This suggests that the balance between phosphorylation and acetylation of Rb2/p130 is essential for its biological function in cell cycle control.

  3. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions.

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas; Zechner, Rudolf; Choudhary, Chunaram

    2015-11-01

    Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation sites, and greater sensitivity of SIRT3-targeted sites to chemical acetylation in vitro and fasting-induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indicate that most mitochondrial acetylation occurs as a low-level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues. PMID:26358839

  4. What Are the Potential Sites of Protein Arylation by N-Acetyl-p-benzoquinone Imine (NAPQI)?

    Leeming, Michael G; Gamon, Luke F; Wille, Uta; Donald, William A; O'Hair, Richard A J

    2015-11-16

    Acetaminophen (paracetamol, APAP) is a safe and widely used analgesic medication when taken at therapeutic doses. However, APAP can cause potentially fatal hepatotoxicity when taken in overdose or in patients with metabolic irregularities. The production of the electrophilic and putatively toxic compound N-acetyl-p-benzoquinone imine (NAPQI), which cannot be efficiently detoxicated at high doses, is implicated in APAP toxicity. Numerous studies have identified that excess NAPQI can form covalent linkages to the thiol side chains of cysteine residues in proteins; however, the reactivity of NAPQI toward other amino acid side chains is largely unexplored. Here, we report a survey of the reactivity of NAPQI toward 11 N-acetyl amino acid methyl esters and four peptides. (1)H NMR analysis reveals that NAPQI forms covalent bonds to the side-chain functional groups of cysteine, methionine, tyrosine, and tryptophan residues. Analogous reaction products were observed when NAPQI was reacted with synthetic model peptides GAIL-X-GAILR for X = Cys, Met, Tyr, and Trp. Tandem mass spectrometry peptide sequencing showed that the NAPQI modification sites are located on the "X" residue in each case. However, when APAP and the GAIL-X-GAILR peptide were incubated with rat liver microsomes that contain many metabolic enzymes, NAPQI formed by oxidative metabolism reacted with GAIL-C-GAILR exclusively. For the peptides where X = Met, Tyr, and Trp, competing reactions between NAPQI and alternative nucleophiles precluded arylation of the target peptide by NAPQI. Although Cys residues are favorably targeted under these conditions, these data suggest that NAPQI can, in principle, also damage proteins at Met, Tyr, and Trp residues. PMID:26523953

  5. Physical and Functional HAT/HDAC Interplay Regulates Protein Acetylation Balance

    Alessia Peserico; Cristiano Simone

    2011-01-01

    The balance between protein acetylation and deacetylation controls several physiological and pathological cellular processes, and the enzymes involved in the maintenance of this equilibrium—acetyltransferases (HATs) and deacetylases (HDACs)—have been widely studied. Presently, the evidences obtained in this field suggest that the dynamic acetylation equilibrium is mostly maintained through the physical and functional interplay between HAT and HDAC activities. This model overcomes the classica...

  6. Proteomic analysis reveals differentially regulated protein acetylation in human amyotrophic lateral sclerosis spinal cord.

    Dong Liu

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord. Histone deacetylase (HDAC inhibitors have neuroprotective effects potentially useful for the treatment of neurodegenerative diseases including ALS; however, the molecular mechanisms underlying their potential efficacy is not well understood. Here we report that protein acetylation in urea-soluble proteins is differently regulated in post-mortem ALS spinal cord. Two-dimensional electrophoresis (2-DE analysis reveals several protein clusters with similar molecular weight but different charge status. Liquid chromatography and tandem mass spectrometry (LC-MS/MS identifies glial fibrillary acidic protein (GFAP as the dominant component in the protein clusters. Further analysis indicates six heavily acetylated lysine residues at positions 89, 153, 189, 218, 259 and 331 of GFAP. Immunoprecipitation followed by Western blotting confirms that the larger form of GFAP fragments are acetylated and upregulated in ALS spinal cord. Further studies demonstrate that acetylation of the proteins additional to GFAP is differently regulated, suggesting that acetylation and/or deacetylation play an important role in pathogenesis of ALS.

  7. Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.

    Ferah Yildirim

    Full Text Available Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT and deacetylase activities (HDAC. Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB-binding protein (CBP as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min subthreshold occlusion of the middle cerebral artery (MCA, followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.

  8. PSG gene expression is up-regulated by lysine acetylation involving histone and nonhistone proteins.

    Soledad A Camolotto

    Full Text Available BACKGROUND: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140, was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA. This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. CONCLUSIONS/SIGNIFICANCE: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.

  9. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas;

    2015-01-01

    Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or...... suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of...... fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation...

  10. Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes

    Pasini, Diego; Malatesta, Martina; Jung, Hye Ryung; Valfridsson, Julian Osmond A; Willer, Anton; Olsson, Linda; Skotte, Julie; Wutz, Anton; Porse, Bo; Jensen, Ole Nørregaard; Helin, Kristian

    2010-01-01

    Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine...... 27 (K27), and it is believed that this activity mediates transcriptional repression. Despite the recent progress in understanding PcG function, the molecular mechanisms by which the PcG proteins repress transcription, as well as the mechanisms that lead to the activation of PcG target genes are....... The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and...

  11. The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis

    Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral nucleocapsid proteins named small and large form hepatitis delta antigen (S-HDAg and L-HDAg). The S-HDAg is essential for viral RNA replication while the L-HDAg is required for viral assembly. In this study, we demonstrated that HDAg are acetylated proteins. Metabolic labeling with [3H]acetate revealed that both forms of HDAg could be acetylated in vivo. The histone acetyltransferase (HAT) domain of cellular acetyltransferase p300 could acetylate the full-length and the N-terminal 88 amino acids of S-HDAg in vitro. By mass spectrometric analysis of the modified protein, Lys-72 of S-HDAg was identified as one of the acetylation sites. Substitution of Lys-72 to Arg caused the mutant S-HDAg to redistribute from the nucleus to the cytoplasm. The mutant reduced viral RNA accumulation and resulted in the earlier appearance of L-HDAg. These results demonstrated that HDAg is an acetylated protein and mutation of HDAg at Lys-72 modulates HDAg subcellular localization and may participate in viral RNA nucleocytoplasmic shuttling and replication

  12. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    Biswas, Arunima; Pasquel, Danielle [Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Tyagi, Rakesh Kumar [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India); Mani, Sridhar, E-mail: sridhar.mani@einstein.yu.edu [Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461 (United States)

    2011-03-18

    Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  13. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    Research highlights: → Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. → PXR undergoes dynamic deacetylation upon ligand-mediated activation. → SIRT1 partially mediates PXR deacetylation. → PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  14. Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

    Chang-Cai Liu

    Full Text Available BACKGROUND: The N-terminal protein processing mechanism (NPM including N-terminal Met excision (NME and N-terminal acetylation (N(α-acetylation represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To reveal the NPM in poplar, we investigated the N(α-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α-acetylated proteins. Most proteins (47, >81% are subjected to N(α-acetylation following the N-terminal removal of Met, indicating that N(α-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α-acetylation (NPM to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs and N-terminal acetyltransferase (Nat enzymes in poplar. The N(α-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. CONCLUSIONS/SIGNIFICANCE: This study represents the first extensive investigation of N(α-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α-acetylation of proteins in poplar.

  15. Chromatin-regulating proteins as targets for cancer therapy

    Oike, Takahiro; Ogiwara, Hideaki; Amornwichet, Napapat; Nakano, Takashi; Kohno, Takashi

    2014-01-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium...

  16. Carbohydrate-linked asparagine-101 of prothrombin contains a metal ion protected acetylation site. Acetylation of this site causes loss of metal ion induced protein fluorescence change

    Prothrombin fragment 1 (prothrombin residues 1-156) contains two acetylation sites that are protected from derivatization by calcium. The first site was protected by only calcium while the second site was protected by magnesium as well. To identify this second acetylation site, fragment 1 was first acetylated with unlabeled reagent in the presence of magnesium. Metal ions were removed, and the protein was acetylated with radiolabeled reagent. The incorporated radiolabel was stable over long periods of time and at acidic or basic pH as long as elevated temperatures were avoided. The radiolabel was removed by treatment of the protein at pH 10 and 50 0C or with 0.2 M hydroxylamine at 50 0C for at least 30 min. Proteolytic degradation of the protein showed that the radioactivity appeared in a tryptic peptide corresponding to residues 94-111 of prothrombin. Amino acid sequence analysis revealed that the radiolabel was associated with an unextracted sequence product. The major radiolabeled product contained Asn101-Ser102 along with the expected chitobiose attached to Asn-101. NMR analysis revealed the presence of three acetate groups which would correspond to two from the chitobiose plus the incorporated acetate residue. Mass spectral analysis showed the correct mass for this glycopeptide plus a single added acetyl group. Amide 1H NMR analysis showed only three amide protons rather than the anticipated four. On the basis of these several observations, it is postulated that the site of acetylation is the β-amide nitrogen of Asn-101. Consequently, these studies showed an unusual chemical reactivity in prothrombin fragment 1. They further show that metal ion binding to prothrombin fragment 1 and subsequent protein fluorescence quenching involve sites ion the kringle region of the protein

  17. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Neto Paiva, Claudia; Torres Bozza, Marcelo [Departamento de Imunologia, Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Rosado Fantappie, Marcelo, E-mail: fantappie@bioqmed.ufrj.br [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil)

    2009-12-25

    Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1{Delta}C) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1{Delta}C were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

  18. Ras protein participated in histone acetylation-mediated cell cycle control in Physarum polycephalum

    LI Xiaoxue; LU Jun; ZHAO Yanmei; WANG Xiuli; HUANG Baiqu

    2005-01-01

    In this paper, we demonstrate that in Physarum polycephalum, a naturally synchronized slime mold, histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), arrestes the cell cycle at the checkpoints of S/G2, G2/M and mitosis exit, and influences the transcription of two ras genes Ppras1 and Pprap1, as well as the Ras protein level. Antibody neutralization experiment using anti-Ras antibody treatment showed that Ras protein played an important role in cell cycle checkpoint control through regulation of the level of Cyclin B1, suggesting that Ras protein might be a key factor for histone acetylation-mediated cell cycle regulation in P. polycephalum.

  19. PSG Gene Expression Is Up-Regulated by Lysine Acetylation Involving Histone and Nonhistone Proteins

    Camolotto, Soledad A.; Racca, Ana C.; Ridano, Magali E.; Genti-Raimondi, Susana; Panzetta-Dutari, Graciela M.

    2013-01-01

    Background Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG) are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the ...

  20. Heat-shock protein 90 promotes nuclear transport of herpes simplex virus 1 capsid protein by interacting with acetylated tubulin.

    Zhong, Meigong; Zheng, Kai; Chen, Maoyun; Xiang, Yangfei; Jin, Fujun; Ma, Kaiqi; Qiu, Xianxiu; Wang, Qiaoli; Peng, Tao; Kitazato, Kaio; Wang, Yifei

    2014-01-01

    Although it is known that inhibitors of heat shock protein 90 (Hsp90) can inhibit herpes simplex virus type 1 (HSV-1) infection, the role of Hsp90 in HSV-1 entry and the antiviral mechanisms of Hsp90 inhibitors remain unclear. In this study, we found that Hsp90 inhibitors have potent antiviral activity against standard or drug-resistant HSV-1 strains and viral gene and protein synthesis are inhibited in an early phase. More detailed studies demonstrated that Hsp90 is upregulated by virus entry and it interacts with virus. Hsp90 knockdown by siRNA or treatment with Hsp90 inhibitors significantly inhibited the nuclear transport of viral capsid protein (ICP5) at the early stage of HSV-1 infection. In contrast, overexpression of Hsp90 restored the nuclear transport that was prevented by the Hsp90 inhibitors, suggesting that Hsp90 is required for nuclear transport of viral capsid protein. Furthermore, HSV-1 infection enhanced acetylation of α-tubulin and Hsp90 interacted with the acetylated α-tubulin, which is suppressed by Hsp90 inhibition. These results demonstrate that Hsp90, by interacting with acetylated α-tubulin, plays a crucial role in viral capsid protein nuclear transport and may provide novel insight into the role of Hsp90 in HSV-1 infection and offer a promising strategy to overcome drug-resistance. PMID:24901434

  1. Targeted Quantitation of Acetylated Lysine Peptides by Selected Reaction Monitoring Mass Spectrometry

    Rardin, Matthew J.; Held, Jason M.; Gibson, Bradford W.

    2013-01-01

    Mass spectrometry (MS) allows for the large-scale identification of multiple peptide analytes in complex mixtures. However, the low abundance of acetylated peptides in the overall mixture requires an enrichment step. After enrichment, the resulting acetylated peptides of interest can be quantitated using selected reaction monitoring (SRM)-MS with stable isotope dilution. Here, we describe the enrichment of lysine acetylated peptides from typsin digested mouse liver mitochondria, and the targe...

  2. Thanatos-associated protein 7 associates with template activating factor-Ibeta and inhibits histone acetylation to repress transcription.

    Macfarlan, Todd; Parker, J Brandon; Nagata, Kyosuke; Chakravarti, Debabrata

    2006-02-01

    The posttranslational modifications of histones on chromatin or a lack thereof is critical in transcriptional regulation. Emerging studies indicate a role for histone-binding proteins in transcriptional activation and repression. We have previously identified template-activating factor-Ibeta (TAF-Ibeta, also called PHAPII, SET, and I(2)(pp2A)) as a component of a cellular complex called inhibitor of acetyltransferases (INHAT) that masks histone acetylation in vitro and blocks histone acetyltransferase (HAT)-dependent transcription in living cells. TAF-Ibeta has also been shown to associate with transcription factors, including nuclear receptors, to regulate their activities. To identify novel interactors of TAF-Ibeta, we employed a yeast two-hybrid screen and identified a previously uncharacterized human protein called thanatos-associated protein-7 (THAP7), a member of a large family of THAP domain-containing putative DNA-binding proteins. In this study we demonstrate that THAP7 associates with TAF-Ibeta in vitro and map their association domains to a C-terminal predicted coiled-coil motif on THAP7 and the central region of TAF-Ibeta. Similarly, stably transfected THAP7 associates with endogenous TAF-Ibeta in intact cells. Like TAF-Ibeta, THAP7 associates with histone H3 and histone H4 and inhibits histone acetylation. The histone-interacting domain of THAP7 is sufficient for this activity in vitro. Promoter-targeted THAP7 can also recruit TAF-Ibeta and silencing mediator of retinoid and thyroid receptors/nuclear hormone receptor corepressor (NCoR) proteins to promoters, and knockdown of TAF-Ibeta by small interfering RNA relieves THAP7-mediated repression, indicating that, like nuclear hormone receptors, THAP7 may represent a novel class of transcription factor that uses TAF-Ibeta as a corepressor to maintain histones in a hypoacetylated, repressed state. PMID:16195249

  3. Development of the intein-mediated method for production of recombinant thymosin β4 from the acetylated in vivo fusion protein.

    Esipov, Roman S; Makarov, Dmitry A; Stepanenko, Vasily N; Miroshnikov, Anatoly I

    2016-06-20

    Thymosin β4 is a 43 amino acid long peptide with an acetylated N-terminal serin that has a high potential as a remedy for healing ulcers, wounds and burns. Although protein biosynthesis offers attractive opportunities in terms of a large-scale production, currently thymosin β4 is mainly produced by chemical synthesis. The problems that hinder the successful commercialization of the biotechnological approach are associated with the small peptides expression and N-terminal acetylation. This work presents an innovative biotechnological method for thymosin β4 production that employs the peptide acetylation in vivo. A genetically engineered construct was created, where the Tβ4 coding sequence fused with the intein Mxe GyrA sequence and chitin-binding domain was combined with the acetyltransferase coding sequence to form a polycistronic construct under a stringent control of T7 promoter. This plasmid construct provided for the expression of the Tβ4-intein fusion protein. In the process of the post-translational modification in vivo formyl methionine was completely removed from the target peptide N-terminus and followed by the Tβ4 precursor N-terminal acetylation. The use of the intein-mediated expression system made it possible to extract thymosin β4 in only 2 chromatographic runs. The method is straightforward to implement and scale up. PMID:27015974

  4. Sensitivity of human lung adenocarcinoma cell lines to targeted inhibition of BET epigenetic signaling proteins

    Lockwood, William W; Zejnullahu, Kreshnik; James E Bradner; Varmus, Harold

    2012-01-01

    Bromodomain and extra terminal domain (BET) proteins function as epigenetic signaling factors that associate with acetylated histones and facilitate transcription of target genes. Inhibitors targeting the activity of BET proteins have shown potent antiproliferative effects in hematological cancers through the suppression of c-MYC and downstream target genes. However, as the epigenetic landscape of a cell varies drastically depending on lineage, transcriptional coactivators such as BETs would ...

  5. Acetylation of TUG protein promotes the accumulation of GLUT4 glucose transporters in an insulin-responsive intracellular compartment.

    Belman, Jonathan P; Bian, Rachel R; Habtemichael, Estifanos N; Li, Don T; Jurczak, Michael J; Alcázar-Román, Abel; McNally, Leah J; Shulman, Gerald I; Bogan, Jonathan S

    2015-02-13

    Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD(+)-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake. PMID:25561724

  6. Acetylation of retinal histones in diabetes increases inflammatory proteins: effects of minocycline and manipulation of histone acetyltransferase (HAT) and histone deacetylase (HDAC).

    Kadiyala, Chandra Sekhar Rao; Zheng, Ling; Du, Yunpeng; Yohannes, Elizabeth; Kao, Hung-Ying; Miyagi, Masaru; Kern, Timothy S

    2012-07-27

    Histone acetylation was significantly increased in retinas from diabetic rats, and this acetylation was inhibited in diabetics treated with minocycline, a drug known to inhibit early diabetic retinopathy in animals. Histone acetylation and expression of inflammatory proteins that have been implicated in the pathogenesis of diabetic retinopathy were increased likewise in cultured retinal Müller glia grown in a diabetes-like concentration of glucose. Both the acetylation and induction of the inflammatory proteins in elevated glucose levels were significantly inhibited by inhibitors of histone acetyltransferase (garcinol and antisense against the histone acetylase, p300) or activators of histone deacetylase (theophylline and resveratrol) and were increased by the histone deacetylase inhibitor, suberolylanilide hydroxamic acid. We conclude that hyperglycemia causes acetylation of retinal histones (and probably other proteins) and that the acetylation contributes to the hyperglycemia-induced up-regulation of proinflammatory proteins and thereby to the development of diabetic retinopathy. PMID:22648458

  7. Bromodomain and extra-terminal (BET) family proteins: New therapeutic targets in major diseases

    Balasundaram Padmanabhan; Shruti Mathur; Manjula Ramu; Shailesh Tripathi

    2016-06-01

    The bromodomains and extra-terminal domain (BET) family proteins recognize acetylated chromatin through their bromodomains (BDs) and helps in regulating gene expression. BDs are chromatin ‘readers’; by interacting with acetylated lysines on the histone tails, they recruit chromatin-regulating proteins on the promoter region to regulate gene expression and repression. Extensive efforts have been employed by the scientific communities worldwide, to identify and develop potential inhibitors of BET family BDs to regulate protein expression by inhibiting acetylated histone (H3/H4) interactions. Several small molecule inhibitors have been reported, which not only have high affinity, but also have high specificity to BET BDs. These developments make BET family proteins to be an important therapeutic targets, for major diseases such as cancer, neurological disorders, obesity and inflammation. Here, we review and discuss the structural biology of BET family BDs and their applications in major diseases.

  8. Patterns of histone acetylation as targets for novel therapeutic approaches in neurological diseases

    Ebrahimi, Azadeh

    2013-01-01

    Neurological diseases, in particular brain tumors and neurodegenerative disorders, cause significant socio-economic burdens on societies. Exploring epigenetic mechanisms in neurological disorders in recent decades has been an emerging tool for describing the pathogenesis of neurological diseases as well as developing new therapeutics. Global histone acetylation is an epigenetic entity whose alternating patterns in various neurological diseases have recently raised special attention concer...

  9. Time-resolved luminescence biosensor for continuous activity detection of protein acetylation-related enzymes based on DNA-sensitized terbium(III) probes.

    Han, Yitao; Li, Hao; Hu, Yufang; Li, Pei; Wang, Huixia; Nie, Zhou; Yao, Shouzhuo

    2015-09-15

    Protein acetylation of histone is an essential post-translational modification (PTM) mechanism in epigenetic gene regulation, and its status is reversibly controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Herein, we have developed a sensitive and label-free time-resolved luminescence (TRL) biosensor for continuous detection of enzymatic activity of HATs and HDACs, respectively, based on acetylation-mediated peptide/DNA interaction and Tb(3+)/DNA luminescent probes. Using guanine (G)-rich DNA-sensitized Tb(3+) luminescence as the output signal, the polycationic substrate peptides interact with DNA with high affinity and subsequently replace Tb(3+), eliminating the luminescent signal. HAT-catalyzed acetylation remarkably reduces the positive charge of the peptides and diminishes the peptide/DNA interaction, resulting in the signal on detection via recovery of DNA-sensitized Tb(3+) luminescence. With this TRL sensor, HAT (p300) can be sensitively detected with a wide linear range from 0.2 to 100 nM and a low detection limit of 0.05 nM. The proposed sensor was further used to continuously monitor the HAT activity in real time. Additionally, the TRL biosensor was successfully applied to evaluating HAT inhibition by two specific inhibitors, anacardic acid and C464, and satisfactory Z'-factors above 0.73 were obtained. Moreover, this sensor is feasible to continuously monitor the HDAC (Sirt1)-catalyzed deacetylation with a linear range from 0.5 to 500 nM and a detection limit of 0.5 nM. The proposed sensor is a convenient, sensitive, and mix-and-read assay, presenting a promising platform for protein acetylation-targeted epigenetic research and drug discovery. PMID:26307596

  10. The biology of lysine acetylation integrates transcriptional programming and metabolism

    Mujtaba Shiraz

    2011-03-01

    Full Text Available Abstract The biochemical landscape of lysine acetylation has expanded from a small number of proteins in the nucleus to a multitude of proteins in the cytoplasm. Since the first report confirming acetylation of the tumor suppressor protein p53 by a lysine acetyltransferase (KAT, there has been a surge in the identification of new, non-histone targets of KATs. Added to the known substrates of KATs are metabolic enzymes, cytoskeletal proteins, molecular chaperones, ribosomal proteins and nuclear import factors. Emerging studies demonstrate that no fewer than 2000 proteins in any particular cell type may undergo lysine acetylation. As described in this review, our analyses of cellular acetylated proteins using DAVID 6.7 bioinformatics resources have facilitated organization of acetylated proteins into functional clusters integral to cell signaling, the stress response, proteolysis, apoptosis, metabolism, and neuronal development. In addition, these clusters also depict association of acetylated proteins with human diseases. These findings not only support lysine acetylation as a widespread cellular phenomenon, but also impel questions to clarify the underlying molecular and cellular mechanisms governing target selectivity by KATs. Present challenges are to understand the molecular basis for the overlapping roles of KAT-containing co-activators, to differentiate between global versus dynamic acetylation marks, and to elucidate the physiological roles of acetylated proteins in biochemical pathways. In addition to discussing the cellular 'acetylome', a focus of this work is to present the widespread and dynamic nature of lysine acetylation and highlight the nexus that exists between epigenetic-directed transcriptional regulation and metabolism.

  11. Comparative covalent protein binding of 2,5-hexanedione and 3-acetyl-2,5-hexanedione in the rat.

    DeCaprio, Anthony P; Kinney, Elizabeth A; LoPachin, Richard M

    2009-01-01

    2,5-Hexanedione (HD) is the metabolite implicated in n-hexane neurotoxicity. This gamma-diketone reacts with protein lysine amines to form 2,5-dimethylpyrrole adducts. Pyrrole adduction of neurofilaments (NF) and/or other axonal proteins was proposed as a critical step in the neuropathy. While pyrrole adduction is widely accepted as necessary, subsequent pyrrole oxidation, which may result in protein cross-linking, was alternatively postulated as the critical mechanistic step. Previous studies have indicated that 3-acetyl-2,5-HD (AcHD), an analogue that forms pyrroles that do not oxidize, was not neurotoxic in rats. However, relative levels of pyrrole adduction of NF or other axonal proteins were not reported. In the present study, groups of 6 male Wistar rats were given saline, [1,6-(14)C]-HD (3 mmol/kg/d), or [5-(14)C]-AcHD (0.1 mmol/kg/d), i.p. for 21 d. HD- and AcHD-treated rats lost 10% and gained 14% body weight, respectively, compared to a 22% gain for control rats. At termination, HD- and AcHD-treated rats exhibited mean scores of 3.5 and 1.4, respectively, for hindlimb weakness (0-5 scale). Incorporation of radiolabel from HD was 27.8 +/- 3.9, 13.9 +/- 2.6, and 7.8 +/- 0.6 nmol/mg in plasma protein, purified globin, and axonal cytoskeletal proteins, respectively, compared to 0.6 +/- 0.1, 1.6 +/- 0.5, and 1.0 +/- 0.1 for AcHD. Binding of HD to the NF-L, -M, and -H subunit proteins from treated animals was 4-, 24-, and 13-fold higher, respectively, that that of AcHD, indicating differing stoichiometry and patterns of NF adduction for the two diketones. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of globin and NF proteins did not demonstrate protein cross-linking for either diketone at the dose levels and time period examined. These results indicate that that the lack of neurotoxicity previously reported for AcHD may reflect differences in adduct levels at critical axonal target sites rather than an inability to form cross

  12. 3β-Acetyl Tormentic Acid (3ATA a Novel Modulator of ABCC Proteins Activity

    Cerli Rocha Gattass

    2012-06-01

    Full Text Available Multidrug resistance (MDR is considered the main cause of cancer chemotherapy failure and patient relapse. The active drug efflux mediated by transporter proteins of the ABC (ATP-binding cassette family is the most investigated mechanism leading to MDR. With the aim of inhibiting this transport and circumventing MDR, a great amount of work has been dedicated to identifying pharmacological inhibitors of specific ABC transporters. We recently showed that 3β-acetyl tormentic acid (3ATA had no effect on P-gp/ABCB1 activity. Herein, we show that 3ATA strongly inhibited the activity of MRP1/ABCC1. In the B16/F10 and Ma104 cell lines, this effect was either 20X higher or similar to that observed with MK571, respectively. Nevertheless, the low inhibitory effect of 3ATA on A549, a cell line that expresses MRP1-5, suggests that it may not inhibit other MRPs. The use of cells transfected with ABCC2, ABCC3 or ABCC4 showed that 3ATA was also able to modulate these transporters, though with an inhibition ratio lower than that observed for MRP1/ABCC1. These data point to 3ATA as a new ABCC inhibitor and call attention to its potential use as a tool to investigate the function of MRP/ABCC proteins or as a co-adjuvant in the treatment of MDR tumors.

  13. Hydrosoluble 50% N-acetylation-thiolated chitosan complex with cobalt as a pH-responsive renal fibrosis targeting drugs.

    Li, Min; Tan, Lishan; Tang, Liangfeng; Li, Aiqing; Hu, Jianqiang

    2016-07-01

    About 50% N-acetylation-thiolated chitosan possessing good water solubility was modified from commercial low-molecular-weight chitosan. Chitosan performed obvious target toward renal tubular epithelial cells, and bivalent cobalt ions improved the renal fibrosis inflammation significantly. There were many complexation sites on chitosan after being modified with sulfydryl. So sulfydryl played a role of connecting bridge between chitosan and cobalt ions. Then, this N-acetylation-thiolated chitosan cobalt (NTCC) nanocomplex was designed. The nanocomplex showed excellent stability under normal physiological conditions, and cobalt would be released from the biomaterials in acidic environment. As it was affected by inflammation, the pH in renal fibrosis lesion region was acidic. So there was a specific drug release process happening in lesion region. And drug release efficiency was determined by acidity, which demonstrated that lower the acidity, the faster and more the cobalt ion release. When this nanocomplex was intraperitoneally injected into ureter-obstructed mice, obvious attenuation of fibrotic progression was shown. It was demonstrated that NTCC exhibited special renal-targeting capacity and could be chosen as drug for treating renal fibrosis. PMID:27115330

  14. Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins

    Cheng Chung-Hsien

    2009-07-01

    Full Text Available Abstract Background Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase, were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D, and the other with five contiguous arginine residues (5R. Results Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac from N-acetyl-D-glucosamine (GlcNAc. Conclusion Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.

  15. Chromatin-regulating proteins as targets for cancer therapy

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. (author)

  16. Chromatin-regulating proteins as targets for cancer therapy.

    Oike, Takahiro; Ogiwara, Hideaki; Amornwichet, Napapat; Nakano, Takashi; Kohno, Takashi

    2014-07-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. PMID:24522270

  17. Acetylation Targets the M2 Isoform of Pyruvate Kinase for Degradation through Chaperone-Mediated Autophagy and Promotes Tumor Growth

    Lv, Lei; Li, Dong; Zhao, Di; Lin, Ruiting; Chu, Yajing; Zhang, Heng; Zha, Zhengyu; Liu, Ying; Li, Zi; Xu, Yanping; Wang, Gang; Huang, Yiran; Xiong, Yue; Guan, Kun-Liang; Lei, Qun-Ying

    2016-01-01

    SUMMARY Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA. PMID:21700219

  18. UDP-N-Acetyl glucosamine pyrophosphorylase as novel target for controlling Aedes aegypti – molecular modeling, docking and simulation studies

    Bhagath Kumar Palaka

    2014-12-01

    Full Text Available Aedes aegypti is a vector that transmits diseases like dengue fever, chikungunya, and yellow fever. It is distributed in all tropical and subtropical regions of the world. According to WHO reports, 40% of the world’s population is currently at risk for dengue fever. As vaccines are not available for such diseases, controlling mosquito population becomes necessary. Hence, this study aims at UDP-N-acetyl glucosamine pyrophosphorylase of Aedes aegypti (AaUAP, an essential enzyme for chitin metabolim in insects, as a drug target. Structure of AaUAP was predicted and validated using in-silico approach. Further, docking studies were performed using a set of 10 inhibitors out of which NAG9 was found to have good docking score, which was further supported by simulation studies. Hence, we propose that NAG9 can be considered as a potential hit in designing new inhibitors to control Aedes aegypti.

  19. CREB-binding protein controls response to cocaine by acetylating histones at the fosB promoter in the mouse striatum

    Levine, Amir A.; Guan, Zhonghui; Barco, Angel; Xu, Shiqin; Kandel, Eric R.; Schwartz, James H.

    2005-01-01

    Remodeling chromatin is essential for cAMP-regulated gene expression, necessary not only for development but also for memory storage and other enduring mental states. Histone acetylation and deacetylation mediate long-lasting forms of synaptic plasticity in Aplysia as well as cognition in mice. Here, we show that histone acetylation by the cAMP-response element binding protein (CREB)-binding protein (CBP) mediates sensitivity to cocaine by regulating expression of the fosB gene and its splice...

  20. The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis.

    P. Marini(GANIL); Li, S J; Gardiol, D; Cronan, J E; De Mendoza, D

    1995-01-01

    The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.

  1. Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

    The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32P-ACC phosphorylated by the casein kinases was identified

  2. Protein targeting to ATP-dependent proteases

    Inobe, Tomonao; Matouschek, Andreas

    2008-01-01

    ATP-dependent proteases control diverse cellular processes by degrading specific regulatory proteins. Understanding how these regulatory proteins are targeted to ATP-dependent proteases is of central importance to understanding their biological role as regulators. Recent work has shown that protein substrates are specifically transferred to ATP-dependent proteases through different routes. These routes can function in parallel or independently. In all of these targeting mechanisms it can be u...

  3. Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction.

    Mizuno, Yuta; Nagano-Shoji, Megumi; Kubo, Shosei; Kawamura, Yumi; Yoshida, Ayako; Kawasaki, Hisashi; Nishiyama, Makoto; Yoshida, Minoru; Kosono, Saori

    2016-02-01

    The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as l-glutamate. During l-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor l-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more

  4. p53 Targets Simian Virus 40 Large T Antigen for Acetylation by CBP

    Poulin, Danielle L.; Kung, Andrew L.; DeCaprio, James A.

    2004-01-01

    Simian virus 40 (SV40) large T antigen (T Ag) interacts with the tumor suppressor p53 and the transcriptional coactivators CBP and p300. Binding of these cellular proteins in a ternary complex has been implicated in T Ag-mediated transformation. It has been suggested that the ability of CBP/p300 to modulate p53 function underlies p53's regulation of cell proliferation and tumorigenesis. In this study, we provide further evidence that CBP activity may be mediated through its synergistic action...

  5. N-acetyl-L-cysteine sensitizes pancreatic cancers to gemcitabine by targeting the NFκB pathway.

    Qanungo, Suparna; Uys, Joachim D; Manevich, Yefim; Distler, Anne M; Shaner, Brooke; Hill, Elizabeth G; Mieyal, John J; Lemasters, John J; Townsend, Danyelle M; Nieminen, Anna-Liisa

    2014-09-01

    First-line therapy for pancreatic cancer is gemcitabine. Although tumors may initially respond to the gemcitabine treatment, soon tumor resistance develops leading to treatment failure. Previously, we demonstrated in human MIA PaCa-2 pancreatic cancer cells that N-acetyl-l-cysteine (NAC), a glutathione (GSH) precursor, prevents NFκB activation via S-glutathionylation of p65-NFκB, thereby blunting expression of survival genes. In this study, we documented the molecular sites of S-glutathionylation of p65, and we investigated whether NAC can suppress NFκB signaling and augment a therapeutic response to gemcitabine in vivo. Mass spectrometric analysis of S-glutathionylated p65-NFκB protein in vitro showed post-translational modifications of cysteines 38, 105, 120, 160 and 216 following oxidative and nitrosative stress. Circular dichroism revealed that S-glutathionylation of p65-NFκB did not change secondary structure of the protein, but increased tryptophan fluorescence revealed altered tertiary structure. Gemcitabine and NAC individually were not effective in decreasing MIA PaCa-2 tumor growth in vivo. However, combination treatment with NAC and gemcitabine decreased tumor growth by approximately 50%. NAC treatment also markedly enhanced tumor apoptosis in gemcitabine-treated mice. Compared to untreated tumors, gemcitabine treatment alone increased p65-NFκB nuclear translocation (3.7-fold) and DNA binding (2.5-fold), and these effects were blunted by NAC. In addition, NAC plus gemcitabine treatment decreased anti-apoptotic XIAP protein expression compared to gemcitabine alone. None of the treatments, however, affected extent of tumor hypoxia, as assessed by EF5 staining. Together, these results indicate that adjunct therapy with NAC prevents NFκB activation and improves gemcitabine chemotherapeutic efficacy. PMID:25257100

  6. Modulation of the cytoplasmic functions of mammalian post-transcriptional regulatory proteins by methylation and acetylation: a key layer of regulation waiting to be uncovered?

    Blee, Tajekesa K P; Gray, Nicola K; Brook, Matthew

    2015-12-01

    Post-transcriptional control of gene expression is critical for normal cellular function and viability and many of the proteins that mediate post-transcriptional control are themselves subject to regulation by post-translational modification (PTM), e.g. phosphorylation. However, proteome-wide studies are revealing new complexities in the PTM status of mammalian proteins, in particular large numbers of novel methylated and acetylated residues are being identified. Here we review studied examples of methylation/acetylation-dependent regulation of post-transcriptional regulatory protein (PTRP) function and present collated PTM data that points to the huge potential for regulation of mRNA fate by these PTMs. PMID:26614674

  7. Differential Effect of Endurance Training on Mitochondrial Protein Damage, Degradation, and Acetylation in the Context of Aging.

    Johnson, Matthew L; Irving, Brian A; Lanza, Ian R; Vendelbo, Mikkel H; Konopka, Adam R; Robinson, Matthew M; Henderson, Gregory C; Klaus, Katherine A; Morse, Dawn M; Heppelmann, Carrie; Bergen, H Robert; Dasari, Surendra; Schimke, Jill M; Jakaitis, Daniel R; Nair, K Sreekumaran

    2015-11-01

    Acute aerobic exercise increases reactive oxygen species and could potentially damage proteins, but exercise training (ET) enhances mitochondrial respiration irrespective of age. Here, we report a differential impact of ET on protein quality in young and older participants. Using mass spectrometry we measured oxidative damage to skeletal muscle proteins before and after 8 weeks of ET and find that young but not older participants reduced oxidative damage to both total skeletal muscle and mitochondrial proteins. Young participants showed higher total and mitochondrial derived semitryptic peptides and 26S proteasome activity indicating increased protein degradation. ET however, increased the activity of the endogenous antioxidants in older participants. ET also increased skeletal muscle content of the mitochondrial deacetylase SIRT3 in both groups. A reduction in the acetylation of isocitrate dehydrogenase 2 was observed following ET that may counteract the effect of acute oxidative stress. In conclusion aging is associated with an inability to improve skeletal muscle and mitochondrial protein quality in response to ET by increasing degradation of damaged proteins. ET does however increase muscle and mitochondrial antioxidant capacity in older individuals, which provides increased buffering from the acute oxidative effects of exercise. PMID:25504576

  8. The PHD finger of p300 influences its ability to acetylate histone and non-histone targets.

    Rack, Johannes G M; Lutter, Timo; Kjæreng Bjerga, Gro Elin; Guder, Corina; Ehrhardt, Christine; Värv, Signe; Ziegler, Mathias; Aasland, Rein

    2014-12-12

    In enzymes that regulate chromatin structure, the combinatorial occurrence of modules that alter and recognise histone modifications is a recurrent feature. In this study, we explored the functional relationship between the acetyltransferase domain and the adjacent bromodomain/PHD finger (bromo/PHD) region of the transcriptional coactivator p300. We found that the bromo/PHD region of p300 can bind to the acetylated catalytic domain in vitro and augment the catalytic activity of the enzyme. Deletion of the PHD finger, but not the bromodomain, impaired the ability of the enzyme to acetylate histones in vivo, whilst it enhanced p300 self-acetylation. A point mutation in the p300 PHD finger that is related to the Rubinstein-Taybi syndrome resulted in increased self-acetylation but retained the ability to acetylate histones. Hence, the PHD finger appears to negatively regulate self-acetylation. Furthermore, our data suggest that the PHD finger has a role in the recruitment of p300 to chromatin. PMID:25158095

  9. Protein search for multiple targets on DNA

    Protein-DNA interactions are crucial for all biological processes. One of the most important fundamental aspects of these interactions is the process of protein searching and recognizing specific binding sites on DNA. A large number of experimental and theoretical investigations have been devoted to uncovering the molecular description of these phenomena, but many aspects of the mechanisms of protein search for the targets on DNA remain not well understood. One of the most intriguing problems is the role of multiple targets in protein search dynamics. Using a recently developed theoretical framework we analyze this question in detail. Our method is based on a discrete-state stochastic approach that takes into account most relevant physical-chemical processes and leads to fully analytical description of all dynamic properties. Specifically, systems with two and three targets have been explicitly investigated. It is found that multiple targets in most cases accelerate the search in comparison with a single target situation. However, the acceleration is not always proportional to the number of targets. Surprisingly, there are even situations when it takes longer to find one of the multiple targets in comparison with the single target. It depends on the spatial position of the targets, distances between them, average scanning lengths of protein molecules on DNA, and the total DNA lengths. Physical-chemical explanations of observed results are presented. Our predictions are compared with experimental observations as well as with results from a continuum theory for the protein search. Extensive Monte Carlo computer simulations fully support our theoretical calculations

  10. Protein search for multiple targets on DNA

    Lange, Martin; Kochugaeva, Maria; Kolomeisky, Anatoly B.

    2015-09-01

    Protein-DNA interactions are crucial for all biological processes. One of the most important fundamental aspects of these interactions is the process of protein searching and recognizing specific binding sites on DNA. A large number of experimental and theoretical investigations have been devoted to uncovering the molecular description of these phenomena, but many aspects of the mechanisms of protein search for the targets on DNA remain not well understood. One of the most intriguing problems is the role of multiple targets in protein search dynamics. Using a recently developed theoretical framework we analyze this question in detail. Our method is based on a discrete-state stochastic approach that takes into account most relevant physical-chemical processes and leads to fully analytical description of all dynamic properties. Specifically, systems with two and three targets have been explicitly investigated. It is found that multiple targets in most cases accelerate the search in comparison with a single target situation. However, the acceleration is not always proportional to the number of targets. Surprisingly, there are even situations when it takes longer to find one of the multiple targets in comparison with the single target. It depends on the spatial position of the targets, distances between them, average scanning lengths of protein molecules on DNA, and the total DNA lengths. Physical-chemical explanations of observed results are presented. Our predictions are compared with experimental observations as well as with results from a continuum theory for the protein search. Extensive Monte Carlo computer simulations fully support our theoretical calculations.

  11. Protein search for multiple targets on DNA

    Lange, Martin [Johannes Gutenberg University, Mainz 55122 (Germany); Department of Chemistry, Rice University, Houston, Texas 77005 (United States); Kochugaeva, Maria [Department of Chemistry, Rice University, Houston, Texas 77005 (United States); Kolomeisky, Anatoly B., E-mail: tolya@rice.edu [Department of Chemistry, Rice University, Houston, Texas 77005 (United States); Center for Theoretical Biological Physics, Rice University, Houston, Texas 77005 (United States)

    2015-09-14

    Protein-DNA interactions are crucial for all biological processes. One of the most important fundamental aspects of these interactions is the process of protein searching and recognizing specific binding sites on DNA. A large number of experimental and theoretical investigations have been devoted to uncovering the molecular description of these phenomena, but many aspects of the mechanisms of protein search for the targets on DNA remain not well understood. One of the most intriguing problems is the role of multiple targets in protein search dynamics. Using a recently developed theoretical framework we analyze this question in detail. Our method is based on a discrete-state stochastic approach that takes into account most relevant physical-chemical processes and leads to fully analytical description of all dynamic properties. Specifically, systems with two and three targets have been explicitly investigated. It is found that multiple targets in most cases accelerate the search in comparison with a single target situation. However, the acceleration is not always proportional to the number of targets. Surprisingly, there are even situations when it takes longer to find one of the multiple targets in comparison with the single target. It depends on the spatial position of the targets, distances between them, average scanning lengths of protein molecules on DNA, and the total DNA lengths. Physical-chemical explanations of observed results are presented. Our predictions are compared with experimental observations as well as with results from a continuum theory for the protein search. Extensive Monte Carlo computer simulations fully support our theoretical calculations.

  12. Targeting IAP proteins in combination with radiotherapy

    The efficacy of radiotherapy critically depends on the activation of intrinsic cell death programs in cancer cells. This implies that evasion of cell death, a hallmark of human cancers, can contribute to radioresistance. Therefore, novel strategies to reactivate cell death programs in cancer cells are required in order to overcome resistance to radiotherapy. Since Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in multiple cancers and block cell death induction at a central point, therapeutic targeting of IAP proteins represents a promising approach to potentiate the efficacy of radiotherapy. The current review discusses the concept of targeting IAP proteins in combination with radiotherapy

  13. Sub-nuclear distribution and mobility of nuclear proteins involved in histone acetylation and pre-mRNA splicing

    The mitotic relationship between levels of highly acetylated chromatin, chromatin condensation, and HAT/HDAC organization was examined. HATs and HDACs were found to dissociate from chromosomes along with a loss of highly acetylated histones in condensed chromatin in mitosis. We demonstrate that, rather than being enzymatically inactivated, HAT and HDAC activities are decreased in mitosis because the enzymes are sequestered to a non-chromatin domain. Highly acetylated histone species reappear coincident with the reassociation of HATs and HDACs in late telophase/early interphase and before reinitiation of transcription. We propose that HATs and HDACs are spatially regulated through the cell cycle and that this regulation influences which chromatin domains are available for acetylation and deacetylation. We examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF:GFP) using timelapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We found that ASF:GFP moves significantly slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of transcription inhibitors and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites. Through a careful analysis of HDAC4 expression we found that HDAC4-containing MAD bodies are not a consistent component of the interphase nucleus. By comparing MAD bodies to PML bodies we found that the assembly, maintenance and distribution of PML bodies is regulated. We investigated the involvement of chromatin condensation in establishing mitotic transcription repression, by analyzing transcriptional activity in

  14. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    Huerlimann, Roger

    2015-07-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  15. Hexavalent Chromium (Cr(VI)) Down-Regulates Acetylation of Histone H4 at Lysine 16 through Induction of Stressor Protein Nupr1

    Chen, Danqi; Kluz, Thomas; Fang, Lei; Zhang, Xiaoru; Sun, Hong; Jin, Chunyuan; Costa, Max

    2016-01-01

    The environmental and occupational carcinogen Hexavalent Chromium (Cr(VI)) has been shown to cause lung cancer in humans when inhaled. In spite of a considerable research effort, the mechanisms of Cr(VI)-induced carcinogenesis remain largely unknown. Nupr1 (nuclear protein 1) is a small, highly basic, and unfolded protein with molecular weight of 8,800 daltons and is induced by a variety of stressors. Studies in animal models have suggested that Nupr1 is a key factor in the development of lung and pancreatic cancers, with little known about the underlying molecular mechanisms. Here we report that the level of Nupr1 is significantly increased in human bronchial epithelial BEAS2B cells following exposure to Cr(VI) through epigenetic mechanisms. Interestingly, Cr(VI) exposure also results in the loss of acetylation at histone H4K16, which is considered a ‘hallmark’ of human cancer. Cr(VI)-induced reduction of H4K16 acetylation appears to be caused by the induction of Nupr1, since (a) overexpression of Nupr1 decreased the levels of both H4K16 acetylation and the histone acetyltransferase MOF (male absent on the first; also known as Kat8, Myst 1), which specifically acetylates H4K16; (b) the loss of acetylation of H4K16 upon Cr(VI) exposure is greatly compromised by knockdown of Nupr1. Moreover, Nupr1-induced reduction of H4K16 acetylation correlates with the transcriptional down-regulation at several genomic loci. Notably, overexpression of Nupr1 induces anchorage-independent cell growth and knockdown of Nupr1 expression prevents Cr(VI)-induced cell transformation. We propose that Cr(VI) induces Nupr1 and rapidly perturbs gene expression by downregulating H4K16 acetylation, thereby contributing to Cr(VI)-induced carcinogenesis. PMID:27285315

  16. Hexavalent Chromium (Cr(VI)) Down-Regulates Acetylation of Histone H4 at Lysine 16 through Induction of Stressor Protein Nupr1.

    Chen, Danqi; Kluz, Thomas; Fang, Lei; Zhang, Xiaoru; Sun, Hong; Jin, Chunyuan; Costa, Max

    2016-01-01

    The environmental and occupational carcinogen Hexavalent Chromium (Cr(VI)) has been shown to cause lung cancer in humans when inhaled. In spite of a considerable research effort, the mechanisms of Cr(VI)-induced carcinogenesis remain largely unknown. Nupr1 (nuclear protein 1) is a small, highly basic, and unfolded protein with molecular weight of 8,800 daltons and is induced by a variety of stressors. Studies in animal models have suggested that Nupr1 is a key factor in the development of lung and pancreatic cancers, with little known about the underlying molecular mechanisms. Here we report that the level of Nupr1 is significantly increased in human bronchial epithelial BEAS2B cells following exposure to Cr(VI) through epigenetic mechanisms. Interestingly, Cr(VI) exposure also results in the loss of acetylation at histone H4K16, which is considered a 'hallmark' of human cancer. Cr(VI)-induced reduction of H4K16 acetylation appears to be caused by the induction of Nupr1, since (a) overexpression of Nupr1 decreased the levels of both H4K16 acetylation and the histone acetyltransferase MOF (male absent on the first; also known as Kat8, Myst 1), which specifically acetylates H4K16; (b) the loss of acetylation of H4K16 upon Cr(VI) exposure is greatly compromised by knockdown of Nupr1. Moreover, Nupr1-induced reduction of H4K16 acetylation correlates with the transcriptional down-regulation at several genomic loci. Notably, overexpression of Nupr1 induces anchorage-independent cell growth and knockdown of Nupr1 expression prevents Cr(VI)-induced cell transformation. We propose that Cr(VI) induces Nupr1 and rapidly perturbs gene expression by downregulating H4K16 acetylation, thereby contributing to Cr(VI)-induced carcinogenesis. PMID:27285315

  17. Targeting protein-protein interactions for parasite control.

    Christina M Taylor

    Full Text Available Finding new drug targets for pathogenic infections would be of great utility for humanity, as there is a large need to develop new drugs to fight infections due to the developing resistance and side effects of current treatments. Current drug targets for pathogen infections involve only a single protein. However, proteins rarely act in isolation, and the majority of biological processes occur via interactions with other proteins, so protein-protein interactions (PPIs offer a realm of unexplored potential drug targets and are thought to be the next-generation of drug targets. Parasitic worms were chosen for this study because they have deleterious effects on human health, livestock, and plants, costing society billions of dollars annually and many sequenced genomes are available. In this study, we present a computational approach that utilizes whole genomes of 6 parasitic and 1 free-living worm species and 2 hosts. The species were placed in orthologous groups, then binned in species-specific orthologous groups. Proteins that are essential and conserved among species that span a phyla are of greatest value, as they provide foundations for developing broad-control strategies. Two PPI databases were used to find PPIs within the species specific bins. PPIs with unique helminth proteins and helminth proteins with unique features relative to the host, such as indels, were prioritized as drug targets. The PPIs were scored based on RNAi phenotype and homology to the PDB (Protein DataBank. EST data for the various life stages, GO annotation, and druggability were also taken into consideration. Several PPIs emerged from this study as potential drug targets. A few interactions were supported by co-localization of expression in M. incognita (plant parasite and B. malayi (H. sapiens parasite, which have extremely different modes of parasitism. As more genomes of pathogens are sequenced and PPI databases expanded, this methodology will become increasingly

  18. Targeting protein-protein interactions as an anticancer strategy

    Ivanov, Andrei A.; Khuri, Fadlo R.; Fu, Haian

    2013-01-01

    The emergence and convergence of cancer genomics, targeted therapies, and network oncology have significantly expanded the landscape of protein-protein interaction (PPI) networks in cancer for therapeutic discovery. Extensive biological and clinical investigations have led to the identification of protein interaction hubs and nodes that are critical for the acquisition and maintaining characteristics of cancer essential for cell transformation. Such cancer enabling PPIs have become promising ...

  19. A fucoidan from Nemacystus decipiens disrupts angiogenesis through targeting bone morphogenetic protein 4.

    Wang, Wucheng; Chen, Huanjun; Zhang, Lei; Qin, Yi; Cong, Qifei; Wang, Peipei; Ding, Kan

    2016-06-25

    A sulfated and acetylated fucoidan, named NDH01, was extracted from seaweed Nemacystus decipiens. NDH01 was composed of mannose, glucuronic acid, fucose, sulfate group and acetyl group in the molar ratio of 3.0: 14.4: 82.6: 34.3: 13.9. The backbone of NDH01 was fucose-free core, composed of α-d-1,2-Manp and β-d-1,4-GlcpA disaccharide repeat unit. The branches were attached at the C3, C4 and C6 of α-d-1,2-Manp. The sidechain was composed of α-l-1,3,4-Fucp, α-l-1,4-Fucp, α-l-1,3-Fucp and α-l-1,4-GlcpA. The sulfate group was linked to C4 of α-l-1,3,4-Fucp, whereas, acetyl group was branched on C2 of α-l-1,2,3-Fucp. NDH01 could disrupt tube formation and inhibit the migration as well as cell growth of human microvascular endothelial cells. Besides, phosphorylation of Smad/1/5/8, Erk and FAK was significantly inhibited by NDH01. Further studies uncovered that NDH01 blocked Smad1/5/8 signaling via interacting with bone morphogenetic protein 4 and downregulating bone morphogenetic protein 4 expression. The results suggested that NDH01 might be an angiogenesis inhibitor through targeting bone morphogenetic protein 4. PMID:27083822

  20. Targeted Delivery of Protein Drugs by Nanocarriers

    Antonella Battisti

    2010-03-01

    Full Text Available Recent advances in biotechnology demonstrate that peptides and proteins are the basis of a new generation of drugs. However, the transportation of protein drugs in the body is limited by their high molecular weight, which prevents the crossing of tissue barriers, and by their short lifetime due to immuno response and enzymatic degradation. Moreover, the ability to selectively deliver drugs to target organs, tissues or cells is a major challenge in the treatment of several human diseases, including cancer. Indeed, targeted delivery can be much more efficient than systemic application, while improving bioavailability and limiting undesirable side effects. This review describes how the use of targeted nanocarriers such as nanoparticles and liposomes can improve the pharmacokinetic properties of protein drugs, thus increasing their safety and maximizing the therapeutic effect.

  1. Dynamics of co-translational protein targeting.

    Elvekrog, Margaret M; Walter, Peter

    2015-12-01

    Most membrane and secretory proteins are delivered co-translationally to protein translocation channels in their destination membrane by the signal recognition particle (SRP) and its receptor. This co-translational molecular machinery is conserved across all kingdoms of life, though it varies in composition and function. Here we report recent progress towards understanding the mechanism of SRP function, focusing on findings about Escherichia coli SRP's conformational dynamics throughout the targeting process. These insights shed light on a key checkpoint in the targeting cycle: how SRP regulates engagement of an actively translating ribosome with the translocation machinery at the membrane. PMID:26517565

  2. Targeted Delivery of Protein Drugs by Nanocarriers

    Antonella Battisti; Federica Chiellini; Roberto Solaro

    2010-01-01

    Recent advances in biotechnology demonstrate that peptides and proteins are the basis of a new generation of drugs. However, the transportation of protein drugs in the body is limited by their high molecular weight, which prevents the crossing of tissue barriers, and by their short lifetime due to immuno response and enzymatic degradation. Moreover, the ability to selectively deliver drugs to target organs, tissues or cells is a major challenge in the treatment of several human diseases, incl...

  3. Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis.

    Burillo, Sergio; Luque, Ignacio; Fuentes, Inmaculada; Contreras, Asunción

    2004-06-01

    PII, one of the most conserved signal transduction proteins, is believed to be a key player in the coordination of nitrogen assimilation and carbon metabolism in bacteria, archaea, and plants. However, the identity of PII receptors remains elusive, particularly in photosynthetic organisms. Here we used yeast two-hybrid approaches to identify new PII receptors and to explore the extent of conservation of PII signaling mechanisms between eubacteria and photosynthetic eukaryotes. Screening of Synechococcus sp. strain PCC 7942 libraries with PII as bait resulted in identification of N-acetyl glutamate kinase (NAGK), a key enzyme in the biosynthesis of arginine. The integrity of Ser49, a residue conserved in PII proteins from organisms that perform oxygenic photosynthesis, appears to be essential for NAGK binding. The effect of glnB mutations on NAGK activity is consistent with positive regulation of NAGK by PII. Phylogenetic and yeast two-hybrid analyses strongly suggest that there was conservation of the NAGK-PII regulatory interaction in the evolution of cyanobacteria and chloroplasts, providing insight into the function of eukaryotic PII-like proteins. PMID:15150219

  4. Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli

    Weinert, Brian T; Iesmantavicius, Vytautas; Wagner, Sebastian A;

    2013-01-01

    Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells in...... acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low...

  5. Motif mediated protein-protein interactions as drug targets.

    Corbi-Verge, Carles; Kim, Philip M

    2016-01-01

    Protein-protein interactions (PPI) are involved in virtually every cellular process and thus represent an attractive target for therapeutic interventions. A significant number of protein interactions are frequently formed between globular domains and short linear peptide motifs (DMI). Targeting these DMIs has proven challenging and classical approaches to inhibiting such interactions with small molecules have had limited success. However, recent new approaches have led to the discovery of potent inhibitors, some of them, such as Obatoclax, ABT-199, AEG-40826 and SAH-p53-8 are likely to become approved drugs. These novel inhibitors belong to a wide range of different molecule classes, ranging from small molecules to peptidomimetics and biologicals. This article reviews the main reasons for limited success in targeting PPIs, discusses how successful approaches overcome these obstacles to discovery promising inhibitors for human protein double minute 2 (HDM2), B-cell lymphoma 2 (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and provides a summary of the promising approaches currently in development that indicate the future potential of PPI inhibitors in drug discovery. PMID:26936767

  6. Autoimmune regulator is acetylated by transcription coactivator CBP/p300

    Saare, Mario, E-mail: mario.saare@ut.ee [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); Rebane, Ana [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); SIAF, Swiss Institute of Allergy and Asthma Research, University of Zuerich, Davos (Switzerland); Rajashekar, Balaji; Vilo, Jaak [BIIT, Bioinformatics, Algorithmics and Data Mining group, Institute of Computer Science, University of Tartu, Tartu (Estonia); Peterson, Paert [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia)

    2012-08-15

    The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes. -- Highlights: Black-Right-Pointing-Pointer AIRE is acetylated by the acetyltransferases p300 and CBP. Black-Right-Pointing-Pointer Acetylation occurs between CARD and SAND domains and within the SAND domain. Black-Right-Pointing-Pointer Acetylation increases the size of AIRE nuclear dots. Black-Right-Pointing-Pointer Acetylation increases AIRE protein stability. Black-Right-Pointing-Pointer AIRE acetylation mimic regulates a different set of AIRE

  7. Antisperm protein targets in azoospermia men

    Mohammad-Sadegh Soltani Zangbar

    2016-01-01

    Full Text Available BACKGROUND: The number of couples that meet the definition of infertility at reproductive ages is increasing worldwide. One of the most known conditions of infertility in males is azoospermia, defined as complete absence of spermatozoa in the semen. Azoospermia manifests in two forms, namely obstructive and non-obstructive azoospermia. Although the presence of antisperm antibody (ASA has been reported in 88% of the patients with obstructive azoospermia (OA, interestingly, there is no data regarding ASA targets in OA individuals. AIM: The present study aimed to identify sperm antibody targets in a group of OA men. SETTINGS AND DESIGN: The present study was carried out on 27 OA infertile men and 27 healthy fertile age-matched males as cases and controls, respectively. SUBJECTS AND METHODS: The sperm proteome was separated using two-dimensional gel electrophoresis technique, transferred onto the polyvinylidene fluoride membrane, and blotted with the sera of a group of OA men. Then, it was compared with the membranes blotted with the sera of a group of healthy fertile men. Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI TOF/TOF mass spectrometry was used to identify the different blotted spots and finally the results of the mass analysis were confirmed using reverse transcriptase polymerase chain reaction method. RESULTS: The results indicated that OA patients might produce antibody against two sperm proteins, Tektin-2 and triose phosphate isomerase. Moreover, the expressions of the two targeted proteins were confirmed at RNA level. CONCLUSIONS: The findings of the present study revealed two functionally important sperm proteins as antibody targets in azoospermic men.

  8. Antibiotics in development targeting protein synthesis.

    Sutcliffe, Joyce A

    2011-12-01

    The resolution of antibiotic-ribosomal subunit complexes and antibacterial-protein complexes at the atomic level has provided new insights into modifications of clinically relevant antimicrobials and provided new classes that target the protein cellular apparatus. New chemistry platforms that use fragment-based drug design or allow novel modifications in known structural classes are being used to design new antibiotics that overcome known resistance mechanisms and extend spectrum and potency by circumventing ubiquitous efflux pumps. This review provides details on seven antibiotics in development for treatment of moderate-to-severe community-acquired bacterial pneumonia and/or acute bacterial skin and skin structure infections: solithromycin, cethromycin, omadacycline, CEM-102, GSK1322322, radezolid, and tedizolid. Two antibiotics of the oxazolidinone class, PF-02341272 and AZD5847, are being developed as antituberculosis agents. Only three antibiotics that target the protein cellular machinery, TP-434, GSK2251052, and plazomicin, have a spectrum that encompasses multidrug-resistant Gram-negative pathogens. These compounds provide hope for treating key pathogens that cause serious disease in both the community and the hospital. PMID:22191530

  9. Accurate in silico identification of species-specific acetylation sites by integrating protein sequence-derived and functional features

    Li, Yuan; Wang, Mingjun; Wang, Huilin; Tan, Hao; Zhang, Ziding; Webb, Geoffrey I.; Song, Jiangning

    2014-07-01

    Lysine acetylation is a reversible post-translational modification, playing an important role in cytokine signaling, transcriptional regulation, and apoptosis. To fully understand acetylation mechanisms, identification of substrates and specific acetylation sites is crucial. Experimental identification is often time-consuming and expensive. Alternative bioinformatics methods are cost-effective and can be used in a high-throughput manner to generate relatively precise predictions. Here we develop a method termed as SSPKA for species-specific lysine acetylation prediction, using random forest classifiers that combine sequence-derived and functional features with two-step feature selection. Feature importance analysis indicates functional features, applied for lysine acetylation site prediction for the first time, significantly improve the predictive performance. We apply the SSPKA model to screen the entire human proteome and identify many high-confidence putative substrates that are not previously identified. The results along with the implemented Java tool, serve as useful resources to elucidate the mechanism of lysine acetylation and facilitate hypothesis-driven experimental design and validation.

  10. Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Marimuthu, Srinivasan; Alfonso, Lloyd F; Bhat, G Jayarama

    2016-05-01

    Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin. PMID:26596838

  11. Protein-targeted corona phase molecular recognition

    Bisker, Gili; Dong, Juyao; Park, Hoyoung D.; Iverson, Nicole M.; Ahn, Jiyoung; Nelson, Justin T.; Landry, Markita P.; Kruss, Sebastian; Strano, Michael S.

    2016-01-01

    Corona phase molecular recognition (CoPhMoRe) uses a heteropolymer adsorbed onto and templated by a nanoparticle surface to recognize a specific target analyte. This method has not yet been extended to macromolecular analytes, including proteins. Herein we develop a variant of a CoPhMoRe screening procedure of single-walled carbon nanotubes (SWCNT) and use it against a panel of human blood proteins, revealing a specific corona phase that recognizes fibrinogen with high selectivity. In response to fibrinogen binding, SWCNT fluorescence decreases by >80% at saturation. Sequential binding of the three fibrinogen nodules is suggested by selective fluorescence quenching by isolated sub-domains and validated by the quenching kinetics. The fibrinogen recognition also occurs in serum environment, at the clinically relevant fibrinogen concentrations in the human blood. These results open new avenues for synthetic, non-biological antibody analogues that recognize biological macromolecules, and hold great promise for medical and clinical applications.

  12. Data for global lysine-acetylation analysis in rice (Oryza sativa).

    Xiong, Yehui; Zhang, Kai; Cheng, Zhongyi; Wang, Guo-Liang; Liu, Wende

    2016-06-01

    Rice is one of the most important crops for human consumption and is a staple food for over half of the world׳s population (Yu et al., 2002) [1]. A systematic identification of the lysine acetylome was performed by our research (Xiong et al., 2016) [2]. Rice plant samples were collected from 5 weeks old seedlings (Oryza sativa, Nipponbare). After the trypsin digestion and immunoaffinity precipitation, LC-MS/MS approach was used to identify acetylated peptides. After the collected MS/MS data procession and GO annotation, the InterProScan was used to annotate protein domain. Subcellular localization of the identified acetylated proteins was predicted by WoLF PSORT. The KEGG pathway database was used to annotate identified acetylated protein interactions, reactions, and relations. The data, supplied in this article, are related to "A comprehensive catalog of the lysine-acetylation targets in rice (O. sativa) based on proteomic analyses" by Xiong et al. (2016) [2]. PMID:26977447

  13. Acetylation-Mediated Suppression of Transcription-Independent Memory: Bidirectional Modulation of Memory by Acetylation

    Katja Merschbaecher; Jakob Haettig; Uli Mueller

    2012-01-01

    Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs), and the antagonistic histone deacetylases (HDACs) play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM). While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact...

  14. Application of the MIDAS approach for analysis of lysine acetylation sites.

    Evans, Caroline A; Griffiths, John R; Unwin, Richard D; Whetton, Anthony D; Corfe, Bernard M

    2013-01-01

    Multiple Reaction Monitoring Initiated Detection and Sequencing (MIDAS™) is a mass spectrometry-based technique for the detection and characterization of specific post-translational modifications (Unwin et al. 4:1134-1144, 2005), for example acetylated lysine residues (Griffiths et al. 18:1423-1428, 2007). The MIDAS™ technique has application for discovery and analysis of acetylation sites. It is a hypothesis-driven approach that requires a priori knowledge of the primary sequence of the target protein and a proteolytic digest of this protein. MIDAS essentially performs a targeted search for the presence of modified, for example acetylated, peptides. The detection is based on the combination of the predicted molecular weight (measured as mass-charge ratio) of the acetylated proteolytic peptide and a diagnostic fragment (product ion of m/z 126.1), which is generated by specific fragmentation of acetylated peptides during collision induced dissociation performed in tandem mass spectrometry (MS) analysis. Sequence information is subsequently obtained which enables acetylation site assignment. The technique of MIDAS was later trademarked by ABSciex for targeted protein analysis where an MRM scan is combined with full MS/MS product ion scan to enable sequence confirmation. PMID:23381851

  15. Targeted proteins for diabetes drug design

    Type 2 diabetes mellitus is a common metabolism disorder characterized by high glucose in the bloodstream, especially in the case of insulin resistance and relative insulin deficiency. Nowadays, it is very common in middle-aged people and involves such dangerous symptoms as increasing risk of stroke, obesity and heart failure. In Vietnam, besides the common treatment of insulin injection, some herbal medication is used but no unified optimum remedy for the disease yet exists and there is no production of antidiabetic drugs in the domestic market yet. In the development of nanomedicine at the present time, drug design is considered as an innovative tool for researchers to study the mechanisms of diseases at the molecular level. The aim of this article is to review some common protein targets involved in type 2 diabetes, offering a new idea for designing new drug candidates to produce antidiabetic drugs against type 2 diabetes for Vietnamese people. (review)

  16. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    Pathak, Ravi [Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Ave, New York, NY 10029 (United States); Philizaire, Marc [Medgar Evers College, City University of New York, 1638 Bedford Ave, 403D, Brooklyn, NY 11225 (United States); Mujtaba, Shiraz, E-mail: smujtaba@mec.cuny.edu [Department of Structural and Chemical Biology, Mount Sinai School of Medicine, 1425 Madison Ave, New York, NY 10029 (United States); Medgar Evers College, City University of New York, 1638 Bedford Ave, 403D, Brooklyn, NY 11225 (United States)

    2015-08-19

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets.

  17. Dichotomy in the Epigenetic Mark Lysine Acetylation is Critical for the Proliferation of Prostate Cancer Cells

    The dynamics of lysine acetylation serve as a major epigenetic mark, which regulates cellular response to inflammation, DNA damage and hormonal changes. Microarray assays reveal changes in gene expression, but cannot predict regulation of a protein function by epigenetic modifications. The present study employs computational tools to inclusively analyze microarray data to understand the potential role of acetylation during development of androgen-independent PCa. The data revealed that the androgen receptor interacts with 333 proteins, out of which at least 92 proteins were acetylated. Notably, the number of cellular proteins undergoing acetylation in the androgen-dependent PCa was more as compared to the androgen-independent PCa. Specifically, the 32 lysine-acetylated proteins in the cellular models of androgen-dependent PCa were mainly involved in regulating stability as well as pre- and post-processing of mRNA. Collectively, the data demonstrate that protein lysine acetylation plays a crucial role during the transition of androgen-dependent to -independent PCa, which importantly, could also serve as a functional axis to unravel new therapeutic targets

  18. Multiphasic interactions between nucleotides and target proteins

    Nissen, Per

    2016-01-01

    The nucleotides guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) bind to target proteins to promote bacterial survival (Corrigan et al. 2016). Thus, the binding of the nucleotides to RsgA, a GTPase, inhibits the hydrolysis of GTP. The dose response, taken to be curvilinear with respect to the logarithm of the inhibitor concentration, is instead much better (P<0.001 when the 6 experiments are combined) represented as multiphasic, with high to exceedingly high absolute r values for the straight lines, and with transitions in the form of non-contiguities (jumps). Profiles for the binding of radiolabeled nucleotides to HprT and Gmk, GTP synthesis enzymes, were, similarly, taken to be curvilinear with respect to the logarithm of the protein concentration. However, the profiles are again much better represented as multiphasic than as curvilinear (the P values range from 0.047 to <0.001 for each of the 8 experiments for binding of ppGpp and pppGpp to HprT). The binding of GTP to HprT and ...

  19. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3.

    Eri Maria Sol

    Full Text Available Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs which are key regulators of many cellular processes. Identifying substrates of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3 by comparing site-specific acetylation in wild-type murine embryonic fibroblasts to Sirt3 knockout cells. We confirm Sirt3-regulated acetylation of several mitochondrial proteins in human cells by comparing acetylation in U2OS cells overexpressing Sirt3 to U2OS cells in which Sirt3 expression was reduced by shRNA. Our data demonstrate that ablation of Sirt3 significantly increases acetylation at dozens of sites on mitochondrial proteins. Substrates of Sirt3 are implicated in various metabolic pathways, including fatty acid metabolism and the tricarboxylic acid cycle. These results imply broader regulatory roles of Sirt3 in the mitochondria by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases.

  20. Identifying drug-target proteins based on network features

    2009-01-01

    Proteins rarely function in isolation inside and outside cells, but operate as part of a highly intercon- nected cellular network called the interaction network. Therefore, the analysis of the properties of drug-target proteins in the biological network is especially helpful for understanding the mechanism of drug action in terms of informatics. At present, no detailed characterization and description of the topological features of drug-target proteins have been available in the human protein-protein interac- tion network. In this work, by mapping the drug-targets in DrugBank onto the interaction network of human proteins, five topological indices of drug-targets were analyzed and compared with those of the whole protein interactome set and the non-drug-target set. The experimental results showed that drug-target proteins have higher connectivity and quicker communication with each other in the PPI network. Based on these features, all proteins in the interaction network were ranked. The results showed that, of the top 100 proteins, 48 are covered by DrugBank; of the remaining 52 proteins, 9 are drug-target proteins covered by the TTD, Matador and other databases, while others have been dem- onstrated to be drug-target proteins in the literature.

  1. Identifying drug-target proteins based on network features

    ZHU MingZhu; GAO Lei; LI Xia; LIU ZhiCheng

    2009-01-01

    Proteins rarely function in isolation Inside and outside cells, but operate as part of a highly Intercon-nected cellular network called the interaction network. Therefore, the analysis of the properties of drug-target proteins in the biological network is especially helpful for understanding the mechanism of drug action In terms of informatice. At present, no detailed characterization and description of the topological features of drug-target proteins have been available in the human protein-protein interac-tion network. In this work, by mapping the drug-targets in DrugBank onto the interaction network of human proteins, five topological indices of drug-targets were analyzed and compared with those of the whole protein interactome set and the non-drug-target set. The experimental results showed that drug-target proteins have higher connectivity and quicker communication with each other in the PPI network. Based on these features, all proteins In the interaction network were ranked. The results showed that, of the top 100 proteins, 48 are covered by DrugBank; of the remaining 52 proteins, 9 are drug-target proteins covered by the TTD, Matador and other databases, while others have been dem-onstrated to be drug-target proteins in the literature.

  2. Wnt Protein Signaling Reduces Nuclear Acetyl-CoA Levels to Suppress Gene Expression during Osteoblast Differentiation.

    Karner, Courtney M; Esen, Emel; Chen, Jiakun; Hsu, Fong-Fu; Turk, John; Long, Fanxin

    2016-06-17

    Developmental signals in metazoans play critical roles in inducing cell differentiation from multipotent progenitors. The existing paradigm posits that the signals operate directly through their downstream transcription factors to activate expression of cell type-specific genes, which are the hallmark of cell identity. We have investigated the mechanism through which Wnt signaling induces osteoblast differentiation in an osteoblast-adipocyte bipotent progenitor cell line. Unexpectedly, Wnt3a acutely suppresses the expression of a large number of genes while inducing osteoblast differentiation. The suppressed genes include Pparg and Cebpa, which encode adipocyte-specifying transcription factors and suppression of which is sufficient to induce osteoblast differentiation. The large scale gene suppression induced by Wnt3a corresponds to a global decrease in histone acetylation, an epigenetic modification that is associated with gene activation. Mechanistically, Wnt3a does not alter histone acetyltransferase or deacetylase activities but, rather, decreases the level of acetyl-CoA in the nucleus. The Wnt-induced decrease in histone acetylation is independent of β-catenin signaling but, rather, correlates with suppression of glucose metabolism in the tricarboxylic acid cycle. Functionally, preventing histone deacetylation by increasing nucleocytoplasmic acetyl-CoA levels impairs Wnt3a-induced osteoblast differentiation. Thus, Wnt signaling induces osteoblast differentiation in part through histone deacetylation and epigenetic suppression of an alternative cell fate. PMID:27129247

  3. Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ.

    Lin, Ying-Hsi; Warren, Chad M; Li, Jieli; McKinsey, Timothy A; Russell, Brenda

    2016-08-01

    The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCɛ (dnPKCɛ) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. PMID:27185186

  4. Identification of sirtuin and its target as the ribosomal protein S4 in Lactobacillus paracasei.

    Atarashi, Hotaka; Kawasaki, Shinji; Niimura, Yoichi; Tanaka, Naoto; Okada, Sanae; Shiwa, Yuh; Endo, Akihito; Nakagawa, Junichi

    2016-01-01

    Sirtuin is a protein with an enzymatic activity of NAD(+)-dependent protein deacetylation. It was first identified in yeast and its homologous genes have been widely found in various organisms. In bacteria, sirtuin gene was first described as cobB, encoding a cobalamin processing enzyme; and later its potential involvement in regulating acetylation levels of metabolic enzymes, transcription factors, chemotactic proteins and others have been reported. In order to study its physiological relevance in probiotic lactic acid bacteria, we analyzed the whole genome of three L. paracasei strains. All strains tested had sirtuin homolog genes designated hereby as sirA, and one of them had an additional gene designated as sirB. Following confirmation of their coding sequences by individual gene cloning, corresponding recombinant proteins have been generated and purified. The enzymatic characterization revealed that the intrinsic NAD(+)-dependent deacetylation activity of LpSirA (protein encoded by sirA) is comparable to human SIRT1. Furthermore, by blocking sirtuin activity using nicotinamide in vivo, together with an in vitro deacetylation reaction using recombinant LpSirA, we identified one of the target proteins in the lactic acid bacteria as the 30S ribosomal protein S4 (rpsD product). PMID:27118078

  5. Spectroscopic study of drug-binding characteristics of unmodified and pNPA-based acetylated human serum albumin: Does esterase activity affect microenvironment of drug binding sites on the protein?

    Moradi, Nastaran [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Faculty of Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ashrafi-Kooshk, Mohammad Reza [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ghobadi, Sirous [Department of Biology, Faculty of Sciences, Razi University, Kermanshah (Iran, Islamic Republic of); Shahlaei, Mohsen [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Faculty of Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Khodarahmi, Reza, E-mail: rkhodarahmi@mbrc.ac.ir [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Faculty of Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of)

    2015-04-15

    Human serum albumin (HSA) is the most prominent extracellular protein in blood plasma. There are several binding sites on the protein which provide accommodation for structurally-unrelated endogenous and exogenous ligands and a wide variety of drugs. “Esterase-like” activity (hydrolysis of p-nitrophenyl esters) by the protein has been also reported. In the current study, we set out to investigate the interaction of indomethacin and ibuprofen with the unmodified and modified HSA (pNPA-modified HSA) using various spectroscopic techniques. Fluorescence data showed that 1:1 binding of drug to HSA is associated with quenching of the protein intrinsic fluorescence. Decrease of protein surface hydrophobicity (PSH), alteration in drug binding affinity and change of the protein stability, after esterase-like activity and permanent acetylation of HSA, were also documented. Analysis of the quenching and thermodynamic parameters indicated that forces involved in drug–HSA interactions change upon the protein modification. - Highlights: • Binding propensity of indomethacin extremely decreased upon the protein acetylation. • There is no ibuprofen binding after protein acetylation. • Protein stability changes upon drug binding as well as protein acetylation. • Drug pharmacokinetics may be influenced under co-administration of HSA-modifier drugs.

  6. Spectroscopic study of drug-binding characteristics of unmodified and pNPA-based acetylated human serum albumin: Does esterase activity affect microenvironment of drug binding sites on the protein?

    Human serum albumin (HSA) is the most prominent extracellular protein in blood plasma. There are several binding sites on the protein which provide accommodation for structurally-unrelated endogenous and exogenous ligands and a wide variety of drugs. “Esterase-like” activity (hydrolysis of p-nitrophenyl esters) by the protein has been also reported. In the current study, we set out to investigate the interaction of indomethacin and ibuprofen with the unmodified and modified HSA (pNPA-modified HSA) using various spectroscopic techniques. Fluorescence data showed that 1:1 binding of drug to HSA is associated with quenching of the protein intrinsic fluorescence. Decrease of protein surface hydrophobicity (PSH), alteration in drug binding affinity and change of the protein stability, after esterase-like activity and permanent acetylation of HSA, were also documented. Analysis of the quenching and thermodynamic parameters indicated that forces involved in drug–HSA interactions change upon the protein modification. - Highlights: • Binding propensity of indomethacin extremely decreased upon the protein acetylation. • There is no ibuprofen binding after protein acetylation. • Protein stability changes upon drug binding as well as protein acetylation. • Drug pharmacokinetics may be influenced under co-administration of HSA-modifier drugs

  7. Vitamin D binding protein-derived macrophage activating factor inhibits human breast cancer cell proliferation and decreases alpha-N-acetyl galactosaminidase in breast cancer patiens.

    L. Thyer; G.Morucci; J.J.V. Branca; E. Wards; Smith, R; Noakes, D.

    2013-01-01

    Objectives: It was demonstrated that vitamin D binding protein-derived macrophage activating factor (GcMAF) decreases serum alpha-N-acetyl galactosaminidase (nagalase) in breast cancer patients; decrease of nagalase was associated with significant improvement of clinical conditions (Int J Cancer. 2008 Jan 15;122(2):461-7). Since nagalase is both an index of immune suppression and of tumour burden, we studied the direct effects of GcMAF on a human breast cancer cell line (MCF-7), and we com...

  8. Chloroplast protein targeting involves localized translation in Chlamydomonas

    Uniacke, James; Zerges, William

    2009-01-01

    The compartmentalization of eukaryotic cells requires that newly synthesized proteins be targeted to the compartments in which they function. In chloroplasts, a few thousand proteins function in photosynthesis, expression of the chloroplast genome, and other processes. Most chloroplast proteins are synthesized in the cytoplasm, imported, and then targeted to a specific chloroplast compartment. The remainder are encoded by the chloroplast genome, synthesized within the organelle, and targeted ...

  9. A Method to determine lysine acetylation stoichiometries

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  10. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    Nissen, Klaus B; Kedström, Linda Maria Haugaard; Wilbek, Theis S;

    2015-01-01

    related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of...... trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG...... linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic...

  11. Bacterial cell division proteins as antibiotic targets

    T. den Blaauwen; J.M. Andreu; O. Monasterio

    2014-01-01

    Proteins involved in bacterial cell division often do not have a counterpart in eukaryotic cells and they are essential for the survival of the bacteria. The genetic accessibility of many bacterial species in combination with the Green Fluorescence Protein revolution to study localization of protein

  12. Identification and Validation of ISG15 Target Proteins.

    Durfee, Larissa A; Huibregtse, Jon M

    2010-01-01

    ISG15 is an interferon-induced ubiquitin-like protein (Ubl) that has antiviral properties. The core E1, E2 and E3 enzymes for conjugation of human ISG15 are Ube1L, UbcH8 and Herc5, all of which are induced at the transcriptional level by Type 1 interferon signaling. Several proteomics studies have, together, identified over 300 cellular proteins as ISG15 targets. These targets include a broad range of constitutively expressed proteins and approximately 15 interferon-induced proteins. This chapter provides an overview of the target identification process and the validation of these targets. We also discuss the limited number of examples where the biochemical effect of ISG15 conjugation on target proteins has been characterized. PMID:21222286

  13. Chromatin proteins and modifications as drug targets

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

    A plethora of groundbreaking studies have demonstrated the importance of chromatin-associated proteins and post-translational modifications of histones, proteins and DNA (so-called epigenetic modifications) for transcriptional control and normal development. Disruption of epigenetic control is a ...

  14. Protein folding as a driving force for dual protein targeting in eukaryotes

    Kalderon, Bella; Pines, Ophry

    2014-01-01

    It is well documented that in eukaryotic cells molecules of one protein can be located in several subcellular locations, a phenomenon termed dual targeting, dual localization, or dual distribution. The differently localized identical or nearly identical proteins are termed “echoforms.” Our conventional definition of dual targeted proteins refers to situations in which one of the echoforms is translocated through/into a membrane. Thus, dual targeted proteins are recognized by at least one orga...

  15. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  16. Principles of protein targeting to the nucleolus

    Martin, R M; Ter-Avetisyan, G.; Herce, H.D.; Ludwig, A.K.; Laettig-Tuennemann, G.; Cardoso, M.C.

    2015-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins insid...

  17. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

    Sol, E-ri Maria; Wagner, Sebastian A; Weinert, Brian T; Kumar, Amit; Kim, Hyun-Seok; Deng, Chu-Xia; Choudhary, Chuna Ram

    2012-01-01

    KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site...

  18. Urban Endocrine Disruptors Targeting Breast Cancer Proteins.

    Montes-Grajales, Diana; Bernardes, Gonçalo J L; Olivero-Verbel, Jesus

    2016-02-15

    Humans are exposed to a huge amount of environmental pollutants called endocrine disrupting chemicals (EDCs). These molecules interfere with the homeostasis of the body, usually through mimicking natural hormones leading to activation or blocking of their receptors. Many of these compounds have been associated with a broad range of diseases including the development or increased susceptibility to breast cancer, the most prevalent cancer in women worldwide, according to the World Health Organization. Thus, this article presents a virtual high-throughput screening (vHTS) to evaluate the affinity of proteins related to breast cancer, such as ESR1, ERBB2, PGR, BCRA1, and SHBG, among others, with EDCs from urban sources. A blind docking strategy was employed to screen each protein-ligand pair in triplicate in AutoDock Vina 2.0, using the computed binding affinities as ranking criteria. The three-dimensional structures were previously obtained from EDCs DataBank and Protein Data Bank, prepared and optimized by SYBYL X-2.0. Some of the chemicals that exhibited the best affinity scores for breast cancer proteins in each category were 1,3,7,8-tetrachlorodibenzo-p-dioxin, bisphenol A derivatives, perfluorooctanesulfonic acid, and benzo(a)pyrene, for catalase, several proteins, sex hormone-binding globulin, and cytochrome P450 1A2, respectively. An experimental validation of this approach was performed with a complex that gave a moderate binding affinity in silico, the sex hormone binding globulin (SHBG), and bisphenol A (BPA) complex. The protein was obtained using DNA recombinant technology and the physical interaction with BPA assessed through spectroscopic techniques. BPA binds on the recombinant SHBG, and this results in an increase of its α helix content. In short, this work shows the potential of several EDCs to bind breast cancer associated proteins as a tool to prioritize compounds to perform in vitro analysis to benefit the regulation or exposure prevention by the

  19. Acetylation of woody lignocellulose: significance and regulation

    Prashant Mohan-Anupama Pawar

    2013-05-01

    Full Text Available Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides, is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose towards improved saccharification. In this review we: 1 summarize literature on lignocellulose acetylation in different tree species, 2 present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, 3 describe plant proteins involved in lignocellulose O-acetylation, 4 give examples of microbial enzymes capable to de-acetylate lignocellulose, and 5 discuss prospects for exploiting these enzymes in planta to modify xylan acetylation.

  20. Comparative Characterization of Hepatic Distribution and mRNA Reduction of Antisense Oligonucleotides Conjugated with Triantennary N-Acetyl Galactosamine and Lipophilic Ligands Targeting Apolipoprotein B.

    Watanabe, Ayahisa; Nakajima, Mado; Kasuya, Takeshi; Onishi, Reina; Kitade, Naohisa; Mayumi, Kei; Ikehara, Tatsuya; Kugimiya, Akira

    2016-05-01

    TriantennaryN-acetyl galactosamine (GalNAc, GN3) and lipophilic ligands such as cholesterol andα-tocopherol conjugations dramatically improve the distribution and efficacy of second-generation antisense oligonucleotides (ASOs) in the whole liver. To characterize ligands for delivery to liver cells based on pharmacokinetics and efficacy, we used a locked nucleic acid gapmer of ASO targeting apolipoprotein B as a model compound and evaluated the amount of ASO and apolipoprotein B mRNA in the whole liver, hepatocytes, and nonparenchymal (NP) cells as well as plasma total cholesterol after administration of ASO conjugated with these ligands to mice. Compared with unconjugated ASO, GN3 conjugation increased the amount (7-fold) and efficacy (more than 10-fold) of ASO in hepatocytes only and showed higher efficacy than the increased rate of the amount of ASO. On the other hand, lipophilic ligand conjugations led to increased delivery (3- to 5-fold) and efficacy (5-fold) of ASO to both hepatocytes and NP cells. GN3 and lipophilic ligand conjugations increased the area under the curve of ASOs and the pharmacodynamic duration but did not change the half-life in hepatocytes and NP cells compared with unconjugated ASO. In the liver, the phosphodiester bond between ASO and these ligands was promptly cleaved to liberate unconjugated ASO. These ligand conjugations reduced plasma total cholesterol compared with unconjugated ASO, although these ASOs were well tolerated with no elevation in plasma transaminases. These findings could facilitate ligand selection tailored to liver cells expressed in disease-related genes and could contribute to the discovery and development of RNA interference-based therapy. PMID:26907624

  1. Acetylation-mediated suppression of transcription-independent memory: bidirectional modulation of memory by acetylation.

    Katja Merschbaecher

    Full Text Available Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs, and the antagonistic histone deacetylases (HDACs play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM. While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact of both, increased and decreased acetylation on formation of appetitive olfactory memory in honeybees. We show that learning-induced changes in the acetylation of histone H3 at aminoacid-positions H3K9 and H3K18 exhibit distinct and different dynamics depending on the training strength. A strong training that induces LTM leads to an immediate increase in acetylation at H3K18 that stays elevated for hours. A weak training, not sufficient to trigger LTM, causes an initial increase in acetylation at H3K18, followed by a strong reduction in acetylation at H3K18 below the control group level. Acetylation at position H3K9 is not affected by associative conditioning, indicating specific learning-induced actions on the acetylation machinery. Elevating acetylation levels by blocking HDACs after conditioning leads to an improved memory. While memory after strong training is enhanced for at least 2 days, the enhancement after weak training is restricted to 1 day. Reducing acetylation levels by blocking HAT activity after strong training leads to a suppression of transcription-dependent LTM. The memory suppression is also observed in case of weak training, which does not require transcription processes. Thus, our findings demonstrate that acetylation-mediated processes act as bidirectional regulators of memory formation that facilitate or suppress memory independent of its transcription-requirement.

  2. Principles of protein targeting to the nucleolus.

    Martin, Robert M; Ter-Avetisyan, Gohar; Herce, Henry D; Ludwig, Anne K; Lättig-Tünnemann, Gisela; Cardoso, M Cristina

    2015-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution. PMID:26280391

  3. A study protocol to investigate the relationship between dietary fibre intake and fermentation, colon cell turnover, global protein acetylation and early carcinogenesis: the FACT study

    A number of studies, notably EPIC, have shown a descrease in colorectal cancer risk associated with increased fibre consumption. Whilst the underlying mechanisms are likely to be multifactorial, production of the short-chain fatty-acid butyrate fro butyratye is frequently cited as a major potential contributor to the effect. Butyrate inhibits histone deacetylases, which work on a wide range of proteins over and above histones. We therefore hypothesized that alterations in the acetylated proteome may be associated with a cancer risk phenotype in the colorectal mucosa, and that such alterations are candidate biomarkers for effectiveness of fibre interventions in cancer prevention. There are two principal arms to this study: (i) a cross-sectional study (FACT OBS) of 90 subjects recruited from gastroenterology clinics and; (ii) an intervention trial in 40 subjects with an 8 week high fibre intervention. In both studies the principal goal is to investigate a link between fibre intake, SCFA production and global protein acetylation. The primary measure is level of faecal butyrate, which it is hoped will be elevated by moving subjects to a high fibre diet. Fibre intakes will be estimated in the cross-sectional group using the EPIC Food Frequency Questionnaire. Subsidiary measures of the effect of butyrate on colon mucosal function and pre-cancerous phenotype will include measures of apoptosis, apoptotic regulators cell cycle and cell division. This study will provide a new level of mechanistic data on alterations in the functional proteome in response to the colon microenvironment which may underwrite the observed cancer preventive effect of fibre. The study may yield novel candidate biomarkers of fibre fermentation and colon mucosal function. Trial Registration Number: ISRCTN90852168

  4. A study protocol to investigate the relationship between dietary fibre intake and fermentation, colon cell turnover, global protein acetylation and early carcinogenesis: the FACT study

    Croucher Lisa J

    2009-09-01

    Full Text Available Abstract Background A number of studies, notably EPIC, have shown a descrease in colorectal cancer risk associated with increased fibre consumption. Whilst the underlying mechanisms are likely to be multifactorial, production of the short-chain fatty-acid butyrate fro butyratye is frequently cited as a major potential contributor to the effect. Butyrate inhibits histone deacetylases, which work on a wide range of proteins over and above histones. We therefore hypothesized that alterations in the acetylated proteome may be associated with a cancer risk phenotype in the colorectal mucosa, and that such alterations are candidate biomarkers for effectiveness of fibre interventions in cancer prevention. Methods an design There are two principal arms to this study: (i a cross-sectional study (FACT OBS of 90 subjects recruited from gastroenterology clinics and; (ii an intervention trial in 40 subjects with an 8 week high fibre intervention. In both studies the principal goal is to investigate a link between fibre intake, SCFA production and global protein acetylation. The primary measure is level of faecal butyrate, which it is hoped will be elevated by moving subjects to a high fibre diet. Fibre intakes will be estimated in the cross-sectional group using the EPIC Food Frequency Questionnaire. Subsidiary measures of the effect of butyrate on colon mucosal function and pre-cancerous phenotype will include measures of apoptosis, apoptotic regulators cell cycle and cell division. Discussion This study will provide a new level of mechanistic data on alterations in the functional proteome in response to the colon microenvironment which may underwrite the observed cancer preventive effect of fibre. The study may yield novel candidate biomarkers of fibre fermentation and colon mucosal function. Trial Registration Trial Registration Number: ISRCTN90852168

  5. DNA-binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene

    A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0-2.5 S, corresponding to a molecular weight of about 20000-25000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100-200 mM NaCl and is relatively independent of temperature and pH. MgCl2 and MnCl2 at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4x10-11 M. There are at least 1x105 DNA-binding protein molecules/HeLa cell. (Auth.)

  6. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  7. Protein kinases are potential targets to treat inflammatory bowel disease

    Lei; Yang; Yutao; Yan

    2014-01-01

    Protein kinases play a crucial role in the pathogenesis of inflammatory bowel disease(IBD), the two main forms of which are ulcerative colitis and Crohn’s dis-ease. In this article, we will review the mechanisms of involvement of protein kinases in the pathogenesis of and intervention against IBD, in terms of their effects on genetics, microbiota, mucous layer and tight junc-tion, and the potential of protein kinases as therapeutic targets against IBD.

  8. Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2

    Schwer, Bjoern; Bunkenborg, Jakob; Verdin, Regis O; Andersen, Jens S; Verdin, Eric

    2006-01-01

    We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 b...

  9. Targeted quantitative mass spectrometric immunoassay for human protein variants

    Nedelkov Dobrin

    2011-04-01

    Full Text Available Abstract Background Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome. Modified proteins also play an important role in biological processes. While sandwich immunoassays are routinely used to determine protein concentrations, they are oblivious to protein variants that may serve as biomarkers with better sensitivity and specificity than their wild-type proteins. Mass spectrometry, coupled to immunoaffinity separations, can provide an efficient mean for simultaneous detection and quantification of protein variants. Results Presented here is a mass spectrometric immunoassay method for targeted quantitative proteomics analysis of protein modifications. Cystatin C, a cysteine proteinase inhibitor and a potential marker for several pathological processes, was used as a target analyte. An internal reference standard was incorporated into the assay, serving as a normalization point for cystatin C quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing cystatin C ELISA. In application, the assay was utilized to determine the individual concentration of several cystatin C variants across a cohort of samples, demonstrating the ability to fully quantify individual forms of post-translationally modified proteins. Conclusions The mass spectrometric immunoassays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity.

  10. Intrinsically Unstructured Proteins: Potential Targets for Drug Discovery

    Pathan Salma

    2009-01-01

    Full Text Available Problem statement: The function of a protein is dependent on its three-dimensional structure. However, numerous proteins lacking intrinsic globular 3D structure under physiological conditions had been recognized. These proteins are frequently involved in some of the most critical cellular control mechanisms and it appears that their rapid turnover, aided by their unstructured nature in the unbound state, provides a level of control that allows rapid and accurate responses of the cell to changing environmental conditions. Approach: A significant number of proteins known to be involved in protein deposition disorders were now considered to Be Intrinsically Unstructured Proteins (IUPs. For example, Aß peptide and tau protein in Alzheimer’s disease, PrP in Prion’s disease and a-Synuclein in Parkinson’s disease. The disorder of intrinsically unstructured proteins (IUP's was crucial to their functions. They may adopt defined but extended structures when bound to cognate ligands. Their amino acid compositions were less hydrophobic than those of soluble proteins. They lack hydrophobic cores and hence did not become insoluble when heated. About 40% of eukaryotic proteins had at least one long (>50 residues disordered region. Roughly 10% of proteins in various genomes had been predicted to be fully disordered. Presently over 100 IUP's had been identified; none are enzymes. Obviously, IUP's were greatly underrepresented in the Protein Data Bank, although there were few cases of an IUP bound to a folded (intrinsically structured protein. Results: The five functional categories for intrinsically unstructured proteins and domains were entropic chains (bristles to ensure spacing, springs, flexible spacers/linkers, effectors (inhibitors and disassemblers, scavengers, assemblers and display sites. These IUPs could serve as potential targets for Structure Based Drug Design (SBDD which stress on the transition

  11. Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30II accessory protein and the induction of oncogenic cellular transformation by p30II/c-MYC

    The human T-cell leukemia retrovirus type-1 (HTLV-1) p30II protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30II interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30II and c-MYC remain to be completely understood. Herein we demonstrate that p30II induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30II in c-myc−/− HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30II is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30II inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30II/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis. - Highlights: • Acetylation of c-MYC is required for oncogenic transformation by HTLV-1 p30II/c-MYC. • Acetylation-defective c-MYC mutants are impaired for foci-formation by p30II/c-MYC. • The HTLV-1 p30II protein induces lysine-acetylation of c-MYC. • p30II is present in c-MYC nucleoprotein complexes in HTLV-1-transformed T-cells. • HTLV-1 p30II inhibits apoptosis in c-MYC-expressing proliferating cells

  12. Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30{sup II} accessory protein and the induction of oncogenic cellular transformation by p30{sup II}/c-MYC

    Romeo, Megan M.; Ko, Bookyung; Kim, Janice; Brady, Rebecca; Heatley, Hayley C.; He, Jeffrey; Harrod, Carolyn K.; Barnett, Braden [Laboratory of Molecular Virology, Department of Biological Sciences, and The Dedman College Center for Drug Discovery, Design, and Delivery, Southern Methodist University, Dallas, TX 75275-0376 (United States); Ratner, Lee [Departments of Medicine and Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Lairmore, Michael D. [University of California-Davis, School of Veterinary Medicine, One Shields Avenue, Davis, CA 95618 (United States); Martinez, Ernest [Department of Biochemistry, University of California, Riverside, CA 92521 (United States); Lüscher, Bernhard [Institute of Biochemistry, Klinikum, RWTH Aachen University, Pauwelsstrasse 30, 52057 Aachen (Germany); Robson, Craig N. [Northern Institute for Cancer Research, Newcastle University, The Medical School, Newcastle upon Tyne, NE2 4HH (United Kingdom); Henriksson, Marie [Department of Microbiology, Cell and Tumor Biology, Karolinska Institutet, Stockholm (Sweden); Harrod, Robert, E-mail: rharrod@smu.edu [Laboratory of Molecular Virology, Department of Biological Sciences, and The Dedman College Center for Drug Discovery, Design, and Delivery, Southern Methodist University, Dallas, TX 75275-0376 (United States)

    2015-02-15

    The human T-cell leukemia retrovirus type-1 (HTLV-1) p30{sup II} protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30{sup II} interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30{sup II} and c-MYC remain to be completely understood. Herein we demonstrate that p30{sup II} induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30{sup II} in c-myc{sup −/−} HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30{sup II} is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30{sup II} inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30{sup II}/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis. - Highlights: • Acetylation of c-MYC is required for oncogenic transformation by HTLV-1 p30{sup II}/c-MYC. • Acetylation-defective c-MYC mutants are impaired for foci-formation by p30{sup II}/c-MYC. • The HTLV-1 p30{sup II} protein induces lysine-acetylation of c-MYC. • p30{sup II} is present in c-MYC nucleoprotein complexes in HTLV-1-transformed T-cells. • HTLV-1 p30{sup II} inhibits apoptosis in c-MYC-expressing proliferating cells.

  13. Aberrant lysine acetylation in tumorigenesis: Implications in the development of therapeutics.

    Kaypee, Stephanie; Sudarshan, Deepthi; Shanmugam, Muthu K; Mukherjee, Debanjan; Sethi, Gautam; Kundu, Tapas K

    2016-06-01

    The 'language' of covalent histone modifications translates environmental and cellular cues into gene expression. This vast array of post-translational modifications on histones are more than just covalent moieties added onto a protein, as they also form a platform on which crucial cellular signals are relayed. The reversible lysine acetylation has emerged as an important post-translational modification of both histone and non-histone proteins, dictating numerous epigenetic programs within a cell. Thus, understanding the complex biology of lysine acetylation and its regulators is essential for the development of epigenetic therapeutics. In this review, we will attempt to address the complexities of lysine acetylation in the context of tumorigenesis, their role in cancer progression and emphasize on the modalities developed to target lysine acetyltransferases towards cancer treatment. PMID:26808162

  14. Target selection by natural and redesigned PUF proteins.

    Porter, Douglas F; Koh, Yvonne Y; VanVeller, Brett; Raines, Ronald T; Wickens, Marvin

    2015-12-29

    Pumilio/fem-3 mRNA binding factor (PUF) proteins bind RNA with sequence specificity and modularity, and have become exemplary scaffolds in the reengineering of new RNA specificities. Here, we report the in vivo RNA binding sites of wild-type (WT) and reengineered forms of the PUF protein Saccharomyces cerevisiae Puf2p across the transcriptome. Puf2p defines an ancient protein family present throughout fungi, with divergent and distinctive PUF RNA binding domains, RNA-recognition motifs (RRMs), and prion regions. We identify sites in RNA bound to Puf2p in vivo by using two forms of UV cross-linking followed by immunopurification. The protein specifically binds more than 1,000 mRNAs, which contain multiple iterations of UAAU-binding elements. Regions outside the PUF domain, including the RRM, enhance discrimination among targets. Compensatory mutants reveal that one Puf2p molecule binds one UAAU sequence, and align the protein with the RNA site. Based on this architecture, we redesign Puf2p to bind UAAG and identify the targets of this reengineered PUF in vivo. The mutant protein finds its target site in 1,800 RNAs and yields a novel RNA network with a dramatic redistribution of binding elements. The mutant protein exhibits even greater RNA specificity than wild type. The redesigned protein decreases the abundance of RNAs in its redesigned network. These results suggest that reengineering using the PUF scaffold redirects and can even enhance specificity in vivo. PMID:26668354

  15. Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein.

    Guangzhi Zhang

    Full Text Available The Gag protein of the mouse mammary tumor virus (MMTV is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV, assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.

  16. Visualization and targeted disruption of protein interactions in living cells

    Herce, Henry D.; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2013-01-01

    Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visual...

  17. Targeting protein kinases in the malaria parasite: update of an antimalarial drug target.

    Zhang, Veronica M; Chavchich, Marina; Waters, Norman C

    2012-01-01

    Millions of deaths each year are attributed to malaria worldwide. Transmitted through the bite of an Anopheles mosquito, infection and subsequent death from the Plasmodium species, most notably P. falciparum, can readily spread through a susceptible population. A malaria vaccine does not exist and resistance to virtually every antimalarial drug predicts that mortality and morbidity associated with this disease will increase. With only a few antimalarial drugs currently in the pipeline, new therapeutic options and novel chemotypes are desperately needed. Hit-to-Lead diversity may successfully provide novel inhibitory scaffolds when essential enzymes are targeted, for example, the plasmodial protein kinases. Throughout the entire life cycle of the malaria parasite, protein kinases are essential for growth and development. Ongoing efforts continue to characterize these kinases, while simultaneously pursuing them as antimalarial drug targets. A collection of structural data, inhibitory profiles and target validation has set the foundation and support for targeting the malarial kinome. Pursuing protein kinases as cancer drug targets has generated a wealth of information on the inhibitory strategies that can be useful for antimalarial drug discovery. In this review, progress on selected protein kinases is described. As the search for novel antimalarials continues, an understanding of the phosphor-regulatory pathways will not only validate protein kinase targets, but also will identify novel chemotypes to thwart malaria drug resistance. PMID:22242850

  18. Complex formation and catalytic activation by the PII signaling protein of N-acetyl-L-glutamate kinase from Synechococcus elongatus strain PCC 7942.

    Maheswaran, Mani; Urbanke, Claus; Forchhammer, Karl

    2004-12-31

    The signal transduction protein P(II) from the cyanobacterium Synechococcus elongatus strain PCC 7942 forms a complex with the key enzyme of arginine biosynthesis, N-acetyl-l-glutamate kinase (NAGK). Here we report the effect of complex formation on the catalytic properties of NAGK. Although pH and ion dependence are not affected, the catalytic efficiency of NAGK is strongly enhanced by binding of P(II), with K(m) decreasing by a factor of 10 and V(max) increasing 4-fold. In addition, arginine feedback inhibition of NAGK is strongly decreased in the presence of P(II), resulting in a tight control of NAGK activity under physiological conditions by P(II). Analysis of the NAGK-P(II) complex suggests that one P(II) trimer binds to one NAGK hexamer with a K(d) of approximately 3 nm. Complex formation is strongly affected by ATP and ADP. ADP is a strong inhibitor of complex formation, whereas ATP inhibits complex formation only in the absence of divalent cations or in the presence of Mg(2+) ions, together with increased 2-oxoglutarate concentrations. Ca(2+) is able to antagonize the negative effect of ATP and 2-oxoglutarate. ADP and ATP exert their adverse effect on NAGK-P(II) complex formation through binding to the P(II) protein. PMID:15502156

  19. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Yuri Pevzner

    2014-05-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

  20. Bacterial Protein Synthesis as a Target for Antibiotic Inhibition.

    Arenz, Stefan; Wilson, Daniel N

    2016-01-01

    Protein synthesis occurs on macromolecular machines, called ribosomes. Bacterial ribosomes and the translational machinery represent one of the major targets for antibiotics in the cell. Therefore, structural and biochemical investigations into ribosome-targeting antibiotics provide not only insight into the mechanism of action and resistance of antibiotics, but also insight into the fundamental process of protein synthesis. This review summarizes the recent advances in our understanding of protein synthesis, particularly with respect to X-ray and cryoelectron microscopy (cryo-EM) structures of ribosome complexes, and highlights the different steps of translation that are targeted by the diverse array of known antibiotics. Such findings will be important for the ongoing development of novel and improved antimicrobial agents to combat the rapid emergence of multidrug resistant pathogenic bacteria. PMID:27481773

  1. The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants

    Soto María

    2009-01-01

    Full Text Available Abstract Background Soil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogenicity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB forms part of a cluster of genes involved in the regulation of pathogenicity factors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene. Results The SMc02161 locus in S. meliloti shows similarities with members of the Major Facilitator Superfamily (MFS of transporters. A S. meliloti null-mutant shows increased sensitivity to chloramphenicol. This indication led us to rename the locus tep1 for transmembrane efflux protein. The lack of tep1 does not affect the appearance of swarming motility. Interestingly, nodule formation efficiency on alfalfa plants is improved in the tep1 mutant during the first days of the interaction though nod gene expression is lower than in the wild type strain. Curiously, a nodC mutation or the addition of N-acetyl glucosamine to the wild type strain lead to similar reductions in nod gene expression as in the tep1 mutant. Moreover, aminosugar precursors of Nod factors inhibit nodulation. Conclusion tep1 putatively encodes a transmembrane protein which can confer chloramphenicol resistance in S. meliloti by expelling the antibiotic outside the bacteria. The improved nodulation of alfalfa but reduced nod gene expression observed in the tep1 mutant suggests that Tep1 transports compounds which influence nodulation. In contrast to Bradyrhizobium japonicum, we show that in S. meliloti there is no feedback regulation

  2. Targeting dengue virus NS4B protein for drug discovery.

    Xie, Xuping; Zou, Jing; Wang, Qing-Yin; Shi, Pei-Yong

    2015-06-01

    The flavivirus nonstructural 4B protein (NS4B) has recently emerged as a valid antiviral target for drug discovery. Here we review (i) the current understanding of the structure and function of DENV NS4B, (ii) the approaches that have been taken to identify NS4B inhibitors, and (iii) the known inhibitors of flavivirus NS4B protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery. PMID:25796970

  3. Mass spectrometry based targeted protein quantification: methods and applications

    Pan, Sheng; Aebersold, Ruedi; Chen, Ru; Rush, John; Goodlett, David R.; McIntosh, Martin W.; Zhang, Jing; Brentnall, Teresa A.

    2009-01-01

    The recent advance in technology for mass spectrometry-based targeted protein quantification has opened new avenues for a broad range of proteomic applications in clinical research. The major breakthroughs are highlighted by the capability of using a “universal” approach to perform quantitative assays for a wide spectrum of proteins with minimum restrictions, and the ease of assembling multiplex detections in a single measurement. The quantitative approach relies on the use of synthetic stabl...

  4. Engineered protein nano-compartments for targeted enzyme localization.

    Swati Choudhary

    Full Text Available Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis.

  5. Mitochondrial genomes are retained by selective constraints on protein targeting.

    Björkholm, Patrik; Harish, Ajith; Hagström, Erik; Ernst, Andreas M; Andersson, Siv G E

    2015-08-18

    Mitochondria are energy-producing organelles in eukaryotic cells considered to be of bacterial origin. The mitochondrial genome has evolved under selection for minimization of gene content, yet it is not known why not all mitochondrial genes have been transferred to the nuclear genome. Here, we predict that hydrophobic membrane proteins encoded by the mitochondrial genomes would be recognized by the signal recognition particle and targeted to the endoplasmic reticulum if they were nuclear-encoded and translated in the cytoplasm. Expression of the mitochondrially encoded proteins Cytochrome oxidase subunit 1, Apocytochrome b, and ATP synthase subunit 6 in the cytoplasm of HeLa cells confirms export to the endoplasmic reticulum. To examine the extent to which the mitochondrial proteome is driven by selective constraints within the eukaryotic cell, we investigated the occurrence of mitochondrial protein domains in bacteria and eukaryotes. The accessory protein domains of the oxidative phosphorylation system are unique to mitochondria, indicating the evolution of new protein folds. Most of the identified domains in the accessory proteins of the ribosome are also found in eukaryotic proteins of other functions and locations. Overall, one-third of the protein domains identified in mitochondrial proteins are only rarely found in bacteria. We conclude that the mitochondrial genome has been maintained to ensure the correct localization of highly hydrophobic membrane proteins. Taken together, the results suggest that selective constraints on the eukaryotic cell have played a major role in modulating the evolution of the mitochondrial genome and proteome. PMID:26195779

  6. Interactions between the Nitrogen Signal Transduction Protein PII and N-Acetyl Glutamate Kinase in Organisms That Perform Oxygenic Photosynthesis

    Burillo, Sergio; Luque, Ignacio; Fuentes, Inmaculada; Contreras, Asunción

    2004-01-01

    PII, one of the most conserved signal transduction proteins, is believed to be a key player in the coordination of nitrogen assimilation and carbon metabolism in bacteria, archaea, and plants. However, the identity of PII receptors remains elusive, particularly in photosynthetic organisms. Here we used yeast two-hybrid approaches to identify new PII receptors and to explore the extent of conservation of PII signaling mechanisms between eubacteria and photosynthetic eukaryotes. Screening of Sy...

  7. ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera.

    Crimmins, Dan L; Brada, Nancy A; Lockwood, Christina M; Griest, Terry A; Waldemer, Rachel J; Cervinski, Mark A; Ohlendorf, Matthew F; McQuillan, Jay J; Ladenson, Jack H

    2010-12-01

    Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33 kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time. PMID:21054278

  8. Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

    N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.

  9. Identification of SUMO target proteins by quantitative proteomics

    Andersen, Jens S; Matic, Ivan; Vertegaal, Alfred C O

    2009-01-01

    , the methodological details of the application of stable isotope labeling of amino acids in cell culture (SILAC) for the identification of target proteins for SUMOs are described. In addition to steady state sumoylation, the sumoylated proteome undergoes dynamic rearrangements in response to a diverse...

  10. Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein

    Salanti, Ali; Clausen, Thomas M.; Agerbæk, Mette Ø.;

    2015-01-01

    Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can...... be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds...... strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification....

  11. 3,2'-Dimethyl-4-aminobiphenyl-DNA adduct formation in tumor target and nontarget organs of rapid and slow acetylator Syrian hamsters cogenic at the NAT2 locus.

    Feng, Y; Jiang, W; Deitz, A C; Hein, D W

    1996-10-01

    DNA adduct formation is an important step in initiation of the carcinogenic process. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is a well-documented multiorgan carcinogenic aromatic amine in rodents. In the present study, DMABP-DNA adduct levels were measured in rapid (Bio. 82.73/H-Pat(r)) and slow (Bio. 82.73/H-Pat(s)) acetylator Syrian hamsters congenic at the NAT2 locus following a single injection of 33 or 100 mg/kg body wt DMABP. Two DNA adducts, N-(deoxyguanosin-8-yl)-DMABP and 5-(deoxyguanosin-N2-yl)-DMABP, were identified and quantitated by 32P-postlabeling assay. After injection of 33 mg/kg, DMABP-DNA adducts were detected in urinary bladder at 6, 18, 24, and 48 hr with adduct levels increasing up to 48 hr postinjection. DMABP-DNA adducts were not detected in liver, colon, and heart. After injection of 100 mg/kg, DMABP-DNA adducts were detected in urinary bladder, liver, prostate, colon, and heart at 48 hr postinjection. DMABP-DNA adduct levels were significantly higher in urinary bladder (primary tumor target organ) than in the other organs of both rapid and slow acetylator congenic hamsters. N-(deoxyguanosin-8-yl)-DMABP levels were significantly higher in liver and prostate than in colon and heart of rapid and slow acetylator congenic hamsters, whereas 5-(deoxyguanosin-N2-yl)-DMABP levels were significantly higher in prostate than in colon and heart of rapid and slow acetylator congenic hamsters. DMABP-DNA adduct levels in each tissue examined did not differ significantly between rapid and slow acetylator hamsters following either 33 or 100 mg/kg injection. The tissue-dependent differences in DMABP-DNA adduct levels observed in the Syrian hamster differ from those reported in the rat and are consistent with previous studies that show DMABP induces primarily urinary bladder tumors in the Syrian hamster. PMID:8887447

  12. Expression and methylation of microsomal triglyceride transfer protein and acetyl-CoA carboxylase are associated with fatty liver syndrome in chicken.

    Liu, Zhen; Li, Qinghe; Liu, Ranran; Zhao, Guiping; Zhang, Yonghong; Zheng, Maiqing; Cui, Huanxian; Li, Peng; Cui, Xiaoyan; Liu, Jie; Wen, Jie

    2016-06-01

    The typical characteristic of fatty liver syndrome (FLS) is an increased hepatic triacylglycerol content, and a sudden decline in egg production often occurs. FLS may develop into fatty liver hemorrhagic syndrome (FLHS), characterized by sudden death from hepatic rupture and hemorrhage. DNA methylation is associated with transcriptional silencing, leading to the etiology and pathogenesis of some animal diseases. The roles of DNA methylation in the genesis of FLS, however, are largely unknown. The lipogenic methyl-deficient diet (MDD) caused FLS similar to human nonalcoholic steatohepatitis (NASH). After 16 Jingxing-Huang (JXH) hens were fed MDD for 10 wk, eight exhibited FLS (designated as FLS-susceptible birds); the remainder, without FLS, served as controls (NFLS). Physiological and biochemical variables, gene expression levels, and DNA methylation were determined in the liver. The development of FLS in JXH hens was accompanied by abnormal lipid accumulation. Relative expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and microsomal triglyceride transfer protein (MTTP) were significantly up-regulated in the FLS group in comparison with the NFLS group. The transcript abundance of sterol regulatory element binding protein 1 (SREBP-1c), stearoyl-CoA desaturase (SCD), liver X receptor alpha (LXRα), peroxisome proliferator-activated receptor alpha (PPARα), and peroxisome proliferator-activated receptor gamma (PPARγ) did not differ between the two groups. Interestingly, MTTP and ACC mRNA abundance were negatively correlated with the level of promoter methylation. The extent of DNA methylation of the cytosine-guanine (CpG) sites in the SREBP-1c, FAS, PPARα, and LXRα promoter regions was also analyzed by direct sequencing but none differed between FLS and NFLS birds. Taken together, these results specify link DNA methylation to the pathogenesis of FLS in chickens. PMID:27083546

  13. Protein disulphide isomerase as a target for nanoparticle-mediated sensitisation of cancer cells to radiation

    Taggart, L. E.; McMahon, S. J.; Butterworth, K. T.; Currell, F. J.; Schettino, G.; Prise, K. M.

    2016-05-01

    Radiation resistance and toxicity in normal tissues are limiting factors in the efficacy of radiotherapy. Gold nanoparticles (GNPs) have been shown to be effective at enhancing radiation-induced cell death, and were initially proposed to physically enhance the radiation dose deposited. However, biological responses of GNP radiosensitization based on physical assumptions alone are not predictive of radiosensitisation and therefore there is a fundamental research need to determine biological mechanisms of response to GNPs alone and in combination with ionising radiation. This study aimed to identify novel mechanisms of cancer cell radiosensitisation through the use of GNPs, focusing on their ability to induce cellular oxidative stress and disrupt mitochondrial function. Using N-acetyl-cysteine, we found mitochondrial oxidation to be a key event prior to radiation for the radiosensitisation of cancer cells and suggests the overall cellular effects of GNP radiosensitisation are a result of their interaction with protein disulphide isomerase (PDI). This investigation identifies PDI and mitochondrial oxidation as novel targets for radiosensitisation.

  14. Visualization and targeted disruption of protein interactions in living cells.

    Herce, Henry D; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M Cristina

    2013-01-01

    Protein-protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein-protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53-HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein-protein interactions in practically any cell type and species. PMID:24154492

  15. Widespread dual targeting of proteins in land plants: When, where, how and why

    Carrie, Chris; Whelan, James

    2013-01-01

    Since the discovery of the first dual targeted protein in plants in 1995 the number of dual targeted proteins in plants has grown to over 250 proteins. Much work and investigations have focused on identifying how or what makes a protein dual targeted. Recently, more research has focused on the evolution and conservation of dual targeting of proteins in plants. This new work has demonstrated that dual targeting arose early within the evolution of plants and because it is rarely lost, once gain...

  16. Targeting membrane proteins for antibody discovery using phage display.

    Jones, Martina L; Alfaleh, Mohamed A; Kumble, Sumukh; Zhang, Shuo; Osborne, Geoffrey W; Yeh, Michael; Arora, Neetika; Hou, Jeff Jia Cheng; Howard, Christopher B; Chin, David Y; Mahler, Stephen M

    2016-01-01

    A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b. PMID:27189586

  17. Human immune cell targeting of protein nanoparticles - caveospheres.

    Glass, Joshua J; Yuen, Daniel; Rae, James; Johnston, Angus P R; Parton, Robert G; Kent, Stephen J; De Rose, Robert

    2016-04-14

    Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells-an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines. PMID:27031090

  18. Human immune cell targeting of protein nanoparticles - caveospheres

    Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

    2016-04-01

    Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

  19. Probing the acetylation code of histone H4.

    Lang, Diana; Schümann, Michael; Gelato, Kathy; Fischle, Wolfgang; Schwarzer, Dirk; Krause, Eberhard

    2013-10-01

    Histone modifications play crucial roles in genome regulation with lysine acetylation being implicated in transcriptional control. Here we report a proteome-wide investigation of the acetylation-dependent protein-protein interactions of the N-terminal tail of histone H4. Quantitative peptide-based affinity MS experiments using the SILAC approach determined the interactomes of H4 tails monoacetylated at the four known acetylation sites K5, K8, K12, and K16, bis-acetylated at K5/K12, triple-acetylated at K8/12/16 and fully tetra-acetylated. A set of 29 proteins was found enriched on the fully acetylated H4 tail while specific binders of the mono and bis-acetylated tails were barely detectable. These observations are in good agreement with earlier reports indicating that the H4 acetylation state establishes its regulatory effects in a cumulative manner rather than via site-specific recruitment of regulatory proteins. PMID:23970329

  20. Drug bioactivation, covalent binding to target proteins and toxicity relevance.

    Zhou, Shufeng; Chan, Eli; Duan, Wei; Huang, Min; Chen, Yu-Zong

    2005-01-01

    A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients

  1. Molecular Structure and Target Recognition of Neuronal Calcium Sensor Proteins

    James Ames

    2012-02-01

    Full Text Available Neuronal calcium sensor (NCS proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1 modulates synaptic activity and neuronal secretion, K+ channel interacting proteins (KChIPs regulate ion channels to control neuronal excitability, and DREAM (KChIP3 is a transcriptional repressor that regulates neuronal gene expression. Here we review the molecular structures of myristoylated forms of NCS1, recoverin, and GCAP1 that all look very different, suggesting that the sequestered myristoyl group helps to refold these highly homologous proteins into very different structures. The molecular structure of NCS target complexes have been solved for recoverin bound to rhodopsin kinase, NCS-1 bound to phosphatidylinositol 4-kinase, and KChIP1 bound to A-type K+ channels. We propose that N-terminal myristoylation is critical for shaping each NCS family member into a different structure, which upon Ca2+-induced extrusion of the myristoyl group exposes a unique set of previously masked residues that interact with a particular physiological target.

  2. From protein catalogues towards targeted proteomics approaches in cereal grains

    Finnie, Christine; Sultan, Abida; Grasser, Klaus D.

    2011-01-01

    Due to their importance for human nutrition, the protein content of cereal grains has been a subject of intense study for over a century and cereal grains were not surprisingly one of the earliest subjects for 2D-gel-based proteome analysis. Over the last two decades, countless cereal grain...... extraction, separation and identification of proteins and peptides is facilitating functional proteomics and analysis of sub-proteomes from small amounts of starting material, such as seed tissues. The combination of proteomics with structural and functional analysis is increasingly applied to target subsets...

  3. Obesity, cancer, and acetyl-CoA metabolism

    Lee, Joyce V.; Shah, Supriya A.; Wellen, Kathryn E.

    2013-01-01

    As rates of obesity soar in the Unites States and around the world, cancer attributed to obesity has emerged as major threat to public health. The link between obesity and cancer can be attributed in part to the state of chronic inflammation that develops in obesity. Acetyl-CoA production and protein acetylation patterns are highly sensitive to metabolic state and are significantly altered in obesity. In this article, we explore the potential role of nutrient-sensitive lysine acetylation in r...

  4. Targeting Protein Kinase C subtypes in pancreatic cancer

    Storz, Peter

    2015-01-01

    In preclinical studies protein kinase C (PKC) enzymes have been implicated in regulating many aspects of pancreatic cancer development and progression. However, clinical phase I or phase II trials with compounds targeting classical PKC isoforms were not successful. Recent studies implicate that mainly atypical and novel PKC enzymes regulate oncogenic signaling pathways in pancreatic cancer. Members of these two subgroups converge signaling induced by mutant Kras, growth factors and inflammato...

  5. p53 Acetylation: Regulation and Consequences

    Reed, Sara M. [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Quelle, Dawn E., E-mail: dawn-quelle@uiowa.edu [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States)

    2014-12-23

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  6. p53 Acetylation: Regulation and Consequences

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer

  7. p53 Acetylation: Regulation and Consequences

    Sara M. Reed

    2014-12-01

    Full Text Available Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  8. Targeting protein kinases to reverse multidrug resistance in sarcoma.

    Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

    2016-02-01

    Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. PMID:26827688

  9. Sequence heterogeneity accelerates protein search for targets on DNA

    The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity, and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry, and heterogeneity of a genome

  10. Sequence heterogeneity accelerates protein search for targets on DNA

    Shvets, Alexey A.; Kolomeisky, Anatoly B., E-mail: tolya@rice.edu [Department of Chemistry and Center for Theoretical Biological Physics, Rice University, Houston, Texas 77005 (United States)

    2015-12-28

    The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity, and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry, and heterogeneity of a genome.

  11. The Effect of Acetyl Salicylic Acid Induced Nitric Oxide Synthesis in the Normalization of Hypertension through the Stimulation of Renal Cortexin Synthesis and by the Inhibition of Dermcidin Isoform 2, A Hypertensive Protein Production

    Ghosh, Rajeshwary; Bank, Sarbashri; Maji, Uttam K.; Bhattacharya, Rabindra; Guha, Santanu; Khan, Nighat N.; Sinha, A. Kumar

    2014-01-01

    Currently, there is no specific medication for essential hypertension (EH), a major form of the condition, in man. As acetyl salicylic acid (aspirin) is reported to stimulate the synthesis of renal (r)-cortexin, an anti-essential hypertensive protein, and, as aspirin is reported to inhibit dermcidin isoform 2 (dermcidin), a causative protein for EH, the role of aspirin in the control of EH in man was studied. Oral administration of 150 mg aspirin/70 kg body weight in subjects with EH was foun...

  12. AlphaSpace: Fragment-Centric Topographical Mapping To Target Protein-Protein Interaction Interfaces.

    Rooklin, David; Wang, Cheng; Katigbak, Joseph; Arora, Paramjit S; Zhang, Yingkai

    2015-08-24

    Inhibition of protein-protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy--that builds on the principles of fragment-based drug discovery (FBDD)--is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein's Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features--alpha-atom and alpha-space--and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors. PMID:26225450

  13. Estimation of benchmark dose as the threshold levels of urinary cadmium, based on excretion of total protein, β 2-microglobulin, and N-acetyl-β-D-glucosaminidase in cadmium nonpolluted regions in Japan

    Previously, we investigated the association between urinary cadmium (Cd) concentration and indicators of renal dysfunction, including total protein, β 2-microglobulin (β 2-MG), and N-acetyl-β-D-glucosaminidase (NAG). In 2778 inhabitants ≥50 years of age (1114 men, 1664 women) in three different Cd nonpolluted areas in Japan, we showed that a dose-response relationship existed between renal effects and Cd exposure in the general environment without any known Cd pollution. However, we could not estimate the threshold levels of urinary Cd at that time. In the present study, we estimated the threshold levels of urinary Cd as the benchmark dose low (BMDL) using the benchmark dose (BMD) approach. Urinary Cd excretion was divided into 10 categories, and an abnormality rate was calculated for each. Cut-off values for urinary substances were defined as corresponding to the 84% and 95% upper limit values of the target population who have not smoked. Then we calculated the BMD and BMDL using a log-logistic model. The values of BMD and BMDL for all urinary substances could be calculated. The BMDL for the 84% cut-off value of β 2-MG, setting an abnormal value at 5%, was 2.4 μg/g creatinine (cr) in men and 3.3 μg/g cr in women. In conclusion, the present study demonstrated that the threshold level of urinary Cd could be estimated in people living in the general environment without any known Cd-pollution in Japan, and the value was inferred to be almost the same as that in Belgium, Sweden, and China

  14. Erythrocyte G protein as a novel target for malarial chemotherapy.

    Sean C Murphy

    2006-12-01

    Full Text Available BACKGROUND: Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte Gs signaling blocked invasion by the human malaria parasite Plasmodium falciparum. METHODS AND FINDINGS: We investigated the erythrocyte guanine nucleotide regulatory protein Gs as a novel antimalarial target. Erythrocyte "ghosts" loaded with a Gs peptide designed to block Gs interaction with its receptors, were blocked in beta-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte Gs is functional and that propranolol, an antagonist of G protein-coupled beta-adrenergic receptors, dampens Gs activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host Gs to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other beta2-antagonists. beta-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection. CONCLUSIONS: Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte Gs antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials.

  15. Probing the acetylation code of histone H4.

    Lang, D; Schümann, M; Gelato, K.; Fischle, W; Schwarzer, D; Krause, E.

    2013-01-01

    Histone modifications play crucial roles in genome regulation with lysine acetylation being implicated in transcriptional control. Here we report a proteome-wide investigation of the acetylation-dependent protein–protein interactions of the N-terminal tail of histone H4. Quantitative peptide-based affinity MS experiments using the SILAC approach determined the interactomes of H4 tails monoacetylated at the four known acetylation sites K5, K8, K12, and K16, bis-acetylated at K5/K12, triple-ace...

  16. Recombinant protein expression by targeting pre-selected chromosomal loci

    Krömer Wolfgang

    2009-12-01

    Full Text Available Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs were targeted by Flp recombinase mediated cassette exchange (RMCE. The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context

  17. Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

    Biely, Peter; Cziszarava, Maria; Agger, Jane W.;

    2014-01-01

    Results The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most promin...

  18. Identification of the lipid droplet targeting domain of the Cidea protein[S

    Christianson, Jennifer L.; Boutet, Emilie; Puri, Vishwajeet; Chawla, Anil; Czech, Michael P.

    2010-01-01

    Cidea, the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) domain-containing protein, is targeted to lipid droplets in mouse adipocytes, where it inhibits triglyceride hydrolysis and promotes lipid storage. In mice, Cidea may prevent lipolysis by binding and shielding lipid droplets from lipase association. Here we demonstrate that human Cidea localizes with lipid droplets in both adipocyte and nonadipocyte cell lines, and we ascribe specific functions to its protein domai...

  19. Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

    Jaume Torres

    2015-06-01

    Full Text Available Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i the envelope protein in coronaviruses and (ii the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity.

  20. Flexibility in targeting and insertion during bacterial membrane protein biogenesis

    The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors

  1. Influence of Translation Initiation on Organellar Protein Targeting in Arabidopsis

    Sally A. Mackenzie

    2011-04-18

    A primary focus of the Mackenzie laboratory is the elucidation of processes and machinery for mitochondrial genome maintenance and transmission in higher plants. We have found that numerous organellar DNA maintenance components in plants appear to be dual targeted to mitochondria and plastids. Of particular interest was the observation that some twin (tandemly arrayed) dual targeting presequences appeared to utilize non-AUG alternative translation initiation, allowing for multiple translation starts at a single gene. Two aspects of this phenomenon were of particular interest: (1) Alternative translation initiation might provide a mechanism to regulate protein targeting temporally and spatially, a possibility that had not been demonstrated previously, and (2) alternative translation initiation might occur in genes involved in nuclear-controlled mitochondrial genome recombination, thought to be exclusively mitochondrial in their function. During the course of this research, we pursued three aims, with an emphasis on two specific genes of interest: POLgamma2, an organellar DNA polymerase, and MSH1, a MutS homolog thought to participate in mitochondrial, but not plastid, genome recombination surveillance. Our aims were to (1) Identify additional genes within Arabidopsis and other genomes that employ non-AUG alternative translation initiation, (2) Locate sequences upstream to the annotated AUG that confer alternative non-AUG translation initiation activity, and (3) Identify cis and trans factors that influence start site selection in genes with non-AUG starts. Toward these ends, we have shown that non-AUG initiation occurs in a number of genes, likely influencing targeting behavior of the protein. We have also shown that start site selection is strongly influenced by Kozak consensus sequence environment, indicating that alternative translation initiation in plants occurs by relaxation of ribosome scanning.

  2. Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

    Baginsky, Sacha

    2016-06-01

    The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation. PMID:26969742

  3. The potential for targeting extracellular LOX proteins in human malignancy

    Mayorca Guiliani, Alejandro Enrique; Erler, Janine T

    2013-01-01

    , is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient...... deaths. However, LOX biosynthesis results in by-product with antiproliferative properties in certain cancers, and LOX enzymes may have different effects depending on the molecular network in which they are active. Therefore, the design of therapies targeting the LOX family needs to be guided by the...

  4. Effects of intersegmental transfers on target location by proteins

    Sheinman, Michael

    2008-01-01

    We study a model for a protein searching for a target, using facilitated diffusion, on a DNA molecule confined in a finite volume. The model includes three distinct pathways for facilitated diffusion: (a) sliding - in which the protein diffuses along the contour of the DNA (b) jumping - where the protein travels between two sites along the DNA by three-dimensional diffusion, and finally (c) intersegmental transfer - which allows the protein to move from one site to another by transiently binding both at the same time. The typical search time is calculated using scaling arguments which are verified numerically. Our results suggest that the inclusion of intersegmental transfer (i) decreases the search time considerably (ii) makes the search time much more robust to variations in the parameters of the model and (iii) that the optimal search time occurs in a regime very different than that found for models which ignore intersegmental transfers. The behavior we find is rich and shows surprising dependencies, for e...

  5. The bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl-CoA carboxylase.

    Gerhardt, Edileusa C M; Rodrigues, Thiago E; Müller-Santos, Marcelo; Pedrosa, Fabio O; Souza, Emanuel M; Forchhammer, Karl; Huergo, Luciano F

    2015-03-01

    Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate-limiting step in fatty acid biosynthesis is catalyzed by a multi-subunit form of the acetyl-CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl-CoA to produce malonyl-CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the PII family. Here we show that the GlnB paralogues of the PII proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC-BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC-BCCP-GlnB interaction and ACC inhibition were relieved by 2-oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2-oxoglutarate-sensitive dissociable regulatory subunit of ACC in Bacteria. PMID:25557370

  6. Targeting soluble proteins to exosomes using a ubiquitin tag.

    Cheng, Yong; Schorey, Jeffery S

    2016-06-01

    As "natural" antigen carriers in the body, exosomes are potential vaccine vectors. A number of animal studies indicate that antigen-containing exosomes can induce a specific immune response which can protect against tumor progression or various infections. Exosomes that carry the protective antigens can be purified from cells that release them including tumor cells, dendritic cells, and macrophages. However, this strategy is restricted to proteins that are naturally targeted to exosomes and is therefore limited in the number of antigens present within exosomes. Therefore, with the goal of developing an exosome-based vaccine that is more flexible in its antigen composition and has the potential to be scalable, we have developed a new approach where recombinant soluble proteins can be packaged into exosomes and released from a transformed cell line. In this study, we determined that a C-terminal fusion of ubiquitin to EGFP, tumor antigenic protein nHer2 and Mycobacterium tuberculosis proteins Ag85B and ESAT6 served as an efficient delivery sequence into exosomes when expressed in a human embryonic kidney (HEK 293) cell line, a cell line widely used in industrial recombinant protein production. Two stably transgenic HEK293 cell lines were generated using a retroviral vector to express the Ag85B-ESAT6 fusion protein either alone or tagged at the C-terminus with ubiquitin. Both transformants released exosomes containing the fusion proteins. However, the concentration of Ag85B and ESAT6 in exosomes was increased approximately 10-fold when they were coupled to ubiquitin. Moreover, when the exosomes were used for immunization, there was a direct correlation between the amount of fusion protein within the exosomes and the number of Ag85B and ESAT6 specific INFɣ-secreting T lymphocytes in the lung and spleen. This suggests that exosomes containing recombinant antigen can be used to elicit a T cell response. In summary our data indicates that a ubiquitin-based exosomal

  7. The growing landscape of lysine acetylation links metabolism and cell signalling

    Choudhary, Chuna Ram; Weinert, Brian Tate; Nishida, Yuya;

    2014-01-01

    Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation......, implicating it in many biological processes through the regulation of protein interactions, activity and localization. In addition, proteins are frequently modified by other types of acylations, such as formylation, butyrylation, propionylation, succinylation, malonylation, myristoylation, glutarylation and...... deacylating enzymes and also highlight the mechanisms by which acetylation regulates various cellular processes....

  8. G protein-coupled receptors as promising cancer targets.

    Liu, Ying; An, Su; Ward, Richard; Yang, Yang; Guo, Xiao-Xi; Li, Wei; Xu, Tian-Rui

    2016-07-01

    G protein-coupled receptors (GPCRs) regulate an array of fundamental biological processes, such as growth, metabolism and homeostasis. Specifically, GPCRs are involved in cancer initiation and progression. However, compared with the involvement of the epidermal growth factor receptor in cancer, that of GPCRs have been largely ignored. Recent findings have implicated many GPCRs in tumorigenesis, tumor progression, invasion and metastasis. Moreover, GPCRs contribute to the establishment and maintenance of a microenvironment which is permissive for tumor formation and growth, including effects upon surrounding blood vessels, signaling molecules and the extracellular matrix. Thus, GPCRs are considered to be among the most useful drug targets against many solid cancers. Development of selective ligands targeting GPCRs may provide novel and effective treatment strategies against cancer and some anticancer compounds are now in clinical trials. Here, we focus on tumor related GPCRs, such as G protein-coupled receptor 30, the lysophosphatidic acid receptor, angiotensin receptors 1 and 2, the sphingosine 1-phosphate receptors and gastrin releasing peptide receptor. We also summarize their tissue distributions, activation and roles in tumorigenesis and discuss the potential use of GPCR agonists and antagonists in cancer therapy. PMID:27000991

  9. Arabidopsis flower development-of protein complexes, targets, and transport.

    Becker, Annette; Ehlers, Katrin

    2016-03-01

    Tremendous progress has been achieved over the past 25 years or more of research on the molecular mechanisms of floral organ identity, patterning, and development. While collections of floral homeotic mutants of Antirrhinum majus laid the foundation already at the beginning of the previous century, it was the genetic analysis of these mutants in A. majus and Arabidopsis thaliana that led to the development of the ABC model of floral organ identity more than 20 years ago. This intuitive model kick-started research focused on the genetic mechanisms regulating flower development, using mainly A. thaliana as a model plant. In recent years, interactions among floral homeotic proteins have been elucidated, and their direct and indirect target genes are known to a large extent. Here, we provide an overview over the advances in understanding the molecular mechanism orchestrating A. thaliana flower development. We focus on floral homeotic protein complexes, their target genes, evidence for their transport in floral primordia, and how these new results advance our view on the processes downstream of floral organ identity, such as organ boundary formation or floral organ patterning. PMID:25845756

  10. Heat-Shock Protein 90-Targeted Nano Anticancer Therapy.

    Rochani, Ankit K; Ravindran Girija, Aswathy; Borah, Ankita; Maekawa, Toru; Sakthi Kumar, D

    2016-04-01

    Suboptimal chemotherapy of anticancer drugs may be attributed to a variety of cellular mechanisms, which synergize to dodge the drug responses. Nearly 2 decades of heat-shock protein 90 (Hsp90)-targeted drug discovery has shown that the mono-therapy with Hsp90 inhibitors seems to be relatively ineffective compared with combination treatment due to several cellular dodging mechanisms. In this article, we have tried to analyze and review the Hsp90 and mammalian target of rapamycin (m-TOR)-mediated drug resistance mechanisms. By using this information we have discussed about the rationale behind use of drug combinations that includes both or any one of these inhibitors for cancer therapy. Currently, biodegradable nano vector (NV)-loaded novel drug delivery systems have shown to resolve the problems of poor bioavailability. NVs of drugs such as paclitaxel, doxorubicin, daunorubicin, and others have been successfully introduced for medicinal use. Hence, looking at the success of NVs, in this article we have also discussed the progress made in the delivery of biodegradable NV-loaded Hsp90 and m-TOR-targeted inhibitors in multiple drug combinations. We have also discussed the possible ways by which the market success of biodegradable NVs can positively impact the clinical trials of anti-Hsp90 and m-TOR combination strategy. PMID:26886301

  11. Differential patterns of histone acetylation in inflammatory bowel diseases

    Adcock Ian M

    2011-01-01

    Full Text Available Abstract Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD to study the expression of acetylated histones (H 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP in dextran sulfate sodium (DSS-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation.

  12. Protein painting reveals solvent-excluded drug targets hidden within native protein–protein interfaces

    Luchini, Alessandra; Espina, Virginia; Liotta, Lance A.

    2014-01-01

    Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein–protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets. PMID:25048602

  13. Structure determination of drug target proteins by neutron crystallography

    High resolution X-ray crystallography provides information for most of the atoms comprising the proteins, with the exception of hydrogen atoms. Whereas, neutron crystallography, which is a powerful technique for locating hydrogen atoms, enables us to obtain accurate atomic positions within proteins. Neutron diffraction data can provide information of the location of hydrogen atoms to the structural information determined by X-ray crystallography. Here, we show the recent results of the structural determination of drug-target proteins, porcine pancreatic elastase and human immuno-deficiency virus type-1 protease by both X-ray and neutron diffraction. The structure of porcine pancreatic elastase with its potent inhibitor was determined to 0.094 nm resolution by X-ray diffraction and 0.165 nm resolution by neutron diffraction. The structure of HIV-PR with its potent inhibitor was also determined to 0.093 nm resolution by X-ray diffraction and 0.19 nm resolution by neutron diffraction. The ionization state and the location of hydrogen atoms of the catalytic residue in these enzymes were determined by neutron diffraction. Furthermore, collaborative use of both X-ray and neutron crystallography to identify the location of ambiguous hydrogen atoms will be shown. (author)

  14. Intracellular imaging of targeted proteins labeled with quantum dots

    We developed a new method for imaging the movement of targeted proteins in living cancer cells with photostable and bright quantum dots (QDs). QDs were conjugated with various molecules and proteins, such as phalloidin, anti-tubulin antibody and kinesin. These bioconjugated QDs were mixed with a transfection reagent and successfully internalized into living cells. The movements of individual QDs were tracked for long periods of time. Phalloidin conjugated QDs bound to actin filaments and showed almost no movement. In contrast, anti-tubulin antibody conjugated QDs bound to microtubules and revealed dynamic movement of microtubules. Kinesin showed an interesting behavior whereby kinesin came to be almost paused briefly for a few seconds and then moved once again. This is in direct contrast to the smoothly continuous movement of kinesin in an in vitro assay. The maximum velocity of kinesin in cells was faster than that in the in vitro assay. These results suggest that intracellular movement of kinesin is different from that in the in vitro assay. This newly described method will be a powerful tool for investigating the functions of proteins in living cells

  15. T3-induced liver AMP-activated protein kinase signaling: Redox dependency and upregulation of downstream targets

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Morales, Paula; Ceballo, Juan; Fischer, Alvaro; Escudero, Nicolás; Escobar, Oscar

    2014-01-01

    AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum β-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca2+-calmodulin-dependent protein kinase kinase-β and transforming growth factor-β-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of β-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury. PMID:25516653

  16. Targeting of p300/CREB binding protein coactivators by simian virus 40 is mediated through p53.

    Borger, Darrell R; DeCaprio, James A

    2006-05-01

    The primary transforming functions of simian virus 40 large T antigen (SV40 LT) are conferred primarily through the binding and inactivation of p53 and the retinoblastoma family members. Normal p53 function requires an association with the CREB binding protein (CBP)/p300 coactivators, and a ternary complex containing SV40 LT, p53, and CBP/p300 has been identified previously. In this report, we have evaluated a secondary function of p53 bound to the SV40 LT complex in mediating the binding of human CBP/p300. We demonstrate that p53 associated with SV40 LT was posttranslationally modified in a manner consistent with the binding of CBP/p300. Furthermore, expression of SV40 LT induced the proportion of p53 phosphorylated on S15. An essential function for p53 in bridging the interaction between SV40 LT and CBP/p300 was identified through the reconstitution of the SV40 LT-CBP/p300 complex upon p53 reexpression in p53-null cells. In addition, the SV40 LT-CBP/p300 complex was disrupted through RNA interference-mediated depletion of endogenous p53. We also demonstrate that SV40 LT was acetylated in a p300- and p53-dependent manner, at least in part through the CH3 domain of p300. Therefore, the binding of p53 serves to modify SV40 LT by targeting CBP and p300 binding to direct the acetylation of SV40 LT. PMID:16611888

  17. G protein coupled receptors as targets for next generation pesticides.

    Audsley, Neil; Down, Rachel E

    2015-12-01

    There is an on-going need for the discovery and development of new pesticides due to the loss of existing products through the continuing development of resistance, the desire for products with more favourable environmental and toxicological profiles and the need to implement the principles of integrated pest management. Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behaviour, including reproduction, osmoregulation, growth and development. Modifying normal receptor function by blocking or over stimulating its actions may either result in the death of a pest or disrupt its normal fitness or reproductive capacity to reduce pest populations. Hence GPCRs offer potential targets for the development of next generation pesticides providing opportunities to discover new chemistries for invertebrate pest control. Such receptors are important targets for pharmaceutical drugs, but are under-exploited by the agro-chemical industry. The octopamine receptor agonists are the only pesticides with a recognized mode of action, as described in the classification scheme developed by the Insecticide Resistance Action Committee, that act via a GPCR. The availability of sequenced insect genomes has facilitated the characterization of insect GPCRs, but the development and utilization of screening assays to identify lead compounds has been slow. Various studies using knock-down technologies or applying the native ligands and/or neuropeptide analogues to pest insects in vivo, have however demonstrated that modifying normal receptor function can have an insecticidal effect. This review presents examples of potential insect neuropeptide receptors that are potential targets for lead compound development, using case studies from three representative pest species, Tribolium castaneum, Acyrthosiphon pisum, and Drosophila suzukii. Functional analysis studies on T. castaneum suggest that GPCRs involved in growth and development (eclosion

  18. N-acetylation and phosphorylation of Sec complex subunits in the ER membrane

    Soromani Christina

    2012-12-01

    Full Text Available Abstract Background Covalent modifications of proteins provide a mechanism to control protein function. Here, we have investigated modifications of the heptameric Sec complex which is responsible for post-translational protein import into the endoplasmic reticulum (ER. It consists of the Sec61 complex (Sec61p, Sbh1p, Sss1p which on its own mediates cotranslational protein import into the ER and the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p. Little is known about the biogenesis and regulation of individual Sec complex subunits. Results We show that Sbh1p when it is part of the Sec61 complex is phosphorylated on T5 which is flanked by proline residues. The phosphorylation site is conserved in mammalian Sec61ß, but only partially in birds, and not in other vertebrates or unicellular eukaryotes, suggesting convergent evolution. Mutation of T5 to A did not affect the ability of mutant Sbh1p to complement the growth defect in a Δsbh1Δsbh2 strain, and did not result in a hypophosphorylated protein which shows that alternate sites can be used by the T5 kinase. A survey of yeast phosphoproteome data shows that Sbh1p can be phosphorylated on multiple sites which are organized in two patches, one at the N-terminus of its cytosolic domain, the other proximal to the transmembrane domain. Surprisingly, although N-acetylation has been shown to interfere with ER targeting, we found that both Sbh1p and Sec62p are cotranslationally N-acetylated by NatA, and N-acetyl-proteome data indicate that Sec61p is modified by the same enzyme. Mutation of the N-acetylation site, however, did not affect Sec62p function in posttranslational protein import into the ER. Disabling NatA resulted in growth retardation, but not in co- or posttranslational translocation defects or instability of Sec62p or Sbh1p. Conclusions We conclude that N-acetylation of transmembrane and tail-anchored proteins does not interfere with their ER-targeting, and that Sbh1p phosphorylation on T5

  19. Acetylated Rhamnogalacturonans from Immature Fruits of Abelmoschus esculentus Inhibit the Adhesion of Helicobacter pylori to Human Gastric Cells by Interaction with Outer Membrane Proteins

    Christian Thöle

    2015-09-01

    Full Text Available Polysaccharide containing extracts from immature fruits of okra (Abelmoschus esculentus are known to exhibit antiadhesive effects against bacterial adhesion of Helicobacter pylori (H. pylori to stomach tissue. The present study investigates structural and functional features of polymers responsible for this inhibition of bacterial attachment to host cells. Ammonium sulfate precipitation of an aqueous extract yielded two fractions at 60% and 90% saturation with significant antiadhesive effects against H. pylori, strain J99, (FE60% 68% ± 15%; FE90% 75% ± 11% inhibition rates after preincubation of the bacteria at 1 mg/mL. Sequential extraction of okra fruits yielded hot buffer soluble solids (HBSS with dose dependent antiadhesive effects against strain J99 and three clinical isolates. Preincubation of H. pylori with HBSS (1 mg/mL led to reduced binding to 3ʹ-sialyl lactose, sialylated Lea and Lex. A reduction of bacterial binding to ligands complementary to BabA and SabA was observed when bacteria were pretreated with FE90%. Structural analysis of the antiadhesive polysaccharides (molecular weight, monomer composition, linkage analysis, stereochemistry, and acetylation indicated the presence of acetylated rhamnogalacturonan-I polymers, decorated with short galactose side chains. Deacetylation of HBSS and FE90% resulted in loss of the antiadhesive activity, indicating esterification being a prerequisite for antiadhesive activity.

  20. N-Myc-interacting protein (NMI) negatively regulates epithelial-mesenchymal transition by inhibiting the acetylation of NF-κB/p65.

    Hou, Jingjing; Wang, Tao; Xie, Qingqing; Deng, Weixian; Yang, James Y; Zhang, Si Qing; Cai, Jian-Chun

    2016-06-28

    The epithelial-mesenchymal transition (EMT) plays an essential role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and tumor progression. However, the mechanisms underlying this process are poorly understood. Many signaling pathways, including the NF-κB signaling pathway, trigger EMT during development and differentiation. In the present study, we report that N-Myc interactor (NMI) inhibits EMT progression by suppressing transcriptional activities of NF-κB in human gastric cancer cells. We show that the expression of NMI is significantly reduced in invasive gastric cancer cells and gastric cancer tissues. Overexpression of NMI inhibited cell migration and invasion, and this inhibition was enhanced after TNF-α stimulation. Tumorigenicity assay in nude mice support the notion that NMI inhibits EMT in cancer cells. Mechanistically, NMI promotes the interaction between NF-κB/p65 and histone deacetylases (HDACs) and inhibits the acetylation and transcriptional activity of p65. The expression of p65 rescues NMI-mediated inhibition of EMT and the inhibition of the acetylation of p65 mediated by NMI is HDACs-dependent. Taken together, these findings suggest that NMI can suppress tumor invasion and metastasis by inhibiting NF-κB pathways, providing an alternative mechanism for EMT inhibition in stomach neoplasm. PMID:27012186

  1. The pros and cons of increased trypsin-to-protein ratio in targeted protein analysis.

    Egeland, Siri Valen; Reubsaet, Léon; Halvorsen, Trine Grønhaug

    2016-05-10

    The effect of increasing the trypsin amount in bottom-up based targeted protein analysis is evaluated. By applying an increased trypsin-to-protein ratio (1:1 (w/w)) after heat denaturation (60°C), reduction and alkylation, the digestion time could be reduced profoundly compared to conventional digestion conditions (ratio 1:40, overnight) without compromising method sensitivity or digestion repeatability. The procedure was obtained after a systematic evaluation of trypsin level and trypsin quality using a set of three model proteins: human chorionic gonadotropin (hCG), bovine serum albumin (BSA) and cytochrome C (CytC). All peptides monitored were produced at similar or higher levels after 45min at trypsin-to-protein ratio 1:1, compared to conventional overnight digestion (exception: CytC using modified trypsin, required up to 4h (at 1:1 ratio) in order achieve this). Peptide decay due to chymotryptic activity was observed at longer digestion times, but the effect was circumvented using digestion times protein hCG to demonstrate its applicability. PMID:26907699

  2. Target highlights in CASP9: Experimental target structures for the critical assessment of techniques for protein structure prediction.

    Kryshtafovych, Andriy; Moult, John; Bartual, Sergio G; Bazan, J Fernando; Berman, Helen; Casteel, Darren E; Christodoulou, Evangelos; Everett, John K; Hausmann, Jens; Heidebrecht, Tatjana; Hills, Tanya; Hui, Raymond; Hunt, John F; Seetharaman, Jayaraman; Joachimiak, Andrzej; Kennedy, Michael A; Kim, Choel; Lingel, Andreas; Michalska, Karolina; Montelione, Gaetano T; Otero, José M; Perrakis, Anastassis; Pizarro, Juan C; van Raaij, Mark J; Ramelot, Theresa A; Rousseau, Francois; Tong, Liang; Wernimont, Amy K; Young, Jasmine; Schwede, Torsten

    2011-01-01

    One goal of the CASP community wide experiment on the critical assessment of techniques for protein structure prediction is to identify the current state of the art in protein structure prediction and modeling. A fundamental principle of CASP is blind prediction on a set of relevant protein targets, that is, the participating computational methods are tested on a common set of experimental target proteins, for which the experimental structures are not known at the time of modeling. Therefore, the CASP experiment would not have been possible without broad support of the experimental protein structural biology community. In this article, several experimental groups discuss the structures of the proteins which they provided as prediction targets for CASP9, highlighting structural and functional peculiarities of these structures: the long tail fiber protein gp37 from bacteriophage T4, the cyclic GMP-dependent protein kinase Iβ dimerization/docking domain, the ectodomain of the JTB (jumping translocation breakpoint) transmembrane receptor, Autotaxin in complex with an inhibitor, the DNA-binding J-binding protein 1 domain essential for biosynthesis and maintenance of DNA base-J (β-D-glucosyl-hydroxymethyluracil) in Trypanosoma and Leishmania, an so far uncharacterized 73 residue domain from Ruminococcus gnavus with a fold typical for PDZ-like domains, a domain from the phycobilisome core-membrane linker phycobiliprotein ApcE from Synechocystis, the heat shock protein 90 activators PFC0360w and PFC0270w from Plasmodium falciparum, and 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae. PMID:22020785

  3. Three novel acetylation sites in the Foxp3 transcription factor regulate the suppressive activity of regulatory T cells

    Kwon, Hye-Sook; Lim, Hyung W; Wu, Jessica; Schnoelzer, Martina; Verdin, Eric; Ott, Melanie

    2012-01-01

    The Foxp3 transcription factor is the master regulator of regulatory T cell (Treg) differentiation and function. Its activity is regulated by reversible acetylation. Using mass spectrometry of immunoprecipitated proteins, we identify three novel acetylation sites in murine Foxp3 (K31, K262, and K267) and the corresponding sites in human FoxP3 proteins. Newly raised modification-specific antibodies against acetylated K31 and K267 confirm acetylation of these residues in murine Tregs. Mutant Fo...

  4. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. PMID:27284048

  5. p53 Acetylation: Regulation and Consequences

    Reed, Sara M.; Quelle, Dawn E.

    2014-01-01

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo ev...

  6. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns

    Lundby, Alicia; Hansen, Kasper Lage; Weinert, Brian Tate; Breinholt Bekker-Jensen, Dorte; Secher, Anna; Skovgaard, Tine; Kelstrup, Christian; Dmytriyev, Anatoliy; Choudhary, Chuna Ram; Lundby, Carsten; Olsen, Jesper Velgaard

    2012-01-01

    Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4...... subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle...

  7. An N-sulfanylethylanilide-based traceable linker for enrichment and selective labelling of target proteins.

    Morisaki, Takuya; Denda, Masaya; Yamamoto, Jun; Tsuji, Daisuke; Inokuma, Tsubasa; Itoh, Kohji; Shigenaga, Akira; Otaka, Akira

    2016-05-25

    An N-sulfanylethylanilide-based traceable linker, developed to facilitate identification of target proteins of bioactive compounds, was introduced into an alkynylated target protein. Subsequent adsorption onto streptavidin beads allowed it to be treated with a cysteine-fluorophore conjugate in the presence of phosphate. This induced the N-S acyl transfer reaction of the N-sulfanylethylanilide unit. The subsequent native chemical ligation of the fluorophore resulted in cleavage of the linker for target elution and fluorescence labelling of the target, allowing it to be distinguished from non-target proteins. PMID:27146590

  8. Targeted quantitative mass spectrometric immunoassay for human protein variants

    Nedelkov Dobrin; Trenchevska Olgica

    2011-01-01

    Abstract Background Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome. Modified proteins also play an important role in biological processes. While sandwich immunoassays are routinely used to determine protein concentrations, they are oblivious to protein variants that may serve as biomarkers with better sensitivity and specificity than their wild-type proteins. Mass spectrometry, coupled to i...

  9. Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling

    Williams, Christopher C.; Jan, Calvin H.; Weissman, Jonathan S.

    2014-01-01

    Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. Most inner membrane proteins were co-translationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of protein...

  10. Dual Targeting of the Protein Methyltransferase PrmA Contributes to Both Chloroplastic and Mitochondrial Ribosomal Protein L11 Methylation in Arabidopsis.

    Mazzoleni, Meryl; Figuet, Sylvie; Martin-Laffon, Jacqueline; Mininno, Morgane; Gilgen, Annabelle; Leroux, Mélanie; Brugière, Sabine; Tardif, Marianne; Alban, Claude; Ravanel, Stéphane

    2015-09-01

    Methylation of ribosomal proteins has long been described in prokaryotes and eukaryotes, but our knowledge about the enzymes responsible for these modifications in plants is scarce. The bacterial protein methyltransferase PrmA catalyzes the trimethylation of ribosomal protein L11 (RPL11) at three distinct sites. The role of these modifications is still unknown. Here, we show that PrmA from Arabidopsis thaliana (AtPrmA) is dually targeted to chloroplasts and mitochondria. Mass spectrometry and enzymatic assays indicated that the enzyme methylates RPL11 in plasto- and mitoribosomes in vivo. We determined that the Arabidopsis and Escherichia coli PrmA enzymes share similar product specificity, making trimethylated residues, but, despite an evolutionary relationship, display a difference in substrate site specificity. In contrast to the bacterial enzyme that trimethylates the ε-amino group of two lysine residues and the N-terminal α-amino group, AtPrmA methylates only one lysine in the MAFCK(D/E)(F/Y)NA motif of plastidial and mitochondrial RPL11. The plant enzyme possibly methylates the N-terminus of plastidial RPL11, whereas mitochondrial RPL11 is N-α-acetylated by an unknown acetyltransferase. Lastly, we found that an Arabidopsis prma-null mutant is viable in standard environmental conditions and no molecular defect could be associated with a lack of RPL11 methylation in leaf chloroplasts or mitochondria. However, the conservation of PrmA during the evolution of photosynthetic eukaryotes together with the location of methylated residues at the binding site of translation factors to ribosomes suggests that RPL11 methylation in plant organelles could be involved, in combination with other post-translational modifications, in optimizing ribosome function. PMID:26116422

  11. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-{kappa}B p65 subunit and cytotoxicity in renal proximal tubule cells

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Lee, Sang Yong [Department of Diagnostic Radiology, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Han, Myung Kwan [Department of Microbiology, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kim, Duk Hoon [Division of Forensic Medicine, National Forensic Service, Seoul (Korea, Republic of); Kim, Won, E-mail: kwon@jbnu.ac.kr [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Cisplatin increases acetylation of NF-{kappa}B p65 subunit in HK2 cells. Black-Right-Pointing-Pointer SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. Black-Right-Pointing-Pointer Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-{kappa}B (NF-{kappa}B) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD{sup +})-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-{kappa}B p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-{kappa}B during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-{kappa}B p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-{kappa}B through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  12. Targeted Interactomics in Plants Through Protein Complex Isolation

    Geert De Jaeger

    2012-01-01

    TAPtag technology is the most widely applied tool to pick up in situ protein interactions in a proteome wide setting.Our research team has developed a versatile TAP technology platform for protein complex isolation from plants.We isolated complexes for hundreds of proteins and extensively demonstrated the power of our technology for protein discovery,functional analysis of proteins and protein complexes,and the modelling of protein networks.Complexes are purified from Arabidopsis cell suspension cultures or seedlings and we are currently translating the technology towards crop plants to bring complex purification in a developmental context.Besides protein complexes,we are deriving protocol variations to isolate chromatin complexes.

  13. Arbitrary protein−protein docking targets biologically relevant interfaces

    Martin Juliette; Lavery Richard

    2012-01-01

    Abstract Background Protein-protein recognition is of fundamental importance in the vast majority of biological processes. However, it has already been demonstrated that it is very hard to distinguish true complexes from false complexes in so-called cross-docking experiments, where binary protein complexes are separated and the isolated proteins are all docked against each other and scored. Does this result, at least in part, reflect a physical reality? False complexes could reflect possible ...

  14. Sequence- and interactome-based prediction of viral protein hotspots targeting host proteins: a case study for HIV Nef.

    Mahdi Sarmady

    Full Text Available Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.

  15. Genomes2Drugs: identifies target proteins and lead drugs from proteome data.

    Toomey, David

    2009-01-01

    BACKGROUND: Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins\\/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and\\/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. METHODOLOGY\\/PRINCIPAL FINDINGS: To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. CONCLUSIONS\\/SIGNIFICANCE: Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under \\'change-of-application\\' patents.

  16. Development of Lentiviral Vectors for Targeted Integration and Protein Delivery.

    Schenkwein, Diana; Ylä-Herttuala, Seppo

    2016-01-01

    The method in this chapter describes the design of human immunodeficiency virus type 1 (HIV-1) integrase (IN)-fusion proteins which we have developed to transport different proteins into the nuclei of lentiviral vector (LV)-transduced cells. The IN-fusion protein cDNA is incorporated into the LV packaging plasmid, which leads to its incorporation into vector particles as part of a large Gag-Pol polyprotein. This specific feature of protein packaging enables also the incorporation of cytotoxic and proapoptotic proteins, such as frequently cutting endonucleases and P53. The vectors can hence be used for various protein transduction needs. An outline of the necessary methods is also given to study the functionality of a chosen IN-fusion protein in a cell culture assay. PMID:27317182

  17. Mitochondrial localization of fission yeast manganese superoxide dismutase is required for its lysine acetylation and for cellular stress resistance and respiratory growth

    Research highlights: → Fission yeast manganese superoxide dismutase (MnSOD) is acetylated. → The mitochondrial targeting sequence (MTS) is required for the acetylation of MnSOD. → The MTS is not crucial for MnSOD activity, but is important for respiratory growth. → Posttranslational regulation of MnSOD differs between budding and fission yeast. -- Abstract: Manganese-dependent superoxide dismutase (MnSOD) is localized in the mitochondria and is important for oxidative stress resistance. Although transcriptional regulation of MnSOD has been relatively well studied, much less is known about the protein's posttranslational regulation. In budding yeast, MnSOD is activated after mitochondrial import by manganese ion incorporation. Here we characterize posttranslational modification of MnSOD in the fission yeast Schizosaccharomyces pombe. Fission yeast MnSOD is acetylated at the 25th lysine residue. This acetylation was diminished by deletion of N-terminal mitochondrial targeting sequence, suggesting that MnSOD is acetylated after import into mitochondria. Mitochondrial localization of MnSOD is not essential for the enzyme activity, but is crucial for oxidative stress resistance and growth under respiratory conditions of fission yeast. These results suggest that, unlike the situation in budding yeast, S. pombe MnSOD is already active even before mitochondrial localization; nonetheless, mitochondrial localization is critical to allow the cell to cope with reactive oxygen species generated inside or outside of mitochondria.

  18. Arbitrary protein−protein docking targets biologically relevant interfaces

    Martin Juliette

    2012-05-01

    Full Text Available Abstract Background Protein-protein recognition is of fundamental importance in the vast majority of biological processes. However, it has already been demonstrated that it is very hard to distinguish true complexes from false complexes in so-called cross-docking experiments, where binary protein complexes are separated and the isolated proteins are all docked against each other and scored. Does this result, at least in part, reflect a physical reality? False complexes could reflect possible nonspecific or weak associations. Results In this paper, we investigate the twilight zone of protein-protein interactions, building on an interesting outcome of cross-docking experiments: false complexes seem to favor residues from the true interaction site, suggesting that randomly chosen partners dock in a non-random fashion on protein surfaces. Here, we carry out arbitrary docking of a non-redundant data set of 198 proteins, with more than 300 randomly chosen "probe" proteins. We investigate the tendency of arbitrary partners to aggregate at localized regions of the protein surfaces, the shape and compositional bias of the generated interfaces, and the potential of this property to predict biologically relevant binding sites. We show that the non-random localization of arbitrary partners after protein-protein docking is a generic feature of protein structures. The interfaces generated in this way are not systematically planar or curved, but tend to be closer than average to the center of the proteins. These results can be used to predict biological interfaces with an AUC value up to 0.69 alone, and 0.72 when used in combination with evolutionary information. An appropriate choice of random partners and number of docking models make this method computationally practical. It is also noted that nonspecific interfaces can point to alternate interaction sites in the case of proteins with multiple interfaces. We illustrate the usefulness of arbitrary docking

  19. Arbitrary protein−protein docking targets biologically relevant interfaces

    Protein-protein recognition is of fundamental importance in the vast majority of biological processes. However, it has already been demonstrated that it is very hard to distinguish true complexes from false complexes in so-called cross-docking experiments, where binary protein complexes are separated and the isolated proteins are all docked against each other and scored. Does this result, at least in part, reflect a physical reality? False complexes could reflect possible nonspecific or weak associations. In this paper, we investigate the twilight zone of protein-protein interactions, building on an interesting outcome of cross-docking experiments: false complexes seem to favor residues from the true interaction site, suggesting that randomly chosen partners dock in a non-random fashion on protein surfaces. Here, we carry out arbitrary docking of a non-redundant data set of 198 proteins, with more than 300 randomly chosen "probe" proteins. We investigate the tendency of arbitrary partners to aggregate at localized regions of the protein surfaces, the shape and compositional bias of the generated interfaces, and the potential of this property to predict biologically relevant binding sites. We show that the non-random localization of arbitrary partners after protein-protein docking is a generic feature of protein structures. The interfaces generated in this way are not systematically planar or curved, but tend to be closer than average to the center of the proteins. These results can be used to predict biological interfaces with an AUC value up to 0.69 alone, and 0.72 when used in combination with evolutionary information. An appropriate choice of random partners and number of docking models make this method computationally practical. It is also noted that nonspecific interfaces can point to alternate interaction sites in the case of proteins with multiple interfaces. We illustrate the usefulness of arbitrary docking using PEBP (Phosphatidylethanolamine binding

  20. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  1. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek; Schölz, Christian; Wagner, Sebastian A; Magnes, Christoph; Zechner, Rudolf; Choudhary, Chuna Ram

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  2. Molecular Structure and Target Recognition of Neuronal Calcium Sensor Proteins

    James Ames; Mitsuhiko Ikura

    2011-01-01

    Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs) both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1) modulates synaptic acti...

  3. Molecular structure and target recognition of neuronal calcium sensor proteins

    Ames, James B.; Lim, Sunghyuk; Ikura, Mitsuhiko

    2012-01-01

    Neuronal calcium sensor (NCS) proteins, a sub-branch of the EF-hand superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs) both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1) modulates synaptic activit...

  4. 14-3-3 proteins as potential therapeutic targets

    Zhao, Jing; Meyerkord, Cheryl L; Du, Yuhong; Khuri, Fadlo R.; Fu, Haian

    2011-01-01

    The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of client proteins in various signaling pathways that control diverse physiological and pathological processes. In response to environmental cues, 14-3-3 proteins orchestrate the highly regulated flow of signals through complex networks of molecular interactions to achieve well-controlled physiological outputs, such as cell proliferation or differentiation. Accumulating evidence now support...

  5. Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin.

    Murahashi, Masataka; Simizu, Siro; Morioka, Masahiko; Umezawa, Kazuo

    2016-08-01

    15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity. PMID:27261432

  6. Comparison of pathways from the geometric targeting method and targeted molecular dynamics in nitrogen regulatory protein C

    Geometric targeting (GT) is a recently introduced method for rapidly generating all-atom pathways from one protein state to another, based on geometric rather than energetic considerations. To generate pathways, a bias is applied that gradually moves atoms toward a target structure, while a set of geometric constraints between atoms is enforced to keep the structure stereochemically acceptable. In this work, we compare conformational pathways generated from GT to pathways from the much more computationally intensive and commonly used targeted molecular dynamics (TMD) technique, for a complicated conformational change in the signaling protein nitrogen regulatory protein C. We show that the all-atom pathways from GT are similar to previously reported TMD pathways for this protein, by comparing motion along six progress variables that describe the various structural changes. The results suggest that for nitrogen regulatory protein C, finding an all-atom pathway is primarily a problem of geometry, and that a detailed force field in this case constitutes an unnecessary extra layer of detail. We also show that the pathway snapshots from GT have good structure quality, by measuring various structure quality metrics. Transient hydrogen bonds detected by the two methods show some similarities but also some differences. The results justify the usage of GT as a rapid, approximate alternative to TMD for generating stereochemically acceptable all-atom pathways in highly constrained protein systems

  7. Rabring7, a Novel Rab7 Target Protein with a RING Finger Motif

    Mizuno, Kouichi; Kitamura, Akiko; Sasaki, Takuya

    2003-01-01

    Rab7, a member of the Rab family small G proteins, has been shown to regulate intracellular vesicle traffic to late endosome/lysosome and lysosome biogenesis, but the exact roles of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins that function in the exocytic/endocytic pathway. We have isolated a new Rab7 target protein, Rabring7 (Rab7-interacting RING finger protein), using a CytoTrap system. It contains an H2 type RING finger mo...

  8. A Universal Method for Fishing Target Proteins from Mixtures of Biomolecules using Isothermal Titration Calorimetry

    The most challenging tasks in biology include the identification of (1) the orphan receptor for a ligand, (2) the ligand for an orphan receptor protein, and (3) the target protein(s) for a given drug or a lead compound that are critical for the pharmacological or side effects. At present, several approaches are available, including cell- or animal-based assays, affinity labeling, solid-phase binding assays, surface plasmon resonance, and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification, or immobilization. Here we demonstrate a new universal method for fishing orphan target proteins from a complex mixture of biomolecules using isothermal titration calorimetry (ITC) as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins, a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization.

  9. Concepts of Protein Sorting or Targeting Signals and Membrane Topology in Undergraduate Teaching

    Tang, Bor Luen; Teng, Felicia Yu Hsuan

    2005-01-01

    The process of protein biogenesis culminates in its correct targeting to specific subcellular locations where it serves a function. Contemporary molecular and cell biology investigations often involve the exogenous expression of epitope- or fluorescent protein-tagged recombinant molecules as well as subsequent analysis of protein-protein…

  10. Acetylation of C/EBPα inhibits its granulopoietic function.

    Bararia, Deepak; Kwok, Hui Si; Welner, Robert S; Numata, Akihiko; Sárosi, Menyhárt B; Yang, Henry; Wee, Sheena; Tschuri, Sebastian; Ray, Debleena; Weigert, Oliver; Levantini, Elena; Ebralidze, Alexander K; Gunaratne, Jayantha; Tenen, Daniel G

    2016-01-01

    CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation. PMID:27005833

  11. Ubiquitination of Notch1 is regulated by MAML1-mediated p300 acetylation of Notch1

    Popko-Scibor, Anita E.; Lindberg, Mikael J.; Hansson, Magnus L.; Holmlund, Teresa [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm (Sweden); Wallberg, Annika E., E-mail: Annika.Wallberg@ki.se [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm (Sweden)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer p300 acetylates conserved lysines within Notch1 C-terminal nuclear localization signal. Black-Right-Pointing-Pointer MAML1 and CSL, components of Notch transcription complex, increase Notch acetylation. Black-Right-Pointing-Pointer MAML1-dependent acetylation of Notch1 by p300 decreases the ubiquitination of Notch1. Black-Right-Pointing-Pointer CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300. -- Abstract: Earlier studies demonstrated the involvement of the p300 histone acetyltransferase in Notch signaling but the precise mechanisms by which p300 might modulate Notch function remains to be investigated. In this study, we show that p300 acetylates Notch1 ICD in cell culture assay and in vitro, and conserved lysines located within the Notch C-terminal nuclear localization signal are essential for Notch acetylation. MAML1 and CSL, which are components of the Notch transcription complex, enhance Notch acetylation and we suggest that MAML1 increases Notch acetylation by potentiating p300 autoacetylation. Furthermore, MAML1-dependent acetylation of Notch1 ICD by p300 decreases the ubiquitination of Notch1 ICD in cellular assays. CDK8 has been shown to target Notch1 for ubiquitination and proteosomal degradation. We show that CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300. Therefore, we speculate that acetylation of Notch1 might be a mechanism to regulate Notch activity by interfering with ubiquitin-dependent pathways.

  12. A structural annotation resource for the selection of putative target proteins in the malaria parasite

    Joubert Fourie

    2008-05-01

    Full Text Available Abstract Background Protein structure plays a pivotal role in elucidating mechanisms of parasite functioning and drug resistance. Moreover, protein structure aids the determination of protein function, which can together with the structure be used to identify novel drug targets in the parasite. However, various structural features in Plasmodium falciparum proteins complicate the experimental determination of protein structures. Limited similarity to proteins in the Protein Data Bank and the shortage of solved protein structures in the malaria parasite necessitate genome-scale structural annotation of P. falciparum proteins. Additionally, the annotation of a range of structural features facilitates the identification of suitable targets for experimental and computational studies. Methods An integrated structural annotation system was developed and applied to P. falciparum, Plasmodium vivax and Plasmodium yoelii. The annotation included searches for sequence similarity, patterns and domains in addition to the following predictions: secondary structure, transmembrane helices, protein disorder, low complexity, coiled-coils and small molecule interactions. Subsequently, candidate proteins for further structural studies were identified based on the annotated structural features. Results The annotation results are accessible through a web interface, enabling users to select groups of proteins which fulfil multiple criteria pertaining to structural and functional features 1. Analysis of features in the P. falciparum proteome showed that protein-interacting proteins contained a higher percentage of predicted disordered residues than non-interacting proteins. Proteins interacting with 10 or more proteins have a disordered content concentrated in the range of 60–100%, while the disorder distribution for proteins having only one interacting partner, was more evenly spread. Conclusion A series of P. falciparum protein targets for experimental structure

  13. Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence

    1996-01-01

    We have identified a novel peroxisomal targeting sequence (PTS) at the extreme COOH terminus of human catalase. The last four amino acids of this protein (-KANL) are necessary and sufficient to effect targeting to peroxisomes in both human fibroblasts and Saccharomyces cerevisiae, when appended to the COOH terminus of the reporter protein, chloramphenicol acetyl transferase. However, this PTS differs from the extensive family of COOH-terminal PTS tripeptides collectively termed PTS1 in two ma...

  14. Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

    Ko, K.; Cashmore, A. R.

    1989-01-01

    Various chimeric precursors and deletions of the 33 kd oxygen-evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase, a stromal protein, resulted in redirection of ma...

  15. An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics

    Vandemoortele, Giel; Staes, An; Gonnelli, Giulia; Samyn, Noortje; De Sutter, Delphine; Vandermarliere, Elien; Timmerman, Evy; Gevaert, Kris; Martens, Lennart; Eyckerman, Sven

    2016-01-01

    The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these ’Proteotypic peptides for Quantification by SRM’ (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins. PMID:27264994

  16. Acetyltransferase and human hemoglobin acetylation

    A minor component of human fetal hemoglobin (Hb F) is acetylated at the amino-terminus of the γ-globin chains. A similar minor component of Hb F is formed during translation of cord blood mRNA in the rabbit reticulocyte lysate system. The acetylation appeared to be enzymatic. This system contains an acetyltransferase capable of acetylating histones and hemoglobins. The enzyme, partially purified by histone-Sepharose affinity chromatography was capable of incorporating labeled acetyl- group from 1-[14C-acetyl]-CoA into both human Hb F0 and HB A0, but at a lower rate than for histones. Characterization of the labeled products indicated that the α-chains of both hemoglobins were being acetylated presumably at a lysyl-residue, but in the case of Hb F0 the amino-terminus of the γ-globin chains was acetylated as well. While histone-Sepharose bound more than 95% of the enzyme, Sepharose linked Hb F0, γ-globin chains, and Hb Bart's bound 14, 5, and 12% of the activity, respectively. Enzyme bound to these resins was not any more active on the hemoglobins than was the enzyme bound to the histone-Sepharose. The histone-Sepharose was also used to detect the enzyme in human cord blood red cells separated by dextran 40 density gradient centrifugation. Activity was found mostly in the young cells, and was directly related to the number of reticulocytes present in any one fraction

  17. SWI/SNF proteins as targets in cancer therapy

    Schiaffino-Ortega, Santiago; Balinas, Carlos; Cuadros Celorrio, Marta; Medina Vico, Pedro

    2014-01-01

    Recent identification of synthetic lethal interactions involving several proteins of the SWI/SNF complex discussed in this Research Highlight has opened the possibility of new cancer therapeutic approaches.

  18. Early secretory antigenic target protein-6/culture filtrate protein-10 fusion protein-specific Th1 and Th2 response and its diagnostic value in tuberculous pleural effusion

    戈启萍

    2013-01-01

    Objective To detect the Th1 and Th2 cell percentage in pleural effusion mononuclear cells (PEMCs) stimulated by early secretory antigenic target protein-6 (ESAT-6) /culture filtrate protein-10 (CFP-10) fusion protein (E/C) with flow cytometry (FCM) ,and to explore the local antigen specific Th1 and Th2 response and

  19. Phytochemical-mediated Protein Expression Profiling and the Potential Applications in Therapeutic Drug Target Identifications.

    Wong, Fai-Chu; Tan, Siok-Thing; Chai, Tsun-Thai

    2016-07-29

    Many phytochemicals derived from edible medicinal plants have been investigated intensively for their various bioactivities. However, the detailed mechanism and their corresponding molecular targets frequently remain elusive. In this review, we present a summary of the research works done on phytochemical-mediated molecular targets, identified via proteomic approach. Concurrently, we also highlighted some pharmaceutical drugs which could be traced back to their origins in phytochemicals. For ease of presentation, these identified protein targets were categorized into two important healthcare-related fields, namely anti-bacterial and anti-cancer research. Through this review, we hope to highlight the usefulness of comparative proteomic as a powerful tool in phytochemical-mediated protein target identifications. Likewise, we wish to inspire further investigations on some of these protein targets identified over the last few years. With contributions from all researchers, the accumulative efforts could eventually lead to the discovery of some target-specific, low-toxicity therapeutic agents. PMID:26193174

  20. A secretory system for bacterial production of high-profile protein targets

    Kotzsch, Alexander; Vernet, Erik; Hammarström, Martin;

    2011-01-01

    Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To...... improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer...... membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system, we clearly...

  1. Biomarkers for ragwort poisoning in horses: identification of protein targets

    Beynon Robert J

    2008-08-01

    Full Text Available Abstract Background Ingestion of the poisonous weed ragwort (Senecio jacobea by horses leads to irreversible liver damage. The principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. In this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods. Results One dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. Using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin. Conclusion These findings demonstrate that pyrrolic metabolites can modify equine plasma proteins. The high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole. This model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock.

  2. Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation

    Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a since threonine as residue 28 instead of the serine(28)-alanine(29) sequence, present in all other known plant and animal H3 histones

  3. The neurobiology of acetyl-L-carnitine.

    Traina, Giovanna

    2016-01-01

    A large body of evidence points to the positive effects of dietary supplementation of acetyl-L-carnitine (ALC). Its use has shown health benefits in neuroinflammation, which is a common denominator in a host of neurodegenerative diseases. ALC is the principal acetyl ester of L-Carnitine (LC), and it plays an essential role in intermediary metabolism, acting as a donor of acetyl groups and facilitating the transfer of fatty acids from cytosol to mitochondria during beta-oxidation. Dietary supplementation of ALC exerts neuroprotective, neurotrophic, antidepressive and analgesic effects in painful neuropathies. ALC also has antioxidant and anti-apoptotic activity. Moreover, ALC exhibits positive effects on mitochondrial metabolism, and shows promise in the treatment of aging and neurodegenerative pathologies by slowing the progression of mental deterioration. In addition, ALC plays neuromodulatory effects on both synaptic morphology and synaptic transmission. These effects are likely due to affects of ALC through modulation of gene expression on several targets in the central nervous system. Here, we review the current state of knowledge on effects of ALC in the nervous system. PMID:27100509

  4. In the absence of cellular poly (A) binding protein, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53

    Highlights: → PABP knock down and cell apoptosis. → Nuclear translocation of GAPDH in PABP depleted cells. → Role of p53 in apoptosis of PABP depleted cells. → Bax translocation and cytochrome C release and caspase 3 activation following PABP depletion. → Association of p53 with Bcl2 and Bax. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) interacts with 3' poly (A) tract of eukaryotic mRNA and is important for both translation and stability of mRNA. Previously, we have shown that depletion of PABP by siRNA prevents protein synthesis and consequently leads to cell death through apoptosis. In the present investigation, we studied the mechanism of cell apoptosis. We show that in the absence of PABP, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53. As a result, p53 translocated to the mitochondria to initiate Bax mediated apoptosis.

  5. In the absence of cellular poly (A) binding protein, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B. [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada); Bag, Jnanankur, E-mail: jbag@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada)

    2011-06-03

    Highlights: {yields} PABP knock down and cell apoptosis. {yields} Nuclear translocation of GAPDH in PABP depleted cells. {yields} Role of p53 in apoptosis of PABP depleted cells. {yields} Bax translocation and cytochrome C release and caspase 3 activation following PABP depletion. {yields} Association of p53 with Bcl2 and Bax. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) interacts with 3' poly (A) tract of eukaryotic mRNA and is important for both translation and stability of mRNA. Previously, we have shown that depletion of PABP by siRNA prevents protein synthesis and consequently leads to cell death through apoptosis. In the present investigation, we studied the mechanism of cell apoptosis. We show that in the absence of PABP, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53. As a result, p53 translocated to the mitochondria to initiate Bax mediated apoptosis.

  6. The Effect of Acetyl Salicylic Acid Induced Nitric Oxide Synthesis in the Normalization of Hypertension through the Stimulation of Renal Cortexin Synthesis and by the Inhibition of Dermcidin Isoform 2, A Hypertensive Protein Production

    Ghosh, Rajeshwary; Bank, Sarbashri; Maji, Uttam K.; Bhattacharya, Rabindra; Guha, Santanu; Khan, Nighat N.; Sinha, A. Kumar

    2014-01-01

    Currently, there is no specific medication for essential hypertension (EH), a major form of the condition, in man. As acetyl salicylic acid (aspirin) is reported to stimulate the synthesis of renal (r)-cortexin, an anti-essential hypertensive protein, and, as aspirin is reported to inhibit dermcidin isoform 2 (dermcidin), a causative protein for EH, the role of aspirin in the control of EH in man was studied. Oral administration of 150 mg aspirin/70 kg body weight in subjects with EH was found to reduce both the elevated systolic and diastolic blood pressures to normal levels within 3 h due to the normalization of dermcidin level in these subjects. The plasma cortexin level at day 0, 1, 30 and 90 were 0.5 pmol/ml, 155.5 pmol/ml, 160.2 pmol/ml, 190.5 pmol/ml respectively with increased NO synthesis (r=+0.994). In vitro studies demonstrated that the incubation of the goat kidney cortex cells with aspirin stimulated (r)-cortexin synthesis due to NO synthesis. It could be suggested that the use of aspirin might control EH in man. PMID:25324696

  7. Amodiaquine-induced toxicity in isolated rat hepatocytes and the cytoprotective effects of taurine and/or N-acetyl cysteine.

    Heidari, R; Babaei, H; Eghbal, M A

    2014-01-01

    Amodiaquine is an antimalarial drug used in the prophylaxis and treatment of this disease. However, hepatotoxicity as a life-threatening adverse effect is associated with its clinical use. We evaluated amodiaquine-induced toxicity in isolated rat hepatocytes as an in vitro model for studying drug-induced hepatotoxicity. This study attempts to investigate the protective effects of taurine and N-acetyl cysteine against the cytotoxicity induced by amodiaquine. Hepatocytes were prepared by the method of collagenase enzyme perfusion via portal vein. This technique is based on liver perfusion with collagenase after removal of calcium ion (Ca(2+)) with a chelator (ethylene glycol tetraacetic acid (EGTA) 0.5 mM). Cells were treated with different concentrations of amodiaquine, taurine and N-acetyl cysteine. Cell death, protein carbonylation, reactive oxygen species formation, lipid peroxidation, and mitochondrial depolarization were assessed as toxicity markers. Amodiaquine cytotoxic mechanism involved protein carbonylation as well as reactive oxygen species formation and lipid peroxidation. In addition, mitochondria seem to be a target for amodiaquine to induce cellular damage. Administration of taurine (200 μM) and/or N-acetyl cysteine (200 μM) reduced oxidative stress, lipid peroxidation and protein carbonylation caused by amodiaquine. Furthermore, amodiaquine-induced mitochondrial injury was significantly mitigated by taurine and/or N-acetyl cysteine. In glutathione-depleted cells, only N-acetyl cysteine protected hepatocytes against amodiaquine, and taurine showed no protective properties in this situation. Taurine and N-acetyl cysteine protect hepatocytes against amodiaquine probably via their antioxidant properties and counteracting oxidative stress. PMID:25657778

  8. Posttranslational Modifications of Chloroplast Proteins: An Emerging Field.

    Lehtimäki, Nina; Koskela, Minna M; Mulo, Paula

    2015-07-01

    Posttranslational modifications of proteins are key effectors of enzyme activity, protein interactions, targeting, and turnover rate, but despite their importance, they are still poorly understood in plants. Although numerous reports have revealed the regulatory role of protein phosphorylation in photosynthesis, various other protein modifications have been identified in chloroplasts only recently. It is known that posttranslational N(α)-acetylation occurs in both nuclear- and plastid-encoded chloroplast proteins, but the physiological significance of this acetylation is not yet understood. Lysine acetylation affects the localization and activity of key metabolic enzymes, and it may work antagonistically or cooperatively with lysine methylation, which also occurs in chloroplasts. In addition, tyrosine nitration may help regulate the repair cycle of photosystem II, while N-glycosylation determines enzyme activity of chloroplastic carbonic anhydrase. This review summarizes the progress in the research field of posttranslational modifications of chloroplast proteins and points out the importance of these modifications in the regulation of chloroplast metabolism. PMID:25911530

  9. Acetylation of Human TCF4 (TCF7L2) Proteins Attenuates Inhibition by the HBP1 Repressor and Induces a Conformational Change in the TCF4::DNA Complex

    Elfert, Susanne; Weise, Andreas; Bruser, Katja; Biniossek, Martin L; Jägle, Sabine; Senghaas, Niklas; Hecht, Andreas

    2013-01-01

    The members of the TCF/LEF family of DNA-binding proteins are components of diverse gene regulatory networks. As nuclear effectors of Wnt/β-catenin signaling they act as assembly platforms for multimeric transcription complexes that either repress or activate gene expression. Previously, it was shown that several aspects of TCF/LEF protein function are regulated by post-translational modification. The association of TCF/LEF family members with acetyltransferases and deacetylases prompted us t...

  10. Epigenetic Regulation of the IL-13-induced Human Eotaxin-3 Gene by CREB-binding Protein-mediated Histone 3 Acetylation*

    Lim, Eun Jin; Lu, Thomas X.; Blanchard, Carine; Rothenberg, Marc E.

    2011-01-01

    The etiology of a variety of chronic inflammatory disorders has been attributed to the interaction of genetic and environmental factors. Herein, we identified a link between epigenetic regulation and IL-13-driven eotaxin-3 in the pathogenesis of chronic allergic inflammation. We first demonstrated that the cAMP-responsive element (CRE) site in the eotaxin-3 promoter affects IL-13-induced eotaxin-3 promoter activity. Furthermore, the CRE-binding protein-binding protein (CBP), a histone acetylt...

  11. Site-specific acetylation of ISWI by GCN5

    Chioda Mariacristina

    2007-08-01

    Full Text Available Abstract Background The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition. Results We found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the Drosophila remodelling ATPase ISWI at a single, conserved lysine, K753, in vivo and in vitro. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF. Conclusion Acetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RKT/SxGx(KacxPR/K differs from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles.

  12. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism.

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-06-14

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR-DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  13. Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

    Shi, Tujin; SUN, XUEFEI; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y; Smith, Richard D.; Tang, Keqi; Camp, David G

    2013-01-01

    We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50–100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depleti...

  14. Heat shock proteins 27, 40, and 70 as combinational and dual therapeutic cancer targets

    McConnell, Jeanette R.; McAlpine, Shelli R.

    2013-01-01

    The heat shock proteins are essential players in the development of cancer and they are prime therapeutic targets. Targeting multiple hsps in dual therapies decreases the likelihood of drug resistance compared to utilizing mono-therapies. Further, employing an hsp inhibitor in combination with another therapy has proven clinically successful. Examples of efficacious strategies include the inhibition of hsp27, which prevents protein aggregation, controlling hsp40’s role as an ATPase modulator,...

  15. Large-scale prediction of drug–target interactions using protein sequences and drug topological structures

    Highlights: ► Drug–target interactions are predicted using an extended SAR methodology. ► A drug–target interaction is regarded as an event triggered by many factors. ► Molecular fingerprint and CTD descriptors are used to represent drugs and proteins. ► Our approach shows compatibility between the new scheme and current SAR methodology. - Abstract: The identification of interactions between drugs and target proteins plays a key role in the process of genomic drug discovery. It is both consuming and costly to determine drug–target interactions by experiments alone. Therefore, there is an urgent need to develop new in silico prediction approaches capable of identifying these potential drug–target interactions in a timely manner. In this article, we aim at extending current structure–activity relationship (SAR) methodology to fulfill such requirements. In some sense, a drug–target interaction can be regarded as an event or property triggered by many influence factors from drugs and target proteins. Thus, each interaction pair can be represented theoretically by using these factors which are based on the structural and physicochemical properties simultaneously from drugs and proteins. To realize this, drug molecules are encoded with MACCS substructure fingerings representing existence of certain functional groups or fragments; and proteins are encoded with some biochemical and physicochemical properties. Four classes of drug–target interaction networks in humans involving enzymes, ion channels, G-protein-coupled receptors (GPCRs) and nuclear receptors, are independently used for establishing predictive models with support vector machines (SVMs). The SVM models gave prediction accuracy of 90.31%, 88.91%, 84.68% and 83.74% for four datasets, respectively. In conclusion, the results demonstrate the ability of our proposed method to predict the drug–target interactions, and show a general compatibility between the new scheme and current SAR

  16. Predicted essential proteins ofPlasmodium falciparum for potential drug targets

    Qing-Feng He; Li Deng; Qin-Ying Xu; Zheng Shao

    2012-01-01

    ABSTRACT Objective:To identify novel drug targets for treatment ofPlasmodium falciparum.Methods:LocalBLASTP were used to find the proteins non-homologous to human essential proteins as novel drug targets. Functional domains of novel drug targets were identified by InterPro and Pfam,3D structures of potential drug targets were predicated by theSWISS-MODELworkspace. Ligands and ligand-binding sites of the proteins were searched byEf-seek.Results:Three essential proteins were identified that might be considered as potential drug targets.AAN37254.1 belonged to1-deoxy-D-xylulose5-phosphate reductoisomerase,CAD50499.1 belonged to chorismate synthase,CAD51220.1 belonged toFAD binging3 family, but the function of CAD51220.1 was unknown. The3D structures, ligands and ligand-binding sites ofAAN37254.1 andCAD50499.1 were successfully predicated.Conclusions:Two of these potential drug targets are key enzymes in2-C-methyl-d-erythritol4-phosphate pathway and shikimate pathway, which are absent in humans, so these two essential proteins are good potential drug targets. The function and3D structures ofCAD50499.1 is still unknown, it still need further study.

  17. Modulation of protein phosphorylation, N-glycosylation and Lys-acetylation in grape (Vitis vinifera) mesocarp and exocarp owing to Lobesia botrana infection

    Melo-Braga, Marcella N; Verano-Braga, Thiago; León, Ileana R;

    2012-01-01

    Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expressio...

  18. Method for Targeted Therapeutic Delivery of Proteins into Cells | NCI Technology Transfer Center | TTC

    The Protein Expression Laboratory at the National Cancer Institute in Frederick, MD is seeking statements of capability or interest from parties interested in collaborative research to further develop a platform technology for the targeted intra-cellular delivery of proteins using virus-like particles (VLPs).

  19. Global analysis of small molecule binding to related protein targets.

    Felix A Kruger

    2012-01-01

    Full Text Available We report on the integration of pharmacological data and homology information for a large scale analysis of small molecule binding to related targets. Differences in small molecule binding have been assessed for curated pairs of human to rat orthologs and also for recently diverged human paralogs. Our analysis shows that in general, small molecule binding is conserved for pairs of human to rat orthologs. Using statistical tests, we identified a small number of cases where small molecule binding is different between human and rat, some of which had previously been reported in the literature. Knowledge of species specific pharmacology can be advantageous for drug discovery, where rats are frequently used as a model system. For human paralogs, we demonstrate a global correlation between sequence identity and the binding of small molecules with equivalent affinity. Our findings provide an initial general model relating small molecule binding and sequence divergence, containing the foundations for a general model to anticipate and predict within-target-family selectivity.

  20. Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain.

    Söderqvist, H; Imreh, G; Kihlmark, M; Linnman, C; Ringertz, N; Hallberg, E

    1997-12-15

    The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121. PMID:9461306

  1. Role of histone acetylation in long-term neurobehavioral effects of neonatal Exposure to sevoflurane in rats.

    Jia, Min; Liu, Wen-Xue; Yang, Jiao-Jiao; Xu, Ning; Xie, Ze-Min; Ju, Ling-Sha; Ji, Mu-Huo; Martynyuk, Anatoly E; Yang, Jian-Jun

    2016-07-01

    Human studies, and especially laboratory studies, provide evidence that early life exposure to general anesthesia may affect neurocognitive development via largely unknown mechanisms. We explored whether hippocampal histone acetylation had a role in neurodevelopmental effects of sevoflurane administered to neonatal rats. Male Sprague-Dawley rats were exposed to 3% sevoflurane or were subjected to maternal separation only for 2h daily at postnatal days 6, 7, and 8. The histone deacetylase inhibitor, sodium butyrate (250mg/kg, intraperitoneally), or saline was administered starting 2h prior to anesthesia or maternal separation and continued daily until the end of behavioral tests, which were performed between postnatal days 33 and 50. Upon completion of the behavioral tests, the brain tissues were harvested for further analysis. Rats neonatally exposed to sevoflurane exhibited decreased freezing time in the fear conditioning contextual test and increased escape latency, decreased time in target quadrant, and number of platform crossings in the Morris water maze test. The sevoflurane-exposed rats had lower hippocampal density of dendritic spines, reduced levels of the brain-derived neurotrophic factor, c-fos protein, microtubule-associated protein 2, synapsin1, postsynaptic density protein 95, pCREB/CREB, CREB binding protein, and acetylated histones H3 and H4, and increased levels of histone deacetylases 3 and 8. These neurobehavioral abnormalities were normalized in the sevoflurane-exposed rats treated with sodium butyrate. Our findings provide evidence that neonatal exposure to sevoflurane induces neurobehavioral abnormalities and long-lasting alterations in histone acetylation; normalization of histone acetylation may alleviate the neurodevelopmental side effects of the anesthetic. PMID:27001149

  2. A secretory system for bacterial production of high-profile protein targets

    Kotzsch, Alexander; Vernet, Erik; Hammarström, Martin; Berthelsen, Jens; Weigelt, Johan; Gräslund, Susanne; Sundström, Michael

    2011-01-01

    Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A co...

  3. The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin

    Küberl, Andreas; Polen, Tino; Bott, Michael

    2016-01-01

    The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded ...

  4. Protein target discovery of drug and its reactive intermediate metabolite by using proteomic strategy

    Lianghai Hu; John Paul Fawcett; Jingkai Gu

    2012-01-01

    Identifying protein targets of bioactive compounds is an effective approach to discover unknown protein functions, identify molecular mechanisms of drug action, and obtain information for optimization of lead compounds. At the same time, metabolic activation of a drug can lead to cytotoxicities. Therefore, it is very important to systemically characterize the drug and its reactive intermediate. Mass spectrometry-based proteomic approach has emerged as the most efficient to study protein funct...

  5. Hide and seek: Identification and confirmation of small molecule protein targets.

    Ursu, Andrei; Waldmann, Herbert

    2015-08-15

    Target identification and confirmation for small molecules is often the rate limiting step in drug discovery. A robust method to identify proteins addressed by small molecules is affinity chromatography using chemical probes. These usually consist of the compound of interest equipped with a linker molecule and a proper tag. Recently, methods emerged that allow the identification of protein targets without prior functionalization of the small molecule of interest. The digest offers an update on the newest developments in the area of target identification with special focus on confirmation techniques. PMID:26115575

  6. Novel Technology for Protein-Protein Interaction-based Targeted Drug Discovery

    Jung Me Hwang

    2011-12-01

    Full Text Available We have developed a simple but highly efficient in-cell protein-protein interaction (PPI discovery system based on the translocation properties of protein kinase C- and its C1a domain in live cells. This system allows the visual detection of trimeric and dimeric protein interactions including cytosolic, nuclear, and/or membrane proteins with their cognate ligands. In addition, this system can be used to identify pharmacological small compounds that inhibit specific PPIs. These properties make this PPI system an attractive tool for screening drug candidates and mapping the protein interactome.

  7. Protein target discovery of drug and its reactive intermediate metabolite by using proteomic strategy

    Lianghai Hu

    2012-04-01

    Full Text Available Identifying protein targets of bioactive compounds is an effective approach to discover unknown protein functions, identify molecular mechanisms of drug action, and obtain information for optimization of lead compounds. At the same time, metabolic activation of a drug can lead to cytotoxicities. Therefore, it is very important to systemically characterize the drug and its reactive intermediate. Mass spectrometry-based proteomic approach has emerged as the most efficient to study protein functions and modifications. This review will discuss method development for the drug target discovery and the application in different fields including the drug toxicity mechanism caused by reactive metabolites.

  8. Plastoglobules: a new address for targeting recombinant proteins in the chloroplast

    Kessler Felix

    2007-01-01

    Full Text Available Abstract Background The potential of transgenic plants for cost-effective production of pharmaceutical molecules is now becoming apparent. Plants have the advantage over established fermentation systems (bacterial, yeast or animal cell cultures to circumvent the risk of pathogen contamination, to be amenable to large scaling up and to necessitate only established farming procedures. Chloroplasts have proven a useful cellular compartment for protein accumulation owing to their large size and number, as well as the possibility for organellar transformation. They therefore represent the targeting destination of choice for recombinant proteins in leaf crops such as tobacco. Extraction and purification of recombinant proteins from leaf material contribute to a large extent to the production costs. Developing new strategies facilitating these processes is therefore necessary. Results Here, we evaluated plastoglobule lipoprotein particles as a new subchloroplastic destination for recombinant proteins. The yellow fluorescent protein as a trackable cargo was targeted to plastoglobules when fused to plastoglobulin 34 (PGL34 as the carrier. Similar to adipocyte differentiation related protein (ADRP in animal cells, most of the protein sequence of PGL34 was necessary for targeting to lipid bodies. The recombinant protein was efficiently enriched in plastoglobules isolated by simple flotation centrifugation. The viability of plants overproducing the recombinant protein was not affected, indicating that plastoglobule targeting did not significantly impair photosynthesis or sugar metabolism. Conclusion Our data identify plastoglobules as a new targeting destination for recombinant protein in leaf crops. The wide-spread presence of plastoglobules and plastoglobulins in crop species promises applications comparable to those of transgenic oilbody-oleosin technology in molecular farming.

  9. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Regina Stoltenburg

    Full Text Available A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  10. Advances in identification and validation of protein targets of natural products without chemical modification.

    Chang, J; Kim, Y; Kwon, H J

    2016-05-01

    Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates. PMID:26964663

  11. Olig1 Acetylation and Nuclear Export Mediate Oligodendrocyte Development.

    Dai, Jinxiang; Bercury, Kathryn K; Jin, Weilin; Macklin, Wendy B

    2015-12-01

    The oligodendrocyte transcription factor Olig1 is critical for both oligodendrocyte development and remyelination in mice. Nuclear to cytoplasmic translocation of Olig1 protein occurs during brain development and in multiple sclerosis, but the detailed molecular mechanism of this translocation remains elusive. Here, we report that Olig1 acetylation and deacetylation drive its active translocation between the nucleus and the cytoplasm in both mouse and rat oligodendrocytes. We identified three functional nuclear export sequences (NES) localized in the basic helix-loop-helix domain and one specific acetylation site at Lys 150 (human Olig1) in NES1. Olig1 acetylation and deacetylation are regulated by the acetyltransferase CREB-binding protein and the histone deacetylases HDAC1, HDAC3, and HDAC10. Acetylation of Olig1 decreased its chromatin association, increased its interaction with inhibitor of DNA binding 2 and facilitated its retention in the cytoplasm of mature oligodendrocytes. These studies establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm and is required for its function in oligodendrocyte maturation. PMID:26631469

  12. Unexpected fold in the circumsporozoite protein target of malaria vaccines

    Doud, Michael B.; Koksal, Adem C.; Mi, Li-Zhi; Song, Gaojie; Lu, Chafen; Springer, Timothy A. (Harvard-Med)

    2012-10-09

    Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an '{alpha}TSR' domain. The {alpha}TSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but {alpha}TSR does not. Interestingly, polymorphic T-cell epitopes map to specialized {alpha}TSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.

  13. Similar pathogen targets in Arabidopsis thaliana and homo sapiens protein networks.

    Paulo Shakarian

    Full Text Available We study the behavior of pathogens on host protein networks for humans and Arabidopsis - noting striking similarities. Specifically, we preform [Formula: see text]-shell decomposition analysis on these networks - which groups the proteins into various "shells" based on network structure. We observe that shells with a higher average degree are more highly targeted (with a power-law relationship and that highly targeted nodes lie in shells closer to the inner-core of the network. Additionally, we also note that the inner core of the network is significantly under-targeted. We show that these core proteins may have a role in intra-cellular communication and hypothesize that they are less attacked to ensure survival of the host. This may explain why certain high-degree proteins are not significantly attacked.

  14. Fatal Intoxication with Acetyl Fentanyl.

    Cunningham, Susan M; Haikal, Nabila A; Kraner, James C

    2016-01-01

    Among the new psychoactive substances encountered in forensic investigations is the opioid, acetyl fentanyl. The death of a 28-year-old man from recreational use of this compound is reported. The decedent was found in the bathroom of his residence with a tourniquet secured around his arm and a syringe nearby. Postmortem examination findings included marked pulmonary and cerebral edema and needle track marks. Toxicological analysis revealed acetyl fentanyl in subclavian blood, liver, vitreous fluid, and urine at concentrations of 235 ng/mL, 2400 ng/g, 131 ng/mL, and 234 ng/mL, respectively. Acetyl fentanyl was also detected in the accompanying syringe. Death was attributed to recreational acetyl fentanyl abuse, likely through intravenous administration. The blood acetyl fentanyl concentration is considerably higher than typically found in fatal fentanyl intoxications. Analysis of this case underscores the need for consideration of a wide range of compounds with potential opioid-agonist activity when investigating apparent recreational drug-related deaths. PMID:26389815

  15. RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses.

    Baker, Oliver; Gupta, Ashish; Obst, Mandy; Zhang, Youming; Anastassiadis, Konstantinos; Fu, Jun; Stewart, A Francis

    2016-01-01

    A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented. PMID:27216209

  16. Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

    Tacchi, Jessica L; Raymond, Benjamin B A; Haynes, Paul A; Berry, Iain J; Widjaja, Michael; Bogema, Daniel R; Woolley, Lauren K; Jenkins, Cheryl; Minion, F Chris; Padula, Matthew P; Djordjevic, Steven P

    2016-02-01

    Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

  17. Pharmacoinformatics elucidation of potential drug targets against migraine to target ion channel protein KCNK18.

    Sehgal, Sheikh Arslan; Hassan, Mubashir; Rashid, Sajid

    2014-01-01

    Migraine, a complex debilitating neurological disorder is strongly associated with potassium channel subfamily K member 18 (KCNK18). Research has emphasized that high levels of KCNK18 may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like migraine. In the present study, a hybrid approach of molecular docking and virtual screening were followed by pharmacophore identification and structure modeling. Screening was performed using a two-dimensional similarity search against recommended migraine drugs, keeping in view the physicochemical properties of drugs. LigandScout tool was used for exploring pharmacophore properties and designing novel molecules. Here, we report the screening of four novel compounds that have showed maximum binding affinity against KCNK18, obtained through the ZINC database, and Drug and Drug-Like libraries. Docking studies revealed that Asp-46, Ile-324, Ile-44, Gly-118, Leu-338, Val-113, and Phe-41 are critical residues for receptor-ligand interaction. A virtual screening approach coupled with docking energies and druglikeness rules illustrated that ergotamine and PB-414901692 are potential inhibitor compounds for targeting KCNK18. We propose that selected compounds may be more potent than the previously listed drug analogs based on the binding energy values. Further analysis of these inhibitors through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful for designing novel therapeutic targets to cure migraine. PMID:24899801

  18. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets.

    Hogan, Gregory J; Brown, Patrick O; Herschlag, Daniel

    2015-11-01

    Reprogramming of a gene's expression pattern by acquisition and loss of sequences recognized by specific regulatory RNA binding proteins may be a major mechanism in the evolution of biological regulatory programs. We identified that RNA targets of Puf3 orthologs have been conserved over 100-500 million years of evolution in five eukaryotic lineages. Focusing on Puf proteins and their targets across 80 fungi, we constructed a parsimonious model for their evolutionary history. This model entails extensive and coordinated changes in the Puf targets as well as changes in the number of Puf genes and alterations of RNA binding specificity including that: 1) Binding of Puf3 to more than 200 RNAs whose protein products are predominantly involved in the production and organization of mitochondrial complexes predates the origin of budding yeasts and filamentous fungi and was maintained for 500 million years, throughout the evolution of budding yeast. 2) In filamentous fungi, remarkably, more than 150 of the ancestral Puf3 targets were gained by Puf4, with one lineage maintaining both Puf3 and Puf4 as regulators and a sister lineage losing Puf3 as a regulator of these RNAs. The decrease in gene expression of these mRNAs upon deletion of Puf4 in filamentous fungi (N. crassa) in contrast to the increase upon Puf3 deletion in budding yeast (S. cerevisiae) suggests that the output of the RNA regulatory network is different with Puf4 in filamentous fungi than with Puf3 in budding yeast. 3) The coregulated Puf4 target set in filamentous fungi expanded to include mitochondrial genes involved in the tricarboxylic acid (TCA) cycle and other nuclear-encoded RNAs with mitochondrial function not bound by Puf3 in budding yeast, observations that provide additional evidence for substantial rewiring of post-transcriptional regulation. 4) Puf3 also expanded and diversified its targets in filamentous fungi, gaining interactions with the mRNAs encoding the mitochondrial electron transport

  19. Localization of Seed Oil Body Proteins in Tobacco Protoplasts Reveals Specific Mechanisms of Protein Targeting to Leaf Lipid Droplets

    Stefania De Domenico; Stefania Bonsegna; Marcello Salvatore Lenucci; Palmiro Poltronieri; Gian Pietro Di Sansebastiano; Angelo Santino

    2011-01-01

    Oleosin,caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies.In the present work,the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy.Our results indicated clear differences in the endocellular localization of the three proteins.Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets (LD),whereas steroleosin,characterized by an N-terminal oil body anchoring domain,was mainly retained in the endoplasmic reticulum (ER).Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosingreen fluorescent protein (GFP) peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein (BiP),were recovered.Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered.Finally,only oleosinand caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids.Taken together,our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.

  20. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses. (paper)

  1. Outer Membrane Targeting of Pseudomonas aeruginosa Proteins Shows Variable Dependence on the Components of Bam and Lol Machineries

    Hoang, Hanh H.; Nickerson, Nicholas N.; Lee, Vincent T.; Kazimirova, Anastasia; Chami, Mohamed; Pugsley, Anthony P.; Lory, Stephen

    2011-01-01

    ABSTRACT In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting...

  2. Identification of proteins targeted by the thioredoxin superfamily in Plasmodium falciparum.

    Nicole Sturm

    2009-04-01

    Full Text Available The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx and glutaredoxin (Grx, as well as plasmoredoxin (Plrx, which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins

  3. Predicting post-translational lysine acetylation using support vector machines

    Gnad, Florian; Ren, Shubin; Choudhary, Chunaram;

    2010-01-01

    spectrometry to identify 3600 lysine acetylation sites on 1750 human proteins covering most of the previously annotated sites and providing the most comprehensive acetylome so far. This dataset should provide an excellent source to train support vector machines (SVMs) allowing the high accuracy in silico...

  4. Heat shock and other apoptosis-related proteins as therapeutic targets in prostate cancer

    Costantine Albany; Noah M Hahn

    2014-01-01

    Defects within apoptotic pathways have been implicated in prostate cancer (PCa) tumorigenesis, metastatic progression and treatment resistance. A hallmark of cancers is the ability to derail apoptosis by inhibiting the apoptotic signal, reducing the expression of apoptotic proteins and/or amplifying survival signals through increased production of antiapoptotic molecule. This review describes associations between heat shock proteins (HSPs) and the human androgen receptor (AR), the role of HSPs and other stress-induced proteins in PCa development and emerging strategies in targeting these protective proteins to treat PCa.

  5. A rapid identification of hit molecules for target proteins via physico-chemical descriptors.

    Mukherjee, Goutam; Jayaram, B

    2013-06-21

    We report here a novel computationally fast protocol (RASPD) for identifying good candidates for any target protein from any molecule/million molecule database. A QSAR-type equation sets up the extent of complementarity of the physico-chemical properties of the target protein and the candidate molecule and an estimate of the binding energy is generated. A correlation coefficient of 0.84 and an average error ±1.45 kcal mol(-1) are obtained for the calculated protein-ligand binding energies against experiment for more than 380 protein-ligand complexes. RASPD is seen to perform better than other popular scoring functions in predicting binding energies. The most interesting feature of this methodology is that it takes only a fraction of a second for calculating the binding energy of any ligand without docking in the active site of the target protein as opposed to several minutes for regular docking and scoring methods, while the accuracy in sorting good candidates remains comparable to that of conventional techniques. An entire million compound library, a (~10(5) compound) natural product library and a (~10(5) compound) NCI database can be scanned against a specified target protein within a few minutes for identifying hit molecules. The RASPD methodology is freely accessible at . PMID:23646352

  6. Proteins with complex architecture as potential targets for drug design: a case study of Mycobacterium tuberculosis.

    Bálint Mészáros

    2011-07-01

    Full Text Available Lengthy co-evolution of Homo sapiens and Mycobacterium tuberculosis, the main causative agent of tuberculosis, resulted in a dramatically successful pathogen species that presents considerable challenge for modern medicine. The continuous and ever increasing appearance of multi-drug resistant mycobacteria necessitates the identification of novel drug targets and drugs with new mechanisms of action. However, further insights are needed to establish automated protocols for target selection based on the available complete genome sequences. In the present study, we perform complete proteome level comparisons between M. tuberculosis, mycobacteria, other prokaryotes and available eukaryotes based on protein domains, local sequence similarities and protein disorder. We show that the enrichment of certain domains in the genome can indicate an important function specific to M. tuberculosis. We identified two families, termed pkn and PE/PPE that stand out in this respect. The common property of these two protein families is a complex domain organization that combines species-specific regions, commonly occurring domains and disordered segments. Besides highlighting promising novel drug target candidates in M. tuberculosis, the presented analysis can also be viewed as a general protocol to identify proteins involved in species-specific functions in a given organism. We conclude that target selection protocols should be extended to include proteins with complex domain architectures instead of focusing on sequentially unique and essential proteins only.

  7. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

  8. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

    2015-06-15

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

  9. Pharmacoinformatics elucidation of potential drug targets against migraine to target ion channel protein KCNK18

    Sehgal SA

    2014-05-01

    Full Text Available Sheikh Arslan Sehgal, Mubashir Hassan, Sajid Rashid National Center for Bioinformatics, Quaid-i-Azam University, Islamabad, Pakistan Abstract: Migraine, a complex debilitating neurological disorder is strongly associated with potassium channel subfamily K member 18 (KCNK18. Research has emphasized that high levels of KCNK18 may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like migraine. In the present study, a hybrid approach of molecular docking and virtual screening were followed by pharmacophore identification and structure modeling. Screening was performed using a two-dimensional similarity search against recommended migraine drugs, keeping in view the physicochemical properties of drugs. LigandScout tool was used for exploring pharmacophore properties and designing novel molecules. Here, we report the screening of four novel compounds that have showed maximum binding affinity against KCNK18, obtained through the ZINC database, and Drug and Drug-Like libraries. Docking studies revealed that Asp-46, Ile-324, Ile-44, Gly-118, Leu-338, Val-113, and Phe-41 are critical residues for receptor–ligand interaction. A virtual screening approach coupled with docking energies and druglikeness rules illustrated that ergotamine and PB-414901692 are potential inhibitor compounds for targeting KCNK18. We propose that selected compounds may be more potent than the previously listed drug analogs based on the binding energy values. Further analysis of these inhibitors through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful for designing novel therapeutic targets to cure migraine. Keywords: migraine, bioinformatics, modeling and docking, KCNK18, TRESK, virtual screening, pharmacoinformatics

  10. Acetylation site specificities of lysine deacetylase inhibitors in human cells

    Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A;

    2015-01-01

    Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain...... acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the...

  11. Peptide aptamer identified by molecular docking targeting translationally controlled tumor protein in leukemia cells.

    Kadioglu, Onat; Efferth, Thomas

    2016-08-01

    Bioinformatics screening and molecular docking analyses were utilized to select high affinity peptides targeting translationally controlled tumor protein (TCTP). Selected peptide aptamers were tested towards cancer cell lines with different levels of TCTP expression. One peptide (WGQWPYHC) revealed specific cytotoxicity according to the TCTP expression in tumor cells without affecting normal cells. Western blot analysis showed peptide-induced down-regulation of TCTP as primary target as well as of cell-cycle related downstream proteins (CDK2, CDK6, Cyclin D3) in MOLT-4 leukemia cells. "WGQWPYHC" deserves further analysis for targeted therapy of TCTP-expressing tumor cells. Graphical abstract Molecular docking on TCTP, cytotoxicity toward MOLT-4 leukemia cell line and downregulation of CDK2, CDK6, CyclinD3 and TCTP proteins. PMID:26972431

  12. Targeting Protein Kinase C Downstream of Growth Factor and Adhesion Signalling

    Dowling, Catríona M., E-mail: Catriona.Dowling@ul.ie; Kiely, Patrick A., E-mail: Catriona.Dowling@ul.ie [Department of Life Sciences, Materials and Surface Science Institute and Stokes Institute, University of Limerick, Limerick 78666 (Ireland); Health Research Institute (HRI), University of Limerick, Limerick 78666 (Ireland)

    2015-07-15

    The signaling outputs of Receptor Tyrosine Kinases, G-protein coupled receptors and integrins converge to mediate key cell process such as cell adhesion, cell migration, cell invasion and cell proliferation. Once activated by their ligands, these cell surface proteins recruit and direct a diverse range of proteins to disseminate the appropriate response downstream of the specific environmental cues. One of the key groups of proteins required to regulate these activities is the family of serine/threonine intracellular kinases called Protein Kinase Cs. The activity and subcellular location of PKCs are mediated by a series of tightly regulated events and is dependent on several posttranslational modifications and the availability of second messengers. Protein Kinase Cs exhibit both pro- and anti-tumorigenic effects making them an interesting target for anti-cancer treatment.

  13. Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

    Tran, Eric; Chinnasamy, Dhanalakshmi; Yu, Zhiya; Morgan, Richard A.; Lee, Chyi-Chia Richard; Restifo, Nicholas P; Rosenberg, Steven A.

    2013-01-01

    Fibroblast activation protein (FAP) is a candidate universal target antigen because it has been reported to be selectively expressed in nearly all solid tumors by a subset of immunosuppressive tumor stromal fibroblasts. We verified that 18/18 human tumors of various histologies contained pronounced stromal elements staining strongly for FAP, and hypothesized that targeting tumor stroma with FAP-reactive T cells would inhibit tumor growth in cancer-bearing hosts. T cells genetically engineered...

  14. Immunotherapy Targeting Pathological Tau Protein in Alzheimer's Disease and Related Tauopathies

    Sigurdsson, Einar M.

    2008-01-01

    Immunotherapies that target the amyloid-β (Aβ) peptide in Alzheimer's disease (AD) have shown promise in animal and human studies. Although the first clinical trial was halted because of adverse reactions, this approach has been refined and additional trials are underway. Another important target in AD is the neurofibrillary tangles, composed primarily of hyperphosphorylated tau proteins, which correlate well with the degree of dementia. As Aβ and tau pathologies are likely synergistic, targe...

  15. Computational Design of Proteins Targeting the Conserved Stem Region of Influenza Hemagglutinin

    Fleishman, Sarel J.; Whitehead, Timothy A.; Ekiert, Damian C.; Dreyfus, Cyrille; Corn, Jacob E.; Strauch, Eva-Maria; Wilson, Ian A.; Baker, David (UWASH); (Scripps)

    2011-09-28

    We describe a general computational method for designing proteins that bind a surface patch of interest on a target macromolecule. Favorable interactions between disembodied amino acid residues and the target surface are identified and used to anchor de novo designed interfaces. The method was used to design proteins that bind a conserved surface patch on the stem of the influenza hemagglutinin (HA) from the 1918 H1N1 pandemic virus. After affinity maturation, two of the designed proteins, HB36 and HB80, bind H1 and H5 HAs with low nanomolar affinity. Further, HB80 inhibits the HA fusogenic conformational changes induced at low pH. The crystal structure of HB36 in complex with 1918/H1 HA revealed that the actual binding interface is nearly identical to that in the computational design model. Such designed binding proteins may be useful for both diagnostics and therapeutics.

  16. Antiapoptotic Bcl-2 protein as a potential target for cancer therapy: A mini review.

    Jagani, Hitesh; Kasinathan, Narayanan; Meka, Sreenivasa Reddy; Josyula, Venkata Rao

    2016-08-01

    Bcl-2, an antiapoptotic protein, is considered as a potential target in cancer treatment since its oncogenic potential has been proven and is well documented. Antisense technology and RNA interference (RNAi) have been used to reduce the expression of the Bcl-2 gene in many types of cancer cells and are effective as adjuvant therapy along with the chemotherapeutic agents. The lack of appropriate delivery systems is considered to be the main hurdle associated with the RNAi. In this review, we discuss the antiapoptotic Bcl-2 protein, its oncogenic potential, and various approaches utilized to target Bcl-2 including suitable delivery systems employed for successful delivery of siRNA. PMID:25801037

  17. Large-scale prediction of drug-target interactions using protein sequences and drug topological structures

    Cao Dongsheng [Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha 410083 (China); Liu Shao [Xiangya Hospital, Central South University, Changsha 410008 (China); Xu Qingsong [School of Mathematical Sciences and Computing Technology, Central South University, Changsha 410083 (China); Lu Hongmei; Huang Jianhua [Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha 410083 (China); Hu Qiannan [Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071 (China); Liang Yizeng, E-mail: yizeng_liang@263.net [Research Center of Modernization of Traditional Chinese Medicines, Central South University, Changsha 410083 (China)

    2012-11-08

    Highlights: Black-Right-Pointing-Pointer Drug-target interactions are predicted using an extended SAR methodology. Black-Right-Pointing-Pointer A drug-target interaction is regarded as an event triggered by many factors. Black-Right-Pointing-Pointer Molecular fingerprint and CTD descriptors are used to represent drugs and proteins. Black-Right-Pointing-Pointer Our approach shows compatibility between the new scheme and current SAR methodology. - Abstract: The identification of interactions between drugs and target proteins plays a key role in the process of genomic drug discovery. It is both consuming and costly to determine drug-target interactions by experiments alone. Therefore, there is an urgent need to develop new in silico prediction approaches capable of identifying these potential drug-target interactions in a timely manner. In this article, we aim at extending current structure-activity relationship (SAR) methodology to fulfill such requirements. In some sense, a drug-target interaction can be regarded as an event or property triggered by many influence factors from drugs and target proteins. Thus, each interaction pair can be represented theoretically by using these factors which are based on the structural and physicochemical properties simultaneously from drugs and proteins. To realize this, drug molecules are encoded with MACCS substructure fingerings representing existence of certain functional groups or fragments; and proteins are encoded with some biochemical and physicochemical properties. Four classes of drug-target interaction networks in humans involving enzymes, ion channels, G-protein-coupled receptors (GPCRs) and nuclear receptors, are independently used for establishing predictive models with support vector machines (SVMs). The SVM models gave prediction accuracy of 90.31%, 88.91%, 84.68% and 83.74% for four datasets, respectively. In conclusion, the results demonstrate the ability of our proposed method to predict the drug-target

  18. Endogenous occurrence of protein S-guanylation in Escherichia coli: Target identification and genetic regulation.

    Tsutsuki, Hiroyasu; Jung, Minkyung; Zhang, Tianli; Ono, Katsuhiko; Ida, Tomoaki; Kunieda, Kohei; Ihara, Hideshi; Akaike, Takaaki; Sawa, Tomohiro

    2016-09-01

    8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated cGMP derivative formed in response to nitric oxide (NO) and reactive oxygen species (ROS). It can cause a post-translational modification (PTM) of protein thiols through cGMP adduction (protein S-guanylation). Accumulating evidence has suggested that, in mammals, S-guanylation of redox-sensor proteins may implicate in regulation of adaptive responses against ROS-associated oxidative stress. Occurrence as well as protein targets of S-guanylation in bacteria remained unknown, however. Here we demonstrated, for the first time, the endogenous occurrence of protein S-guanylation in Escherichia coli (E. coli). Western blotting using anti-S-guanylation antibody clearly showed that multiple proteins were S-guanylated in E. coli. Interestingly, some of those proteins were more intensely S-guanylated when bacteria were cultured under static culture condition than shaking culture condition. It has been known that E. coli is deficient of guanylate cyclase, an enzyme indispensable for 8-nitro-cGMP formation in mammals. We found that adenylate cyclase from E. coli potentially catalyzed 8-nitro-cGMP formation from its precursor 8-nitroguanosine 5'-triphosphate. More importantly, E. coli lacking adenylate cyclase showed significantly reduced formation of S-guanylated proteins. Our S-guanylation proteomics successfully identified S-guanylation protein targets in E. coli, including chaperons, ribosomal proteins, and enzymes which associate with protein synthesis, redox regulation and metabolism. Understanding of functional impacts for protein S-guanylation in bacterial signal transduction is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of pathogenic bacterial infections. PMID:27473654

  19. A dysregulated acetyl/SUMO switch of FXR promotes hepatic inflammation in obesity.

    Kim, Dong-Hyun; Xiao, Zhen; Kwon, Sanghoon; Sun, Xiaoxiao; Ryerson, Daniel; Tkac, David; Ma, Ping; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Zhou, Edward; Xu, H Eric; Palvimo, Jorma J; Chen, Lin-Feng; Kemper, Byron; Kemper, Jongsook Kim

    2015-01-13

    Acetylation of transcriptional regulators is normally dynamically regulated by nutrient status but is often persistently elevated in nutrient-excessive obesity conditions. We investigated the functional consequences of such aberrantly elevated acetylation of the nuclear receptor FXR as a model. Proteomic studies identified K217 as the FXR acetylation site in diet-induced obese mice. In vivo studies utilizing acetylation-mimic and acetylation-defective K217 mutants and gene expression profiling revealed that FXR acetylation increased proinflammatory gene expression, macrophage infiltration, and liver cytokine and triglyceride levels, impaired insulin signaling, and increased glucose intolerance. Mechanistically, acetylation of FXR blocked its interaction with the SUMO ligase PIASy and inhibited SUMO2 modification at K277, resulting in activation of inflammatory genes. SUMOylation of agonist-activated FXR increased its interaction with NF-κB but blocked that with RXRα, so that SUMO2-modified FXR was selectively recruited to and trans-repressed inflammatory genes without affecting FXR/RXRα target genes. A dysregulated acetyl/SUMO switch of FXR in obesity may serve as a general mechanism for diminished anti-inflammatory response of other transcriptional regulators and provide potential therapeutic and diagnostic targets for obesity-related metabolic disorders. PMID:25425577

  20. MOTIPS: Automated Motif Analysis for Predicting Targets of Modular Protein Domains

    Tonikian Raffi

    2010-05-01

    Full Text Available Abstract Background Many protein interactions, especially those involved in signaling, involve short linear motifs consisting of 5-10 amino acid residues that interact with modular protein domains such as the SH3 binding domains and the kinase catalytic domains. One straightforward way of identifying these interactions is by scanning for matches to the motif against all the sequences in a target proteome. However, predicting domain targets by motif sequence alone without considering other genomic and structural information has been shown to be lacking in accuracy. Results We developed an efficient search algorithm to scan the target proteome for potential domain targets and to increase the accuracy of each hit by integrating a variety of pre-computed features, such as conservation, surface propensity, and disorder. The integration is performed using naïve Bayes and a training set of validated experiments. Conclusions By integrating a variety of biologically relevant features to predict domain targets, we demonstrated a notably improved prediction of modular protein domain targets. Combined with emerging high-resolution data of domain specificities, we believe that our approach can assist in the reconstruction of many signaling pathways.

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

    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

  2. Regulation of SUMO2 Target Proteins by the Proteasome in Human Cells Exposed to Replication Stress

    Bursomanno, Sara; McGouran, Joanna F; Kessler, Benedikt M;

    2015-01-01

    In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role of the...... genome instability, which is suggested to drive tumorigenesis and possibly aging, our data will facilitate future functional studies in the fields of DNA metabolism and cancer biology....

  3. Exosomal protein interactors as emerging therapeutic targets in urothelial bladder cancer

    Background: Exosomes are rich sources of biological material (proteins and nucleic acids) secreted by both tumor and normal cells, and found in urine of urinary bladder cancer patients. Objective: The objective of the study was to identify interacting exosomal proteins in bladder cancer for future use in targeted therapy. Methods: The Exocarta database (www.exocarta.org) was mined for urinary bladder cancer specific exosomal proteins. The urinary bladder cancer specific exosomal proteins (n = 248) were analyzed to identify enriched pathways by Onto-tool Pathway Express (http://vortex.cs.wayne.edu/ ontoexpress). Results: Enriched pathways included cellular architecture, motility, cell to cell adhesion, tumorigenesis and metastasis. Proteins in the 9 top-ranked pathways included CTNNA1 (alpha-catenin), CTNNB1 (beta-catenin), VSAP, ITGA4, PAK1, DDR1, CDC42, RHOA, NRAS, RHO, PIK3AR1, MLC1, MMRN1, and CTTNBP2 and network analysis revealed 10 important hub proteins and identified inferred interactor NF2. Conclusions: The importance of identifying interactors is that that they can be used as targets for therapy, for example, using Bevacizumab (avastin - an angiogenesis inhibitor) against NF2 to inhibit protein-protein interactions will inhibit tumor growth and progression by hindering the exosome biogenesis

  4. Acetylation of TAFI68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription

    Muth, Viola; Nadaud, Sophie; Grummt, Ingrid; Voit, Renate

    2001-01-01

    Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAFI68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, an...

  5. The garlic compound ajoene targets protein folding in the endoplasmic reticulum of cancer cells.

    Kaschula, Catherine H; Hunter, Roger; Cotton, Jonathan; Tuveri, Rossana; Ngarande, Ellen; Dzobo, Kevin; Schäfer, Georgia; Siyo, Vuyolwethu; Lang, Dirk; Kusza, Daniel A; Davies, Bronwen; Katz, Arieh A; Parker, M Iqbal

    2016-08-01

    Ajoene is a natural allylsulfur compound found in crushed garlic that arrests growth and induces apoptosis in cancer cells. To gain mechanistic insights into the cytotoxicity of ajoene in cancer cells, two fluorescently labelled ajoene analogs with dansyl- (DP) and fluorescein- (FOX) tags were synthesized. The tagged ajoenes were found to retain their activity at inhibiting proliferation and inducing apoptosis in MDA-MB-231 human breast-cancer and WHCO1 human esophageal-cancer cells. Both tagged ajoenes localized to the endoplasmic reticulum (ER) in MDA-MB-231 cells as observed by live cell confocal laser scanning microscopy (CLSM) and confirmed by generating an MDA-MB-231 cell line expressing yellow fluorescent protein (YFP) in the ER. DP appears to S-thiolate multiple protein targets in MDA-MB-231 cells as observed by immunoblotting under non-reducing conditions only; and a competition assay demonstrated that DP and Z-ajoene in fact share the same target. Ajoene S-thiolation interfered with protein folding and led to an accumulation of misfolded protein aggregates and activated the unfolded protein response (UPR). Consistent with this mechanism, increased levels of GRP78 and total ubiquitinated proteins were observed; and an ER-folded protein, type-1 collagen, was tracked to the proteasome following ajoene treatment. The intracellular protein aggregates were observed by CLSM and transmission electron microscopy (TEM). This is the first time that ajoene has been shown to target protein folding in the ER of cancer cells. © 2015 Wiley Periodicals, Inc. PMID:26207910

  6. Co-evolution of SNF spliceosomal proteins with their RNA targets in trans-splicing nematodes.

    Strange, Rex Meade; Russelburg, L Peyton; Delaney, Kimberly J

    2016-08-01

    Although the mechanism of pre-mRNA splicing has been well characterized, the evolution of spliceosomal proteins is poorly understood. The U1A/U2B″/SNF family (hereafter referred to as the SNF family) of RNA binding spliceosomal proteins participates in both the U1 and U2 small interacting nuclear ribonucleoproteins (snRNPs). The highly constrained nature of this system has inhibited an analysis of co-evolutionary trends between the proteins and their RNA binding targets. Here we report accelerated sequence evolution in the SNF protein family in Phylum Nematoda, which has allowed an analysis of protein:RNA co-evolution. In a comparison of SNF genes from ecdysozoan species, we found a correlation between trans-splicing species (nematodes) and increased phylogenetic branch lengths of the SNF protein family, with respect to their sister clade Arthropoda. In particular, we found that nematodes (~70-80 % of pre-mRNAs are trans-spliced) have experienced higher rates of SNF sequence evolution than arthropods (predominantly cis-spliced) at both the nucleotide and amino acid levels. Interestingly, this increased evolutionary rate correlates with the reliance on trans-splicing by nematodes, which would alter the role of the SNF family of spliceosomal proteins. We mapped amino acid substitutions to functionally important regions of the SNF protein, specifically to sites that are predicted to disrupt protein:RNA and protein:protein interactions. Finally, we investigated SNF's RNA targets: the U1 and U2 snRNAs. Both are more divergent in nematodes than arthropods, suggesting the RNAs have co-evolved with SNF in order to maintain the necessarily high affinity interaction that has been characterized in other species. PMID:27450547

  7. Contractile actin cables induced by Bacillus anthracis lethal toxin depend on the histone acetylation machinery.

    Rolando, Monica; Stefani, Caroline; Doye, Anne; Acosta, Maria I; Visvikis, Orane; Yevick, Hannah G; Buchrieser, Carmen; Mettouchi, Amel; Bassereau, Patricia; Lemichez, Emmanuel

    2015-10-01

    It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier. PMID:26403219

  8. Ku70 acetylation mediates neuroblastoma cell death induced by histone deacetylase inhibitors

    Subramanian, Chitra; Opipari, Anthony W.; Bian, Xin; Castle, Valerie P; Kwok, Roland P S

    2005-01-01

    Histone deacetylase inhibitors (HDACIs) are therapeutic drugs that inhibit deacetylase activity, thereby increasing acetylation of many proteins, including histones. HDACIs have antineoplastic effects in preclinical and clinical trials and are being considered for cancers with unmet therapeutic need, including neuroblastoma (NB). Uncertainty of how HDACI-induced protein acetylation leads to cell death, however, makes it difficult to determine which tumors are likely to be responsive to these ...

  9. Expression of protein-tyrosine phosphatases in the major insulin target tissues

    Norris, K; Norris, F; Kono, D H;

    1997-01-01

    Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a repre...

  10. Orphan G protein receptor GPR55 as an emerging target in cancer therapy and management

    G protein-coupled receptors (GPCRs) modulate a vast array of cellular processes. The current review gives an overview of the general characteristics of GPCRs and their role in physiological conditions. In addition, it describes the current knowledge of the physiological and pathophysiological functions of GPR55, an orphan GPCR, and how it can be exploited as a therapeutic target to combat various cancers

  11. DRUG TARGETING TO THE KIDNEY WITH LOW-MOLECULAR-WEIGHT PROTEINS

    FRANSSEN, EJF; MOOLENAAR, F; DEZEEUW, D; MEIJER, DKF

    1993-01-01

    This paper reviews the design of a drug targeting strategy for renal specific delivery and endorenal release of drugs using low-molecular-weight proteins (LMWPs). In general, LMWPs are known to be filtered and subsequently reabsorbed by the proximal tubular cells of the kidneys. Within these cells L

  12. Separation of 211At from irradiated bismuth target and labelling of a model protein

    Astatine-211 is a promising radionuclide for therapeutic use in nuclear medicine. An efficient method (70%) for radiochemical separation from the irradiated Bi-target has been described. The level of Bi contamination in the purified 2'11At-solution was estimated. Finally labelling of a model protein has been tried. (author). 14 refs

  13. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  14. Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development.

    Song, Hye-Won; Cauffman, Karen; Chan, Agnes P; Zhou, Yi; King, Mary Lou; Etkin, Laurence D; Kloc, Malgorzata

    2007-07-01

    The early development of metazoans is mainly regulated by differential translation and localization of maternal mRNAs in the embryo. In general, these processes are orchestrated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated region (UTR) of their target RNAs. Hermes is an RNA-binding protein, which contains a single RNA recognition motif (RRM) and is found in various vertebrate species from fish to human. In Xenopus laevis, Hermes mRNA and protein are localized in the vegetal region of oocytes. A subpopulation of Hermes protein is concentrated in a specific structure in the vegetal cortex, called the germ plasm (believed to contain determinants of the germ cell fate) where Hermes protein co-localizes with Xcat2 and RINGO/Spy mRNAs. The level of total Hermes protein decreases during maturation. The precocious depletion of Hermes protein by injection of Hermes antisense morpholino oligonucleotide (HE-MO) accelerates the process of maturation and results in cleavage defects in vegetal blastomeres of the embryo. It is known that several maternal mRNAs including RINGO/Spy and Mos are regulated at the translational level during meiotic maturation and early cleavage in Xenopus. The ectopic expression of RINGO/Spy or Mos causes resumption of meiotic maturation and cleavage arrests, which resemble the loss of Hermes phenotypes. We found that the injection of HE-MO enhances the acceleration of maturation caused by the injection of RINGO/Spy mRNA, and that Hermes protein is present as mRNP complex containing RINGO/Spy, Mos, and Xcat2 mRNAs in vivo. We propose that as an RNA-binding protein, Hermes may be involved in maturation, cleavage events at the vegetal pole and germ cell development by negatively regulating the expression of RINGO/Spy, Mos, and Xcat2 mRNAs. PMID:17309605

  15. Structure of the signal transduction protein TRAP (target of RNAIII-activating protein)

    The 1.85 Å resolution structure of the signal transduction protein TRAP is presented. The overall fold of TRAP is an unsymmetrical eight-stranded β-barrel with five helices. The crystal structure of the signal transduction protein TRAP is reported at 1.85 Å resolution. The structure of TRAP consists of a central eight-stranded β-barrel flanked asymmetrically by helices and is monomeric both in solution and in the crystal structure. A formate ion was found bound to TRAP identically in all four molecules in the asymmetric unit

  16. A sol-gel-integrated protein array system for affinity analysis of aptamer-target protein interaction.

    Ahn, Ji-Young; Kim, Eunkyung; Kang, Jeehye; Kim, Soyoun

    2011-06-01

    A sol-gel microarray system was developed for a protein interaction assay with high activity. Comparing to 2-dimensional microarray surfaces, sol-gel can offer a more dynamic and broad range for proteins. In the present study, this sol-gel-integrated protein array was used in binding affinity analysis for aptamers. Six RNA aptamers and their target protein, yeast TBP (TATA-binding protein), were used to evaluate this method. A TBP-containing sol-gel mixture was spotted using a dispensing workstation under high-humidity conditions and each Cy-3-labeled aptamer was incubated. The dissociation constants (K(d)) were calculated by plotting the fluorescent intensity of the bound aptamers as a function of the TBP concentrations. The K(d) value of the control aptamer was found to be 8 nM, which agrees well with the values obtained using the conventional method, electric mobility shift assay. The sol-gel-based binding affinity measurements fit well with conventional binding affinity measurements, suggesting their possible use as an alternative to the conventional method. In addition, aptamer affinity measurements by the sol-gel-integrated protein chip make it possible to develop a simple high-throughput affinity method for screening high-affinity aptamers. PMID:21749295

  17. Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination

    Highlights: → Tob/BTG family proteins are monoubiquitinated in the absence of E3s in vitro. → Monoubiquitination sites of Tob are identified by mass spectrometry. → The monoubiquitination event correlates with lower levels of polyubiquitination. -- Abstract: Tob belongs to the anti-proliferative Tob/BTG protein family. The expression level of Tob family proteins is strictly regulated both transcriptionally and through post-translational modification. Ubiquitin (Ub)/proteosome-dependent degradation of Tob family proteins is critical in controlling cell cycle progression and DNA damage responses. Various Ub ligases (E3s) are responsible for degradation of Tob protein. Here, we show that Tob family proteins undergo monoubiquitination even in the absence of E3s in vitro. Determination of the ubiquitination site(s) in Tob by mass spectrometric analysis revealed that two lysine residues (Lys48 and Lys63) located in Tob/BTG homology domain are ubiquitinated. A mutant Tob, in which both Lys48 and Lys63 are substituted with alanine, is more strongly polyubiquitinated than wild-type Tob in vivo. These data suggest that monoubiquitination of Tob family proteins confers resistance against polyubiquitination, which targets proteins for degradation. The strategy for regulating the stability of Tob family proteins suggests a novel role for monoubiquitination.

  18. Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies*

    Littleton, Edward; Dreger, Mathias; Palace, Jackie; Vincent, Angela

    2009-01-01

    There is increasing interest in the role of antibodies targeting specific membrane proteins in neurological and other diseases. The target(s) of these pathogenic antibodies is known in a few diseases, usually when candidate cell surface proteins have been tested. Approaches for identifying new antigens have mainly resulted in the identification of antibodies to intracellular proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating antibodies. To identify cell surface antigens, we developed a “conformational membrane antigen isolation and identification” strategy. First, a cell line is identified that reacts with patient sera but not with control sera. Second, intact cells are exposed to sera to allow the binding of presumptive autoantibodies to their cell surface targets. After washing off non-bound serum components, the cells are lysed, and immune complexes are precipitated. Third, the bound surface antigen is identified by mass spectrometry. As a model system we used a muscle cell line, TE671, that endogenously expresses muscle-specific tyrosine receptor kinase (MuSK) and sera or plasmas from patients with a subtype of the autoimmune disease myasthenia gravis in which patients have autoantibodies against MuSK. MuSK was robustly detected as the only membrane protein in immunoprecipitates from all three patient samples tested and not from the three MuSK antibody-negative control samples processed in parallel. Of note, however, there were many intracellular proteins found in the immunoprecipitates from both patients and controls, suggesting that these were nonspecifically immunoprecipitated from cell extracts. The conformational membrane antigen isolation and identification technique should be of value for the detection of highly relevant antigenic targets in the growing number of suspected antibody-mediated autoimmune disorders. The

  19. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers.

    Fuchs, Hendrik; Weng, Alexander; Gilabert-Oriol, Roger

    2016-01-01

    The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments. PMID:27376327

  20. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

    Hendrik Fuchs

    2016-07-01

    Full Text Available The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

  1. Swelling of acetylated wood in organic liquids

    Obataya, E; Obataya, Eiichi; Gril, Joseph

    2005-01-01

    To investigate the affinity of acetylated wood for organic liquids, Yezo spruce wood specimens were acetylated with acetic anhydride, and their swelling in various liquids were compared to those of untreated specimens. The acetylated wood was rapidly and remarkably swollen in aprotic organic liquids such as benzene and toluene in which the untreated wood was swollen only slightly and/or very slowly. On the other hand, the swelling of wood in water, ethylene glycol and alcohols remained unchanged or decreased by the acetylation. Consequently the maximum volume of wood swollen in organic liquids was always larger than that in water. The effect of acetylation on the maximum swollen volume of wood was greater in liquids having smaller solubility parameters. The easier penetration of aprotic organic liquids into the acetylated wood was considered to be due to the scission of hydrogen bonds among the amorphous wood constituents by the substitution of hydroxyl groups with hydrophobic acetyl groups.

  2. Sorting and targeting of melanosomal membrane proteins: signals, pathways, and mechanisms.

    Setaluri, V

    2000-06-01

    Newly synthesized melanosomal proteins, like many other cellular proteins, traverse through a series of intracellular compartments en route to melanosomes. Entry and exit of proteins through these compartments is orchestrated by cellular sorting machinery that recognize specific sorting signals. Melanosomal membrane proteins begin their intracellular journey upon co-translational importation into the endoplasmic reticulum (ER). The biosynthetic output of tyrosinase, the key melanogenic enzyme, appears to be regulated by quality-control events at the ER, the 'port of entry' to the secretory pathway. Following maturation in the ER and through the Golgi, the sorting of these proteins in the trans-Golgi network for intracellular retention and transport along endosome/lysosome pathway requires cytoplasmically exposed signals. A di-leucine motif, present in the cytoplasmic tails of most melanosomal proteins, and its interaction with adaptor protein (AP) complexes, specifically AP-3, are critical for these events. Defects in sorting signals and the cytosolic components that interact with these signals result in a number of murine coat color phenotypes and cause human pigmentary disorders. Thus, missense or frame-shift mutations that produce truncated tyrosinase lacking the melanosomal sorting signal(s) appear to be responsible for murine platinum coat color phenotypes and a proportion of human oculocutaneous albinism-1; mutations in AP-3 appear to be responsible for the mocha phenotype in mice and Hermansky-Pudlak-like syndrome in man. Additional signals and sorting steps downstream of AP-3 appear to be required for endosomal sorting and targeting proteins to melanosomes. Signals and mechanisms that sequester melanosomal proteins from endosomes/lysosomes are not understood. Potential candidates that mediate such processes include proteins encoded by lyst and pallid genes. The common occurrence of abnormalities in melanosomes in many storage-pool disorders suggests that

  3. Matrix metalloproteinase-mediation of tumor targeting human recombinant tumor necrosis factor-α fusion protein.

    Ren, Hui; Shao, Xin; Zeng, Liang; Wang, Fa; Huang, Di-Nan; Hou, Gan

    2015-08-01

    The aim of the present study was to use genetic engineering in order to establish an efficient tumor necrosis factor (TNF)-α fusion protein with low toxicity, which may be used to target tumors. Four types of matrix metalloproteinase (MMP)-mediated tumor targeting human recombinant TNF-α (rhTNF-α) fusion protein vectors were constructed. These were subsequently introduced into Escherichia coli. rhTNF-α fusion protein with a glutathione S-transferase (GST)-tag was purified using GST resin affinity chromatography, and GST-tags were digested using factor Xa. The cytotoxic effects of the fusion protein on L929 cells were determined using MTT assays. At a concentration of 1 pM, the GST-tagged fusion protein exerted no cytotoxic effects on the cells, compared with the negative control cells (P=0.975>0.05). However, at a concentration of 1000 pM, the deblocking fusion protein exerted greater cytotoxic effects on L929 cells, compared with positive control cells (Peffects on healthy cells. PMID:25891416

  4. Histone Acetylation in Drug Addiction

    Renthal, William; Nestler, Eric J.

    2009-01-01

    Regulation of chromatin structure through post-translational modifications of histones (e.g. acetylation) has emerged as an important mechanism to translate a variety of environmental stimuli, including drugs of abuse, into specific changes in gene expression. Since alterations in gene expression are thought to contribute to the development and maintenance of the addicted state, recent efforts are aimed at identifying how drugs of abuse alter chromatin structure and the enzymes which regulate...

  5. Protein Targets of Isoniazid-Reactive Metabolites in Mouse Liver in Vivo.

    Koen, Yakov M; Galeva, Nadezhda A; Metushi, Imir G; Uetrecht, Jack; Hanzlik, Robert P

    2016-06-20

    Isoniazid (INH) has been a first-line drug for the treatment of tuberculosis for more than 40 years. INH is well-tolerated by most patients, but some patients develop hepatitis that can be severe in rare cases or after overdose. The mechanisms underlying the hepatotoxicity of INH are not known, but covalent binding of reactive metabolites is known to occur in animals and is suspected in human cases. A major unresolved question is the identity of the liver proteins that are modified by INH metabolites. Treating mice with INH leads to accumulation of isonicotinoyl-lysine residues on numerous proteins in the hepatic S9 fraction. Analysis of this fraction by SDS-PAGE followed by tryptic digestion of bands and LC-MS/MS revealed a single adducted peptide derived from d-dopachrome decarboxylase. When a tryptic digest of whole S9 was applied to anti-INH antibody immobilized on beads, only 12 peptides were retained, 5 of which clearly contained isonicotinoyl-lysine adducts and could be confidently assigned to 5 liver proteins. In another experiment, undigested S9 fractions from INA-treated and untreated (UT) mice were adsorbed in parallel on anti-INA beads and the retained proteins were digested and analyzed by LC-MS/MS. The INA-S9 digest showed 1 adducted peptide that was associated with a unique protein whose identity was corroborated by numerous nonadducted peptides in the digest and 13 other proteins identified only by multiple nonadducted peptides. None of these 14 proteins was associated with any peptides present in the UT-S9 fraction. Overall, we identified 7 mouse liver proteins that became adducted by INH metabolites in vivo. Of these 7 INH target proteins, only 2 have been previously reported as targets of any reactive metabolite in vivo. PMID:27097313

  6. Self-assembled "dock and lock" system for linking payloads to targeting proteins.

    Backer, Marina V; Patel, Vimal; Jehning, Brian T; Backer, Joseph M

    2006-01-01

    Random conjugation of therapeutic or diagnostic payloads to targeting proteins generates functionally heterogeneous products. Conjugation of payloads to an adapter that binds to a peptide tag engineered into a targeting protein provides an alternative strategy. To progress into clinical development, an adapter/docking tag system should include humanized components and be stable in circulation. We describe here an adapter/docking tag system based on mutated fragments of human RNase I that spontaneously bind to each other and form a conjugate with a disulfide bond between complimentary cysteine residues. This self-assembled "dock and lock" system utilizes the previously described fusion C-tag, a 1-15 aa fragment of human RNase I with the R4C amino acid substitution, and a newly engineered adapter protein (Ad-C), a 21-127-aa fragment of human RNase I with the V118C substitution. Two vastly different C-tagged recombinant proteins, human vascular endothelial growth factor (VEGF) and a 254-aa long N-terminal fragment of anthrax lethal factor (LFn), retain functional activities after spontaneous conjugation of Ad-C to N-terminal or C-terminal C-tag, respectively. Ad-C modified with pegylated phospolipid and inserted into the lipid membrane of drug-loaded liposomes (Doxil) retained the ability to conjugate C-tagged proteins, yielding targeted liposomes decorated with functionally active proteins. To further optimize the system, we engineered an adapter with an additional cysteine residue at position 88 for site-specific modification, conjugated it to C-tagged VEGF, and labeled with a near-infrared fluorescent dye Cy5.5, yielding a unique functionally active probe for in vivo molecular imaging. We expect that this self-assembled "dock and lock" system will provide new opportunities for using functionally active proteins for biomedical purposes. PMID:16848397

  7. Pharmacological Targeting of AMP-Activated Protein Kinase and Opportunities for Computer-Aided Drug Design.

    Miglianico, Marie; Nicolaes, Gerry A F; Neumann, Dietbert

    2016-04-14

    As a central regulator of metabolism, the AMP-activated protein kinase (AMPK) is an established therapeutic target for metabolic diseases. Beyond the metabolic area, the number of medical fields that involve AMPK grows continuously, expanding the potential applications for AMPK modulators. Even though indirect AMPK activators are used in the clinics for their beneficial metabolic outcome, the few described direct agonists all failed to reach the market to date, which leaves options open for novel targeting methods. As AMPK is not actually a single molecule and has different roles depending on its isoform composition, the opportunity for isoform-specific targeting has notably come forward, but the currently available modulators fall short of expectations. In this review, we argue that with the amount of available structural and ligand data, computer-based drug design offers a number of opportunities to undertake novel and isoform-specific targeting of AMPK. PMID:26510622

  8. Protein kinase Cs in lung cancer: A promising target for therapies

    Chenfang Fan

    2013-01-01

    Full Text Available Lung cancer has been identified as one of the most deadly oncologies. The most influential causes for disease progression include smoking, genetic mutation and inflammatory lung diseases. Conventional therapies for lung cancer including chemo and radio-treatments often cause serious adverse effects. The advent of novel therapeutics that specifically target signalling pathways activated by genetic alterations has revolutionized the way patients with lung cancer are treated. These are comprised of various molecular targets on its carcinogen signalling pathways, among which the protein kinase C (PKC family is a promising target. The 12 isotypes in the family demonstrate complex interactions. This inter-linked signalling loop has added complexity of developing effective therapies. An improved understanding of different molecules involved in these signalling pathways will provide several profound implications, ranging from preclinical work on the mechanisms to trial design. Therapies developed targeting individual/multiple PKCs combined with conventional strategies offer promising future combating cancer.

  9. Study on Wangzaozin-A-Inducing Cancer Apoptosis and Its Theoretical Protein Targets.

    Chen, Jing; Wang, Shixia; Lu, Xiaoquan

    2016-08-01

    Wangzaozin A is the most representative cytotoxic C-20-nonoxide compound of Isodon plants of Labiatae. The protein targets of the 2 isomers (X and X') of Wangzaozin A are, respectively, extracted from target fishing dock Web site. Each isomer has 23 targets with good energy scores. The binding modes of each isomer and its targets are, respectively, analyzed by Dock. The energy score of the binding mode between the isomer X and the inositol-1(or 4)-monophosphatase is the smallest. The apoptosis, antiproliferative, and lethal effects of human gastric cancer cells induced by Wangzaozin A are assessed by trypan blue exclusion, hoechst33258 stain in SGC-7901, and flow cytometric measurement. The mechanism of Wangzaozin-A-inducing cancer apoptosis is analyzed. Wangzaozin A inhibits the growth of human gastric cancer SGC-7901 cell lines at the lower concentration (8.0 µmol/L). PMID:26082454

  10. Global Decrease of Histone H3K27 Acetylation in ZEB1-Induced Epithelial to Mesenchymal Transition in Lung Cancer Cells

    The epithelial to mesenchymal transition (EMT) enables epithelial cells with a migratory mesenchymal phenotype. It is activated in cancer cells and is involved in invasion, metastasis and stem-like properties. ZEB1, an E-box binding transcription factor, is a major suppressor of epithelial genes in lung cancer. In the present study, we show that in H358 non-small cell lung cancer cells, ZEB1 downregulates EpCAM (coding for an epithelial cell adhesion molecule), ESRP1 (epithelial splicing regulatory protein), ST14 (a membrane associated serine protease involved in HGF processing) and RAB25 (a small G-protein) by direct binding to these genes. Following ZEB1 induction, acetylation of histone H4 and histone H3 on lysine 9 (H3K9) and 27 (H3K27) was decreased on ZEB1 binding sites on these genes as demonstrated by chromatin immunoprecipitation. Of note, decreased H3K27 acetylation could be also detected by western blot and immunocytochemistry in ZEB1 induced cells. In lung cancers, H3K27 acetylation level was higher in the tumor compartment than in the corresponding stroma where ZEB1 was more often expressed. Since HDAC and DNA methylation inhibitors increased expression of ZEB1 target genes, targeting these epigenetic modifications would be expected to reduce metastasis

  11. Specific nongluten proteins of wheat are novel target antigens in celiac disease humoral response.

    Huebener, Sina; Tanaka, Charlene K; Uhde, Melanie; Zone, John J; Vensel, William H; Kasarda, Donald D; Beams, Leilani; Briani, Chiara; Green, Peter H R; Altenbach, Susan B; Alaedini, Armin

    2015-01-01

    While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, α-amylase/protease inhibitors, globulins, and farinins. Assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens. The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat. PMID:25329597

  12. Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

    Rebekka Müller

    Full Text Available Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM. Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

  13. Effects of endogenous proteins and microRNA target sequence in a positive feedback system.

    Kanda, Genki N; Togashi, Ryohei; Harashima, Hideyoshi; Kamiya, Hiroyuki

    2012-01-01

    A positive feedback system, using GAL4-vp16 (a fusion protein of yeast GAL4 and herpes simplex virus vp16) as an activator and firefly luciferase as a reporter, maintained luciferase expression for 7 d in mice. However, the luciferase expression decreased after 7 d, and this phenomenon could be caused by immunoreactions against these exogenous proteins. This hypothesis was examined by the following three strategies, designed to avoid the putative immunoreactions: (i) use of the endogenous secreted alkaline phosphatase (SEAP) protein as a reporter, (ii) replacement of vp16 with endogenous transcription factors, and (iii) insertion of the target sequence of microRNA expressed in cells of hematopoietic origin, to suppress GAL4-vp16 expression in antigen-presenting cells. The results obtained in this study suggested that silencing would be induced by mechanism(s) besides immunoreactions against reporter and activator proteins. PMID:22975505

  14. Targeted Proteomics of Metabolic Pathways and Protein Turnover Analysis in Plants

    A.Harvey Millar; Clark Nelson; Lei Li; Nicolas L.Taylor; Ricarda Fenske

    2012-01-01

    Shotgun approaches have dominated proteome studies as discovery tools to find changes in protein abundance.However,they often only provide a mosaic image of the proteome response and they focus mainly on the proteins that are changing in abundance to find biological insights.Using mass spectrometry for targeted identification of changes in whole biochemical pathways and analysing protein synthesis and degradation rates with stable isotope labelling provide an added depth of biological insights.These can help us to uncover the costs of protein production in plants and the role of specific pathways in responding to harsh or nutrient depleted environments.By adapting the selected reaction monitoring and the progressive 15N incorporation stu-dies we have developed in model plants,we are beginning to discover the power of these systems to analyse the response of barley to low nitrogen,rice to low phosphate and wheat to saline conditions.

  15. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

    Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

    2011-01-01

    intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding......In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce...... sequence allowed tuning of protein expression over ~300-fold with preservation of amino acid identity. This approach is simple and should be generally applicable in bacteria. The data support that features in the 5' mRNA coding region near the AUG start codon are key in determining translation output...

  16. Sialic Acid within the Glycosylphosphatidylinositol Anchor Targets the Cellular Prion Protein to Synapses.

    Bate, Clive; Nolan, William; McHale-Owen, Harriet; Williams, Alun

    2016-08-12

    Although the cellular prion protein (PrP(C)) is concentrated at synapses, the factors that target PrP(C) to synapses are not understood. Here we demonstrate that exogenous PrP(C) was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out((0/0)) mice. The targeting of PrP(C) to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrP(C) to synapses. Isolated GPIs (derived from PrP(C)) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrP(C) to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrP(C) acts as a synapse homing signal. PMID:27325697

  17. Spring Viremia of Carp Virus N Protein Suppresses Fish IFNφ1 Production by Targeting the Mitochondrial Antiviral Signaling Protein.

    Lu, Long-Feng; Li, Shun; Lu, Xiao-Bing; LaPatra, Scott E; Zhang, Nu; Zhang, Xu-Jie; Chen, Dan-Dan; Nie, Pin; Zhang, Yong-An

    2016-05-01

    For a virus to replicate efficiently, it must try and inhibit host IFN expression because IFN is an important host defense at early stages after viral infection. For aquatic viruses, the mechanisms used to escape the hosts IFN system are still unclear. In this study, we show that the N protein of spring viremia of carp virus (SVCV) inhibits zebrafish IFNφ1 production by degrading the mitochondrial antiviral signaling protein (MAVS). First, the upregulation of IFNφ1 promoter activity stimulated by polyinosinic:polycytidylic acid, retinoic acid-inducible gene I (RIG-I) or MAVS was suppressed by the SVCV infection. However, the upregulation by the downstream factor of the RIG-I-like receptor signaling pathway, TANK-binding kinase 1, was not affected. Notably, at the protein level, MAVS decreased remarkably when cells were infected with SVCV. Second, consistent with the result of the SVCV infection, overexpression of the N protein of SVCV blocked the IFNφ1 transcription activated by MAVS and downregulated MAVS expression at the protein level but not at the mRNA level. Further analysis demonstrated that the N protein targeted MAVS for K48-linked ubiquitination, which promoted the degradation of MAVS. These data indicated that fish MAVS could be degraded by the N protein of SVCV through the ubiquitin-proteasome pathway. To our knowledge, this is the first article of a fish RIG-I-like receptor pathway interfered by an aquatic virus in an ubiquitin-proteasome manner, suggesting that immune evasion of a virus also exists in lower vertebrates. PMID:26994222

  18. Identification of target genes of transcription factor activator protein 2 gamma in breast cancer cells

    Activator protein 2 gamma (AP-2γ) is a member of the transcription factor activator protein-2 (AP-2) family, which is developmentally regulated and plays a role in human neoplasia. AP-2γ has been found to be overexpressed in most breast cancers, and have a dual role to inhibit tumor initiation and promote tumor progression afterwards during mammary tumorigensis. To identify the gene targets that mediate its effects, we performed chromatin immunoprecipitation (ChIP) to isolate AP-2γ binding sites on genomic DNA from human breast cancer cell line MDA-MB-453. 20 novel DNA fragments proximal to potential AP-2γ targets were obtained. They are categorized into functional groups of carcinogenesis, metabolism and others. A combination of sequence analysis, reporter gene assays, quantitative real-time PCR, electrophoretic gel mobility shift assays and immunoblot analysis further confirmed the four AP-2γ target genes in carcinogenesis group: ErbB2, CDH2, HPSE and IGSF11. Our results were consistent with the previous reports that ErbB2 was the target gene of AP-2γ. Decreased expression and overexpression of AP-2γ in human breast cancer cells significantly altered the expression of these four genes, indicating that AP-2γ directly regulates them. This suggested that AP-2γ can coordinate the expression of a network of genes, involving in carcinogenesis, especially in breast cancer. They could serve as therapeutic targets against breast cancers in the future

  19. Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

    Andrew Pierce

    Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

  20. Quantitative imaging of protein targets in the human brain with PET

    Gunn, Roger N.; Slifstein, Mark; Searle, Graham E.; Price, Julie C.

    2015-11-01

    PET imaging of proteins in the human brain with high affinity radiolabelled molecules has a history stretching back over 30 years. During this period the portfolio of protein targets that can be imaged has increased significantly through successes in radioligand discovery and development. This portfolio now spans six major categories of proteins; G-protein coupled receptors, membrane transporters, ligand gated ion channels, enzymes, misfolded proteins and tryptophan-rich sensory proteins. In parallel to these achievements in radiochemical sciences there have also been significant advances in the quantitative analysis and interpretation of the imaging data including the development of methods for image registration, image segmentation, tracer compartmental modeling, reference tissue kinetic analysis and partial volume correction. In this review, we analyze the activity of the field around each of the protein targets in order to give a perspective on the historical focus and the possible future trajectory of the field. The important neurobiology and pharmacology is introduced for each of the six protein classes and we present established radioligands for each that have successfully transitioned to quantitative imaging in humans. We present a standard quantitative analysis workflow for these radioligands which takes the dynamic PET data, associated blood and anatomical MRI data as the inputs to a series of image processing and bio-mathematical modeling steps before outputting the outcome measure of interest on either a regional or parametric image basis. The quantitative outcome measures are then used in a range of different imaging studies including tracer discovery and development studies, cross sectional studies, classification studies, intervention studies and longitudinal studies. Finally we consider some of the confounds, challenges and subtleties that arise in practice when trying to quantify and interpret PET neuroimaging data including motion artifacts

  1. Quantitative imaging of protein targets in the human brain with PET

    PET imaging of proteins in the human brain with high affinity radiolabelled molecules has a history stretching back over 30 years. During this period the portfolio of protein targets that can be imaged has increased significantly through successes in radioligand discovery and development. This portfolio now spans six major categories of proteins; G-protein coupled receptors, membrane transporters, ligand gated ion channels, enzymes, misfolded proteins and tryptophan-rich sensory proteins. In parallel to these achievements in radiochemical sciences there have also been significant advances in the quantitative analysis and interpretation of the imaging data including the development of methods for image registration, image segmentation, tracer compartmental modeling, reference tissue kinetic analysis and partial volume correction. In this review, we analyze the activity of the field around each of the protein targets in order to give a perspective on the historical focus and the possible future trajectory of the field. The important neurobiology and pharmacology is introduced for each of the six protein classes and we present established radioligands for each that have successfully transitioned to quantitative imaging in humans. We present a standard quantitative analysis workflow for these radioligands which takes the dynamic PET data, associated blood and anatomical MRI data as the inputs to a series of image processing and bio-mathematical modeling steps before outputting the outcome measure of interest on either a regional or parametric image basis. The quantitative outcome measures are then used in a range of different imaging studies including tracer discovery and development studies, cross sectional studies, classification studies, intervention studies and longitudinal studies. Finally we consider some of the confounds, challenges and subtleties that arise in practice when trying to quantify and interpret PET neuroimaging data including motion artifacts

  2. Construction and evaluation of novel fusion proteins for targeted delivery of micro particles to cellulose surfaces.

    Lewis, William; Keshavarz-Moore, Eli; Windust, John; Bushell, Donna; Parry, Neil

    2006-07-01

    The use of IgG antibodies and fragments has been limited to specific sectors of the biotechnology industry due to the high cost of producing large batches of product necessary for alternative applications. A novel class of Camelid antibodies, known as V(HH) offer a more economical opportunity to meet a wider application in industry. In this study, we report the evaluation of four llama V(HH)-cellulose binding domain fusion proteins displaying varying formats of V(HH) and CBD domains. Proteins were characterized in a targeted particle delivery system as a method of delivering agents such as perfume to laundry in the wash cycle. Fusion proteins were shown to be stable at high pH and in the presence of a detergent base. They were also shown to bind effectively to both the designated antigen, the azo-dye reactive-red 6 (either conjugated to BSA or attached to coacervate microparticles), and cellulose. Binding strength differences were observed between the different fusion protein formats using surface plasmon resonance. The effect of key laundry ingredients was also studied. Combining the fusion proteins and particles into a delivery and deposition study generated clear microscopy evidence for bifunctionality. Confirmation of this was validated by GC-MS analysis of retained fragrance. This research, reporting the construction and characterization of a variety of fusion proteins, illustrates that the single multidomain fusion protein route offers a new technology for successful targeted delivery of encapsulated benefit agents. Furthermore, the potential to modify or select for proteins to recognize a wide range of surfaces is also possible. PMID:16673421

  3. Targeting leukemic fusion proteins with small interfering RNAs: recent advances and therapeutic potentials

    Maria THOMAS; Johann GREIL; Olaf HEIDENREICH

    2006-01-01

    RNA interference has become an indispensable research tool to study gene functions in a wide variety of organisms.Because of their high efficacy and specificity,RNA interference-based approaches may also translate into new therapeutic strategies to treat human diseases.In particular,oncogenes such as leukemic fusion proteins,which arise from chromosomal translocations,are promising targets for such gene silencing approaches,because they are exclusively expressed in precancerous and cancerous tissues,and because they are frequently indispensable for maintaining the malignant phenotype.This review summarizes recent developments in targeting leukemia-specific genes and discusses problems and approaches for possible clinical applications.

  4. Targeting the Vasculature of Colorectal Carcinoma with a Fused Protein of (RGD)3-tTF

    Zheng-jie Huang; Yilin Zhao; Wei-yuan Luo; Jun You; Shui-wen Li; Wen-cheng Yi; Sheng-yu Wang; Jiang-hua Yan; Qi Luo

    2013-01-01

    Purpose. Truncated tissue factor (tTF) fusion protein targeting tumor vasculature can induce tumor vascular thrombosis and necrosis. Here, we generated (RGD)3-tTF in which three arginine-glycine-aspartic (RGD) targeting integrin α v β 3 and tTF induce blood coagulation in tumor vessels. Methods. The bioactivities of (RGD)3-tTF including coagulation activity, FX activation, and binding with integrin α v β 3 were performed. The fluorescent labeled (RGD)3-tTF was intravenously injected into tumo...

  5. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted. PMID:26028028

  6. DMPD: Protein kinase C epsilon: a new target to control inflammation andimmune-mediated disorders. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 14643884 Protein kinase C epsilon: a new target to control inflammation andimmune-mediated diso...g) (.html) (.csml) Show Protein kinase C epsilon: a new target to control inflammation andimmune-mediated diso...l inflammation andimmune-mediated disorders. Authors Aksoy E, Goldman M, Willems F. Publication Int J Bioche

  7. The BET family of proteins targets Moloney Murine Leukemia Virus integration near transcription start sites

    De Rijck, Jan; de Kogel, Christine; Demeulemeester, Jonas; Vets, Sofie; Ashkar, Sara El; Malani, Nirav; Bushman, Frederic D; Landuyt, Bart; Husson, Steven J.; Busschots, Katrien; Gijsbers, Rik; Debyser, Zeger

    2014-01-01

    Summary A hallmark of retroviral replication is integration of the viral genome in the host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to Murine Leukemia Virus (MLV)-derived vector integration, hamper their application. Here we show that bromodomain and extraterminal (BET) proteins (BRD2, BRD3 and BRD4) and MLV integrase specifically interact and co-localize within the nucleus of the cell. Inhibition of the BET proteins chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75, retargets MLV integration away from TSS and into the body of actively transcribed genes, conform to the Human Immunodeficiency Virus (HIV) integration pattern. Together these data validate BET proteins as MLV integration targeting factors. PMID:24183673

  8. Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions.

    Chakraborty, Sandeep; Rao, Basuthkar J; Asgeirsson, Bjarni; Dandekar, Abhaya

    2014-01-01

    Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The neutralizing antibody (KZ52) derived from a human survivor of the 1995 Kikwit outbreak recognizes a protein epitope on this AH, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' ( http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html). PMID:25717367

  9. Chemical Genetics of Acetyl-CoA Carboxylases

    Xuyu Zu

    2013-01-01

    Full Text Available Chemical genetic studies on acetyl-CoA carboxylases (ACCs, rate-limiting enzymes in long chain fatty acid biosynthesis, have greatly advanced the understanding of their biochemistry and molecular biology and promoted the use of ACCs as targets for herbicides in agriculture and for development of drugs for diabetes, obesity and cancers. In mammals, ACCs have both biotin carboxylase (BC and carboxyltransferase (CT activity, catalyzing carboxylation of acetyl-CoA to malonyl-CoA. Several classes of small chemicals modulate ACC activity, including cellular metabolites, natural compounds, and chemically synthesized products. This article reviews chemical genetic studies of ACCs and the use of ACCs for targeted therapy of cancers.

  10. Progress in the development of therapeutic antibodies targeting prion proteins and β-amyloid peptides

    2010-01-01

    Prion diseases and Alzheimer’s disease (AD) are characterized by protein misfolding, and can lead to dementia. However, prion diseases are infectious and transmissible, while AD is not. The similarities and differences between these diseases have led researchers to perform comparative studies. In the last 2 decades, progress has been made in immunotherapy using anti-prion protein and anti-β-amyloid antibodies. In this study, we review new ideas and strategies for therapeutic antibodies targeting prion diseases and AD through conformation dependence.

  11. A COMPLEX OF INTERMEDIATE FILAMENT PROTEIN-DNA: A TARGET FOR AUTOANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS?

    阎锡德; S.Kuhn; P.Traub

    1995-01-01

    Autoantibodies in systemic lupus erythematosus which cross-react with double stranded DNA and in-termediate filament proteins are frequently reported.However,little is Known about the origin and the target of these antibodies.In this paper,a polyspecific monoclonal antibody,XY12,produced by the im-munization of genetically non-autoimmune mice with a DNA-protein complex is detsiled.Its antigen bind-ing patterms are very similar to the autoantibodies.The data suggest that these autoantibodies may be trig-gered by a circulating nucleoprotein.

  12. Protein lysine methyltransferase G9a acts on non-histone targets

    Rathert, Philipp; Dhayalan, Arunkumar; Murakami, Marie; Zhang, Xing; Tamas, Raluca; Jurkowska, Renata; Komatsu, Yasuhiko; Shinkai, Yoichi; Cheng, Xiaodong; Jeltsch, Albert

    2008-01-01

    By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-h...

  13. Preferential binding of growth inhibitory prostaglandins by the target protein of a carcinogen.

    S H Khan; Sorof, S

    1990-01-01

    Liver fatty acid binding protein (L-FABP) is the principal target protein of the hepatic carcinogen N-(2-fluorenyl)acetamide (2-acetylaminofluorene) in rat liver. In addition, the cyclopentenone prostaglandins (PG), PGA, PGJ2, and delta 12-PGJ2, inhibit the growth of many cell types in vitro. This report describes the preferential binding of the growth inhibitory prostaglandins by L-FABP and the reversible inhibition of thymidine incorporation into DNA by PGA2 and delta 12-PGJ2 in primary cul...

  14. CHARACTERIZATION OF A NOVEL RADIOTRACER TARGETING SYNAPTIC VESICLE PROTEIN 2A (SV2A)

    Warnock, Geoffrey; Aerts, Joël; Bahri, Mohamed Ali; Bretin, Florian; Buchanan, T; Klitgaard, H; Mestdagh, N; A. Valade; Mercier, J.; Seret, Alain; Luxen, André; Salmon, Eric; Plenevaux, Alain

    2012-01-01

    Synaptic vesicle protein 2A (SV2A) has been identified as the binding site of the antiepileptic levetiracetam (Keppra) [1]. SV2 proteins are critical for proper nervous system function and have been demonstrated to be involved in vesicle trafficking. Their implication in epilepsy makes them an interesting therapeutic target, and the widespread distribution of SV2A in particular may provide an opportunity to develop a PET-based measure of neuronal function in brain diseases. [18F]UCB-H i...

  15. Disorder Prediction Methods, Their Applicability to Different Protein Targets and Their Usefulness for Guiding Experimental Studies

    Jennifer D. Atkins

    2015-08-01

    Full Text Available The role and function of a given protein is dependent on its structure. In recent years, however, numerous studies have highlighted the importance of unstructured, or disordered regions in governing a protein’s function. Disordered proteins have been found to play important roles in pivotal cellular functions, such as DNA binding and signalling cascades. Studying proteins with extended disordered regions is often problematic as they can be challenging to express, purify and crystallise. This means that interpretable experimental data on protein disorder is hard to generate. As a result, predictive computational tools have been developed with the aim of predicting the level and location of disorder within a protein. Currently, over 60 prediction servers exist, utilizing different methods for classifying disorder and different training sets. Here we review several good performing, publicly available prediction methods, comparing their application and discussing how disorder prediction servers can be used to aid the experimental solution of protein structure. The use of disorder prediction methods allows us to adopt a more targeted approach to experimental studies by accurately identifying the boundaries of ordered protein domains so that they may be investigated separately, thereby increasing the likelihood of their successful experimental solution.

  16. Accurate microRNA target prediction correlates with protein repression levels

    Simossis Victor A

    2009-09-01

    Full Text Available Abstract Background MicroRNAs are small endogenously expressed non-coding RNA molecules that regulate target gene expression through translation repression or messenger RNA degradation. MicroRNA regulation is performed through pairing of the microRNA to sites in the messenger RNA of protein coding genes. Since experimental identification of miRNA target genes poses difficulties, computational microRNA target prediction is one of the key means in deciphering the role of microRNAs in development and disease. Results DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction. Conclusion Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at http://www.microrna.gr/microT

  17. Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases.

    Zeke, András; Bastys, Tomas; Alexa, Anita; Garai, Ágnes; Mészáros, Bálint; Kirsch, Klára; Dosztányi, Zsuzsanna; Kalinina, Olga V; Reményi, Attila

    2015-11-01

    Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles. PMID:26538579

  18. Identification of cell surface proteins as potential immunotherapy targets in twelve pediatric cancers

    RimasJOrentas

    2012-12-01

    Full Text Available Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in twelve pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene, in order to categorize transcripts by their subcellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22. For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18, MTDH (metadherin, and GPC2 (glypican-2. These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues.

  19. Acetylation of GATA-3 affects T-cell survival and homing to secondary lymphoid organs

    Yamagata, Tetsuya; Mitani, Kinuko; Oda, Hideaki; Suzuki, Takahiro; Honda, Hiroaki; Asai, Takashi; Maki, Kazuhiro; Nakamoto, Tetsuya; Hirai, Hisamaru

    2000-01-01

    Acetylation of a transcription factor has recently been shown to play a significant role in gene regulation. Here we show that GATA-3 is acetylated in T cells and that a mutation introduced into amino acids 305–307 (KRR-GATA3) creates local hypoacetylation in GATA-3. Remarkably, KRR-GATA3 possesses the most potent suppressive effect when compared with other mutants that are disrupted in putative acetylation targets. Expressing this mutant in peripheral T cells results in defective T-cell homi...

  20. Sushi Domain-Containing Protein 3: A Potential Target for Breast Cancer.

    Yu, Zhenghong; Jiang, Enze; Wang, Xinxing; Shi, Yaqin; Shangguan, Anna Junjie; Zhang, Luo; Li, Jie

    2015-06-01

    Aromatase inhibitors (AIs) are the most effective endocrine treatment for estrogen receptor α-positive (ERα+) postmenopausal breast cancer. Identification of biomarkers that are able to predict AIs responsiveness of patients is a key for successful treatment. The currently used biomarkers for tamoxifen responsiveness, which including ERα as well as progesterone receptor can only predict part of the potential responders to AIs treatment. Sushi domain-containing protein 3 (SUSD3) is a potential novel biomarker of AIs responsiveness. The lack of SUSD3 expression in breast cancer tissue can be an important predictor for non-responsiveness to AI. Here we reviewed the property and function of SUSD3, its usage as a biomarker and the practicability for SUSD3 to become a target for immune therapy. We suggest this protein can be potentially measured or targeted for prevention, diagnostic, and therapeutic purposes for estrogen or progesterone-dependent disorders including breast cancer in women. PMID:25556073

  1. Targeted reengineering of protein geranylgeranyltransferase type I selectivity functionally implicates active-site residues in protein-substrate recognition.

    Gangopadhyay, Soumyashree A; Losito, Erica L; Hougland, James L

    2014-01-21

    Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins. PMID:24344934

  2. The Leishmania infantum PUF proteins are targets of the humoral response during visceral leishmaniasis

    Requena Jose M

    2010-01-01

    Full Text Available Abstract Background RNA-binding proteins of the PUF family share a conserved domain consisting of tandemly repeated 36-40 amino acid motifs (typically eight known as Puf repeats. Proteins containing tandem repeats are often dominant targets of humoral responses during infectious diseases. Thus, we considered of interest to analyze whether Leishmania PUF proteins result antigenic during visceral leishmaniasis (VL. Findings Here, employing whole-genome databases, we report the composition, and structural features, of the PUF family in Leishmania infantum. Additionally, the 10 genes of the L. infantum PUF family were cloned and used to express the Leishmania PUFs in bacteria as recombinant proteins. Finally, the antigenicity of these PUF proteins was evaluated by determining levels of specific antibodies in sera from experimentally infected hamsters. The Leishmania PUFs were all recognized by the sera, even though with different degree of reactivity and/or frequency of recognition. The reactivity of hamster sera against recombinant LiPUF1 and LiPUF2 was particularly prominent, and these proteins were subsequently assayed against sera from human patients. High antibody responses against rLiPUF1 and rLiPUF2 were found in sera from VL patients, but these proteins resulted also recognized by sera from Chagas' disease patients. Conclusion Our results suggest that Leishmania PUFs are targets of the humoral response during L. infantum infection and may represent candidates for serodiagnosis and/or vaccine reagents; however, it should be kept in mind the cross-reactivity of LiPUFs with antibodies induced against other trypanosomatids such as Trypanosoma cruzi.

  3. Acetylation regulates DNA repair mechanisms in human cells.

    Piekna-Przybylska, Dorota; Bambara, Robert A; Balakrishnan, Lata

    2016-06-01

    The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation. PMID:27104361

  4. Candidate Single Nucleotide Polymorphism Markers for Arsenic Responsiveness of Protein Targets

    Graham-Evans, Barbara E.; Udensi, Udensi K.; Tchounwou, Paul B.; Rajendram V. Rajnarayanan; Anyanwu, Matthew N; Cohly, Hari H.P.; Isokpehi, Raphael D.

    2010-01-01

    Arsenic is a toxic metalloid that causes skin cancer and binds to cysteine residues—a property that could be used to infer arsenic responsiveness of a target protein. Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) result in amino acid substitutions and may alter arsenic binding with cysteine residues. Thus, the objective of this investigation was to identify and analyze nsSNPs that lead to substitutions to or from cysteine residues as an indication of increased or decreased arsenic r...

  5. TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA targeting proteins

    Doyle, Erin L.; Stoddard, Barry L.; Voytas, Daniel F.; Bogdanove, Adam J

    2013-01-01

    Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria in the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenue...

  6. Pragmatic approach to nutrition in the ICU: Expert opinion regarding which calorie protein target

    Singer, Pierre; Hiesmayr, Michael; Biolo, Gianni; Felbinger, Thomas W.; Berger, Mette M; Goeters, Christiane; Kondrup, Jens; Wunder, Christian; Pichard, Claude

    2014-01-01

    Background & aims: Since the publications of the ESPEN guidelines on enteral and parenteral nutrition in ICU, numerous studies have added information to assist the nutritional management of critically ill patients regarding the recognition of the right population to feed, the energy-protein targeting, the route and the timing to start. Methods: We reviewed and discussed the literature related to nutrition in the ICU from 2006 until October 2013. Results: To identify safe, minimal and maximal ...

  7. Mitochondrial fusion and fission proteins: Novel therapeutic targets for combating cardiovascular disease.

    Hall, A.; Burke, N; Dongworth, R.; Hausenloy, D.

    2014-01-01

    Mitochondria are no longer considered to be solely the static powerhouses of the cell. While they are undoubtedly essential to sustaining life and meeting the energy requirements of the cell through oxidative phosphorylation, they are now regarded as highly dynamic organelles with multiple funtions, playing key roles in cell survival and death. In this review, we discuss the emerging role of mitochondrial fusion and fission proteins, as novel therapeutic targets for treating a wide range of c...

  8. Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles

    Vannucci L

    2012-03-01

    Full Text Available Luca Vannucci1,*, Elisabetta Falvo2,*, Manuela Fornara3, Patrizio Di Micco4, Oldrich Benada1, Jiri Krizan1, Jan Svoboda1, Katarina Hulikova-Capkova1, Veronica Morea3, Alberto Boffi4,5, Pierpaolo Ceci3 1Institute of Microbiology, Academy of Sciences of the Czech Republic, VVI, Prague, Czech Republic; 2Regina Elena Cancer Institute, Pharmacokinetic/Pharmacogenomic Unit, 3National Research Council of Italy, Institute of Molecular Biology and Pathology, 4Department of Biochemical Sciences “A Rossi Fanelli”, University of Rome “Sapienza”, 5Center for Life Nano Science at Sapienza, Italian Institute of Technology, Rome, Italy *These two authors contributed equally to this workBackground: Nanoparticle-based systems are promising for the development of imaging and therapeutic agents. The main advantage of nanoparticles over traditional systems lies in the possibility of loading multiple functionalities onto a single molecule, which are useful for therapeutic and/or diagnostic purposes. These functionalities include targeting moieties which are able to recognize receptors overexpressed by specific cells and tissues. However, targeted delivery of nanoparticles requires an accurate system design. We present here a rationally designed, genetically engineered, and chemically modified protein-based nanoplatform for cell/tissue-specific targeting.Methods: Our nanoparticle constructs were based on the heavy chain of the human protein ferritin (HFt, a highly symmetrical assembly of 24 subunits enclosing a hollow cavity. HFt-based nanoparticles were produced using both genetic engineering and chemical functionalization methods to impart several functionalities, ie, the α-melanocyte-stimulating hormone peptide as a melanoma-targeting moiety, stabilizing and HFt-masking polyethylene glycol molecules, rhodamine fluorophores, and magnetic resonance imaging agents. The constructs produced were extensively characterized by a number of physicochemical

  9. Targeting low-density lipoprotein receptors with protein-only nanoparticles

    Low-density lipoprotein receptors (LDLR) are appealing cell surface targets in drug delivery, as they are expressed in the blood–brain barrier (BBB) endothelium and are able to mediate transcytosis of functionalized drugs for molecular therapies of the central nervous system (CNS). On the other hand, brain-targeted drug delivery is currently limited, among others, by the poor availability of biocompatible vehicles, as most of the nanoparticles under development as drug carriers pose severe toxicity issues. In this context, protein nanoparticles offer functional versatility, easy and cost-effective bioproduction, and full biocompatibility. In this study, we have designed and characterized several chimerical proteins containing different LDLR ligands, regarding their ability to bind and internalize target cells and to self-organize as viral mimetic nanoparticles of about 18 nm in diameter. While the self-assembling of LDLR-binding proteins as nanoparticles positively influences cell penetration in vitro, the nanoparticulate architecture might be not favoring BBB crossing in vivo. These findings are discussed in the context of the use of nanostructured materials as vehicles for the systemic treatment of CNS diseases

  10. Targeting low-density lipoprotein receptors with protein-only nanoparticles

    Xu, Zhikun [Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (Spain); Céspedes, María Virtudes [CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) (Spain); Unzueta, Ugutz [Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (Spain); Álamo, Patricia [CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) (Spain); Pesarrodona, Mireia [Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (Spain); Mangues, Ramón [CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) (Spain); Vázquez, Esther; Villaverde, Antonio, E-mail: antoni.villaverde@uab.cat; Ferrer-Miralles, Neus, E-mail: neus.ferrer@uab.cat [Universitat Autònoma de Barcelona, Institut de Biotecnologia i de Biomedicina (Spain)

    2015-03-15

    Low-density lipoprotein receptors (LDLR) are appealing cell surface targets in drug delivery, as they are expressed in the blood–brain barrier (BBB) endothelium and are able to mediate transcytosis of functionalized drugs for molecular therapies of the central nervous system (CNS). On the other hand, brain-targeted drug delivery is currently limited, among others, by the poor availability of biocompatible vehicles, as most of the nanoparticles under development as drug carriers pose severe toxicity issues. In this context, protein nanoparticles offer functional versatility, easy and cost-effective bioproduction, and full biocompatibility. In this study, we have designed and characterized several chimerical proteins containing different LDLR ligands, regarding their ability to bind and internalize target cells and to self-organize as viral mimetic nanoparticles of about 18 nm in diameter. While the self-assembling of LDLR-binding proteins as nanoparticles positively influences cell penetration in vitro, the nanoparticulate architecture might be not favoring BBB crossing in vivo. These findings are discussed in the context of the use of nanostructured materials as vehicles for the systemic treatment of CNS diseases.

  11. Nonstructural Proteins Are Preferential Positive Selection Targets in Zika Virus and Related Flaviviruses.

    Sironi, Manuela; Forni, Diego; Clerici, Mario; Cagliani, Rachele

    2016-09-01

    The Flavivirus genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barré syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian

  12. Orphan G protein-coupled receptors (GPCRs):biological functions and potential drug targets

    Xiao-long TANG; Ying WANG; Da-li LI; Jian LUO; Ming-yao LIU

    2012-01-01

    The superfamily of G protein-coupled receptors (GPCRs) includes at least 800 seven-transmembrane receptors that participate in diverse physiological and pathological functions.GPCRs are the most successful targets of modern medicine,and approximately 36%of marketed pharmaceuticals target human GPCRs.However,the endogenous ligands of more than 140 GPCRs remain unidentified,leaving the natural functions of those GPCRs in doubt.These are the so-called orphan GPCRs,a great source of drug targets.This review focuses on the signaling transduction pathways of the Adhesion GPCR family,the LGR subfamily,and the PSGR subfamily,and their potential functions in immunology,development,and cancers.In this review,we present the current approaches and difficulties of orphan GPCR deorphanization and characterization.

  13. Hitting the Sweet Spot: Glycans as Targets of Fungal Defense Effector Proteins

    Markus Künzler

    2015-05-01

    Full Text Available Organisms which rely solely on innate defense systems must combat a large number of antagonists with a comparatively low number of defense effector molecules. As one solution of this problem, these organisms have evolved effector molecules targeting epitopes that are conserved between different antagonists of a specific taxon or, if possible, even of different taxa. In order to restrict the activity of the defense effector molecules to physiologically relevant taxa, these target epitopes should, on the other hand, be taxon-specific and easily accessible. Glycans fulfill all these requirements and are therefore a preferred target of defense effector molecules, in particular defense proteins. Here, we review this defense strategy using the example of the defense system of multicellular (filamentous fungi against microbial competitors and animal predators.

  14. Dual Targeting of a Mitochondrial Protein: The Case Study of Cytochrome C1

    Anja R(o)diger; Bianca Baudisch; Uwe Langner; Ralf Bernd Kl(o)sgen

    2011-01-01

    As a result of the endosymbiotic gene transfer, the majority of proteins of mitochondria and chloroplasts is encoded in the nucleus and synthesized in the cytosol as precursor molecules carrying N-terminal transit peptides for the transport into the respective target organelle. In most instances, transport takes place into either mitochondria or chlor-oplasts, although a few examples of dual targeting into both organelles have been described. Here, we show by a com-bination of three different experimental strategies that also cytochrome c of potato, a component of the respiratory electron transport chain, is imported not only into mitochondria, but also into plastids. In organello import experiments with isolated mitochondria and chloroplasts, which were analyzed in both single and mixed organelle assays, demonstrate that the processing products accumulating after import within the two endosymbiotic organelles are different in size. Dual targeting of cytochrome c is observed also in vivo, after biolistic transformation of leaf epidermal cells with suitable reporter constructions. Finally, Western analyses employing cytochrome c-specific antiserum provide evidence that the protein accumulates in significant amounts in mitochondria and chloroplasts of both pea and spinach. The possible consequences of our findings on the relevance of the dual targeting phenomenon are discussed.

  15. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  16. A simple yeast-based strategy to identify host cellular processes targeted by bacterial effector proteins.

    Eran Bosis

    Full Text Available Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.

  17. Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

    Mondal, Anupam; Bhattacherjee, Arnab

    2015-01-01

    DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely. PMID:26400158

  18. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-11-01

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

  19. [Epigenetics and drug addiction: a focus on MeCP2 and on histone acetylation].

    Zwiller, Jean

    2015-04-01

    Chronic drug exposure alters gene expression in the brain, which is believed to underlie compulsive drug seeking and drug taking behavior. Recent evidence shows that drug-induced long-term neuroadaptations in the brain are mediated in part by epigenetic mechanisms. By remodeling chromatin, this type of regulation contributes to drug-induced synaptic plasticity that translates into behavioral modifications. How drug-induced alterations in DNA methylation regulate gene expression is reviewed here, with a focus on MeCP2, a protein binding methylated DNA. The importance of histone modifications, especially acetylation is also discussed, with an emphasis on the effects of inhibitors of histone deacetylases on drug-induced behavioral changes. The precise identification of the epigenetic mechanisms that are under the control of drugs of abuse may help to uncover novel targets for the treatment of drug seeking and relapse. PMID:25958763

  20. Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes.

    Yunwon Moon

    Full Text Available This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2 and severe hypoxia (0.1% O2. We found that chenodeoxy cholic acid (CDCA reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR, a CDCA receptor and its target gene, Small heterodimer partner (SHP are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.

  1. Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes.

    Moon, Yunwon; Choi, Su Mi; Chang, Soojeong; Park, Bongju; Lee, Seongyeol; Lee, Mi-Ock; Choi, Hueng-Sik; Park, Hyunsung

    2015-01-01

    This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2) and severe hypoxia (0.1% O2). We found that chenodeoxy cholic acid (CDCA) reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR), a CDCA receptor and its target gene, Small heterodimer partner (SHP) are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein. PMID:26098428

  2. In vitro bioactive properties of intact and enzymatically hydrolysed whey protein: targeting the enteroinsular axis.

    Power-Grant, O; Bruen, C; Brennan, L; Giblin, L; Jakeman, P; FitzGerald, R J

    2015-03-01

    Enzymatically hydrolysed milk proteins have a variety of biofunctional effects some of which may be beneficial in the management of type 2 diabetes mellitus. The purpose of this study was to evaluate the effect of commercially available intact and hydrolysed whey protein ingredients (DH 32, DH 45) on markers of the enteroinsular axis (glucagon like peptide-1 secretion, dipeptidyl peptidase IV inhibition, insulin secretion and antioxidant activity) before and after simulated gastrointestinal digestion (SGID). A whey protein hydrolysate, DH32, significantly enhanced (P whey protein hydrolysates inhibited dipeptidyl peptidase IV activity, yielding half maximal inhibitory concentration values (IC50) of 1.5 ± 0.1 and 1.1 ± 0.1 mg mL(-1) for the DH 32 and DH 45, samples respectively, and were significantly more potent than the intact whey (P whey protein significantly enhanced (P antioxidant activity compared to intact whey, as measured by the oxygen radical absorbance capacity assay (ORAC). This antioxidant activity was maintained (DH 32, P > 0.05) or enhanced (DH 45, P whey stimulated GLP-1 secretion from enteroendocrine cells compared to vehicle control (P whey proteins and peptides can act through multiple targets within the enteroinsular axis and as such may have glucoregulatory potential. PMID:25666373

  3. The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin.

    Küberl, Andreas; Polen, Tino; Bott, Michael

    2016-04-26

    The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation. PMID:27078093

  4. Vacuolar targeting of r-proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice.

    Palaniswamy, Harunipriya; Syamaladevi, Divya P; Mohan, Chakravarthi; Philip, Anna; Petchiyappan, Anushya; Narayanan, Subramonian

    2016-02-01

    Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well-developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high-value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar-targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N-terminal 78-bp-long putative vacuolar-targeting sequence from the N-terminal domain of unknown function (DUF) in Triticum aestivum 6-SFT (sucrose: fructan 6-fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar-targeting determinants at the N-terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His-tagged β-glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar-targeting determinant could localize recombinant proteins (r-proteins) to the vacuole in high concentrations and such targeted r-proteins can be purified from the juice with a few simple steps. PMID:26183462

  5. Targeted Protein Degradation by Salmonella under Phagosome-Mimicking Culture Conditions Investigated Using Comparative Peptidomics

    Manes, Nathan P.; Gustin, Jean K.; Rue, Joanne; Mottaz, Heather M.; Purvine, Samuel O.; Norbeck, Angela D.; Monroe, Matthew E.; Zimmer, Jennifer S.; Metz, Thomas O.; Adkins, Joshua N.; Smith, Richard D.; Heffron, Fred

    2007-04-01

    The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Due to the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g., contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study Salmonella enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal compartment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified 5,163 distinct peptides originating from 682 proteins and the data clearly indicated that compared to cells cultured in the rich medium, Salmonella cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed by using bottom-up proteomics, and when the peptidomic and proteomic data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.

  6. The BET Family of Proteins Targets Moloney Murine Leukemia Virus Integration near Transcription Start Sites

    Jan De Rijck

    2013-11-01

    Full Text Available A hallmark of retroviral replication is integration of the viral genome into host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to murine leukemia virus (MLV-derived vector integration, hamper their application. Here, we show that bromodomain and extraterminal (BET proteins (BRD2, BRD3, and BRD4 and MLV integrase specifically interact and colocalize within the nucleus of the cell. Inhibition of the BET proteins’ chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75 retargets MLV integration away from transcription start sites and into the body of actively transcribed genes, conforming to the HIV integration pattern. Together, these data validate BET proteins as MLV integration targeting factors.

  7. The Pneumovirinae fusion (F) protein: A common target for vaccines and antivirals.

    Melero, José A; Mas, Vicente

    2015-11-01

    The Pneumovirinae fusion (F) protein mediates fusion of the virus and cell membrane, an essential step for entry of the viral genome in the cell cytoplasm and initiation of a new infectious cycle. Accordingly, potent inhibitors of virus infectivity have been found among antibodies and chemical compounds that target the Pneumovirinae F protein. Recent developments in structure-based vaccines have led to a deeper understanding of F protein antigenicity, unveiling new conformations and epitopes which should assist in development of efficacious vaccines. Similarly, structure-based studies of potent antiviral inhibitors have provided information about their mode of action and mechanisms of resistance. The advantages and disadvantages of the different options to battle against important pathogens, such as human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are summarized and critically discussed in this review. PMID:25738581

  8. Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25

    Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the mechanism of Nop25 localization in the nucleolus. Deletion experiments of Nop25 amino acid sequence showed Nop25 to contain a nuclear targeting sequence in the N-terminal and a nucleolar targeting sequence in the C-terminal. By expressing derivative peptides from the C-terminal as GFP-fusion proteins in the cells, a lysine and arginine residue-enriched peptide (KRKHPRRAQDSTKKPPSATRTSKTQRRRR) allowed a GFP-fusion protein to be transported and fully retained in the nucleolus. When the peptide was fused with cMyc epitope and expressed in the cells, a cMyc epitope was then detected in the nucleolus. Nop25 did not localize in the nucleolus by deletion of the peptide from Nop25. Furthermore, deletion of a subdomain (KRKHPRRAQ) in the peptide or amino acid substitution of lysine and arginine residues in the subdomain resulted in the loss of Nop25 nucleolar localization. These results suggest that the lysine and arginine residue-enriched peptide is the most prominent nucleolar targeting sequence of Nop25 and that the long stretch of basic residues might play an important role in the nucleolar localization of Nop25. Although Nop25 contained putative SUMOylation, phosphorylation and glycosylation sites, the amino acid substitution in these sites had no effect on the nucleolar localization, thus suggesting that these post-translational modifications did not contribute to the localization of Nop25 in the nucleolus. The treatment of the cells, which expressed a GFP-fusion protein with a nucleolar targeting sequence of Nop25, with RNase A resulted in a complete dislocation of the protein from the nucleolus. These data suggested that the nucleolar targeting sequence might therefore play an important role in the binding of Nop25 to RNA molecules and that the RNA binding of Nop25 might be essential for the nucleolar localization of Nop25

  9. Nuclear matrix associated protein PML: an arsenic trioxide apoptosis therapeutic target protein in HepG2 cells

    于鼎; 王子慧; 朱立元; 邱殷庆

    2003-01-01

    Objective To investigate arsenic trioxide (As2O3)-induced apoptosis and the effects on cell nuclear matrix related protein promyelocytic leukaemia (PML). Methods HepG2 cells were cultured in MEM medium and treated with 0.5, 2, 5 and 10 μmol/L As2O3 for either 24 h or 96 h at each concentration. In situ terminal deoxynucleotidyl transferase (TdT) labeling (TUNEL) and DNA ladders were used to detect apoptosis. Confocal microscopy and Western blotting were used to observe the expression of PML. Results The growth rates of HepG2 cells were slower in the As2O3 treated than the untreated control group. DNA ladder and TUNEL positive apoptotic cells could be detected in As2O3 treated groups. The expression of PML decreased in HepG2 cells with 2 μmol/L As2O3 treatment. Confocal images demonstrated that the expression of PML protein in HepG2 cell nuclei decreased after treatment with 2 μmol/L As2O3, and micropunctates characteristic of PML protein in HepG2 cell nuclei disappeared after treatment with 5 μmol/L As2O3.Conclusions Our results show that arsenic trioxide can significantly inhibit the growth of HepG2 cells in vitro. As2O3 induces apoptosis in HepG2 tumor cells in a time and concentration dependent manner. As2O3 may degrade the PML protein in HepG2 cell nuclei. The decreased expression of PML in As2O3 treated tumor cells is most likely to be caused by apoptosis. Nuclear matrix associated protein PML could be the target of As2O3 therapy.

  10. Identification of the Target Proteins of Rosiglitazone in 3T3-L1 Adipocytes through Proteomic Analysis of Cytosolic and Secreted Proteins

    Hwang, Hyun-Ho; Moon, Pyong-Gon; Lee, Jeong-Eun; Kim, Jung-Guk; LEE, WAN; Ryu, Sung-Ho; Baek, Moon-Chang

    2011-01-01

    Rosiglitazone, one of the thiazolidinedione (TZD), is an oral antidiabetic drug that activates a gamma isoform of peroxisome proliferator-activated receptor (PPARγ). To identify target proteins induced by rosiglitazone in adipocytes, we first performed simultaneous in-depth proteomic profiling of cytosolic proteins and secreted proteins (secretome) from 3T3-L1 adipocytes using a label-free quantification method with nano-UPLC MS/MS. In total, we identified 646 proteins from 3T3-L1 adipocytes,...

  11. The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate

    Clemens Schmeitzl

    2015-08-01

    Full Text Available Deoxynivalenol (DON is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON, 15-acetyl-DON (15-ADON and 3,15-diacetyl-DON (3,15-diADON, and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G and of 15-acetyl-DON-3-sulfate (15-ADON3S as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G. This study highlights significant differences in the metabolization of DON and its acetylated derivatives.

  12. Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration.

    Siudeja, Katarzyna; Srinivasan, Balaji; Xu, Lanjun; Rana, Anil; de Jong, Jannie; Nollen, Ellen A A; Jackowski, Suzanne; Sanford, Lynn; Hayflick, Susan; Sibon, Ody C M

    2011-12-01

    Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are transferred to lysine residues of proteins, in a reaction regulated by acetyltransferases. The tight balance between acetyltransferases and their antagonistic counterparts histone deacetylases is a well-known determining factor for the acetylation status of proteins. However, the influence of Coenzyme A levels on protein acetylation is unknown. Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models. We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival. Decreased protein acetylation and the concurrent phenotypes are partly rescued by pantethine and HDAC inhibitors, suggesting possible directions for future PKAN therapy development. PMID:21998097

  13. UNIQUE ACETYLATION OF OLIGOSACCHARIDES BY TRICHODERMA REESEI ACETYL ESTERASE IN WATER - VINYL ACETATE MIXTURE

    Purified T. reesei RUT C-30 acetyl esterase catalyzes acetyl transfer to a variety of carbohydrates in water in the presence of vinyl acetate as the acetyl group donor. The degree of conversion and the number of formed acetates depended on the acceptor used. With some acceptors, such as methyl or ...

  14. A comparative genomic analysis of targets of Hox protein Ultrabithorax amongst distant insect species.

    Prasad, Naveen; Tarikere, Shreeharsha; Khanale, Dhanashree; Habib, Farhat; Shashidhara, L S

    2016-01-01

    In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is dependent on the function of the Hox protein Ultrabithorax (Ubx). Here we compare Ubx-mediated regulation of wing patterning genes between the honeybee, Apis mellifera, the silkmoth, Bombyx mori and Drosophila. Orthologues of Ubx are expressed in the third thoracic segment of Apis and Bombyx, although they make functional hindwings. When over-expressed in transgenic Drosophila, Ubx derived from Apis or Bombyx could suppress wing development, suggesting evolutionary changes at the level of co-factors and/or targets of Ubx. To gain further insights into such events, we identified direct targets of Ubx from Apis and Bombyx by ChIP-seq and compared them with those of Drosophila. While majority of the putative targets of Ubx are species-specific, a considerable number of wing-patterning genes are retained, over the past 300 millions years, as targets in all the three species. Interestingly, many of these are differentially expressed only between wing and haltere in Drosophila but not between forewing and hindwing in Apis or Bombyx. Detailed bioinformatics and experimental validation of enhancer sequences suggest that, perhaps along with other factors, changes in the cis-regulatory sequences of earlier targets contribute to diversity in Ubx function. PMID:27296678

  15. A designed recombinant fusion protein for targeted delivery of siRNA to the mouse brain.

    Haroon, Mohamed Mohamed; Dar, Ghulam Hassan; Jeyalakshmi, Durga; Venkatraman, Uthra; Saba, Kamal; Rangaraj, Nandini; Patel, Anant Bahadur; Gopal, Vijaya

    2016-04-28

    RNA interference represents a novel therapeutic approach to modulate several neurodegenerative disease-related genes. However, exogenous delivery of siRNA restricts their transport into different tissues and specifically into the brain mainly due to its large size and the presence of the blood-brain barrier (BBB). To overcome these challenges, we developed here a strategy wherein a peptide known to target specific gangliosides was fused to a double-stranded RNA binding protein to deliver siRNA to the brain parenchyma. The designed fusion protein designated as TARBP-BTP consists of a double-stranded RNA-binding domain (dsRBD) of human Trans Activation response element (TAR) RNA Binding Protein (TARBP2) fused to a brain targeting peptide that binds to monosialoganglioside GM1. Conformation-specific binding of TARBP2 domain to siRNA led to the formation of homogenous serum-stable complex with targeting potential. Further, uptake of the complex in Neuro-2a, IMR32 and HepG2 cells analyzed by confocal microscopy and fluorescence activated cell sorting, revealed selective requirement of GM1 for entry. Remarkably, systemic delivery of the fluorescently labeled complex (TARBP-BTP:siRNA) in ΑβPP-PS1 mouse model of Alzheimer's disease (AD) led to distinctive localization in the cerebral hemisphere. Further, the delivery of siRNA mediated by TARBP-BTP led to significant knockdown of BACE1 in the brain, in both ΑβPP-PS1 mice and wild type C57BL/6. The study establishes the growing importance of fusion proteins in delivering therapeutic siRNA to brain tissues. PMID:26948382

  16. Integrin-mediated targeting of protein polymer nanoparticles carrying a cytostatic macrolide

    Shi, Pu

    Cytotoxicity, low water solubility, rapid clearance from circulation, and offtarget side-effects are common drawbacks of conventional small-molecule drugs. To overcome these shortcomings, many multifunctional nanocarriers have been proposed to enhance drug delivery. In concept, multifunctional nanoparticles might carry multiple agents, control release rate, biodegrade, and utilize target-mediated drug delivery; however, the design of these particles presents many challenges at the stage of pharmaceutical development. An emerging solution to improve control over these particles is to turn to genetic engineering. Genetically engineered nanocarriers are precisely controlled in size and structure and can provide specific control over sites for chemical attachment of drugs. Genetically engineered drug carriers that assemble nanostructures including nanoparticles and nanofibers can be polymeric or nonpolymeric. This chapter summarizes the recent development of applications in drug and gene delivery utilizing nanostructures of polymeric genetically engineered drug carriers such as elastin-like polypeptides, silk-like polypeptides, and silk-elastin-like protein polymers, and non-polymeric genetically engineered drug carriers such as vault proteins and viral proteins. This chapter explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for

  17. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins.

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L; Mackey, David

    2016-05-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B' regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B' subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B' subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B' subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B' subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  18. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins.

    Lin Jin

    2016-05-01

    Full Text Available Bacterial AvrE-family Type-III effector proteins (T3Es contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A heterotrimeric enzyme complexes via direct interaction with B' regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000, associates with specific PP2A B' subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B' subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B' subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B' subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family.

  19. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  20. Acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism

    The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [3H]CoA were found to produce acetyl-[3H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [3H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [3H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate

  1. Target Detection Assay (TDA): a versatile procedure to determine DNA binding sites as demonstrated on SP1 protein.

    Thiesen, H J; Bach, C.

    1990-01-01

    We developed a rapid method designated Target Detection Assay (TDA) to determine DNA binding sites for putative DNA binding proteins. A purified, functionally active DNA binding protein and a pool of random double-stranded oligonucleotides harbouring PCR primer sites at each end are included the TDA cycle which consists of four separate steps: a DNA protein incubation step, a protein DNA complex separation step, a DNA elution step and a polymerase chain reaction (PCR) DNA amplification step. ...

  2. Growth arrest specific protein 6 participates in DOCA-induced target-organ damage.

    Park, Joon-Keun; Theuer, Stefanie; Kirsch, Torsten; Lindschau, Carsten; Klinge, Uwe; Heuser, Arnd; Plehm, Ralph; Todiras, Mihai; Carmeliet, Peter; Haller, Hermann; Luft, Friedrich C; Muller, Dominik N; Fiebeler, Anette

    2009-08-01

    Growth arrest-specific protein 6 (Gas 6) is involved in inflammatory kidney diseases, vascular remodeling, cell adhesion, and thrombus formation. We explored a role for Gas 6 in aldosterone-induced target organ damage. We observed that Gas 6 was upregulated in rats with high aldosterone levels. Mineralocorticoid receptor blockade prevented target organ damage and decreased the elevated Gas 6 expression. Vascular smooth muscle cells given aldosterone increased their Gas 6 expression in vitro. To test the pathophysiological relevance, we investigated the effects of deoxycorticosterone acetate (DOCA) on Gas 6 gene-deleted ((-/-)) mice. After 6 weeks DOCA, Gas 6(-/-) mice developed similar telemetric blood pressure elevations compared to wild-type mice but were protected from cardiac hypertrophy. Cardiac expression of interleukin 6 and collagen IV was blunted in Gas 6(-/-) mice, indicating reduced inflammation and fibrosis. Gas 6(-/-) mice also had an improved renal function with reduced albuminuria, compared to wild-type mice. Renal fibrosis and fibronectin deposition in the kidney were also reduced. Gas 6 deficiency reduces the detrimental effects of aldosterone on cardiac and renal remodeling independent of blood pressure reduction. Gas 6 appears to play a role in mineralocorticoid receptor-mediated target organ damage. Furthermore, because warfarin interferes with Gas 6 protein expression, the findings could be of clinical relevance for anticoagulant choices. PMID:19564549

  3. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Unzueta U

    2012-08-01

    Full Text Available Ugutz Unzueta,1–3 María Virtudes Céspedes,3,4 Neus Ferrer-Miralles,1–3 Isolda Casanova,3,4 Juan Cedano,5 José Luis Corchero,1–3 Joan Domingo-Espín,1–3 Antonio Villaverde,1–3 Ramón Mangues,3,4 Esther Vázquez1–31Institut de Biotecnologia i de Biomedicina, 2Departamento de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 3CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, Barcelona, 4Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Laboratory of Immunology, Regional Norte, Universidad de la Republica, Salto, UruguayBackground: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4 is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing.Results: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer.Conclusion: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles

  4. Construction, Expression and Characterization of a Chimeric Protein Targeting Carcinoembryonic Antigen in Lung Cancer

    LI Yang; HUA Shu-cheng; MA Cheng-yuan; YU Zhen-xiang; XU Li-jun; LI Dan; SUN Li-li; LI Xiao; PENG Li-ping

    2011-01-01

    The carcinoembryonic antigen(CEA) is an oncofetal glycoprotein known as an important clinical tumor marker and is overexpressed in several types of tumors, including colorectal and lung carcinomas. We constructed a chimeric protein that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A(SEA) to the C-terminus of an anti-CEA single-chain disulfide-stabilized Fv(scdsFv) antibody (single-chain-C-terminus/SEA, SC-C/SEA). The SC-C/SEA protein was expressed in Escherichia coli(E. coli), refolded, and purified on an immobilized Ni2+ affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analysis reveal that the target protein was expressed sufficiently. We used immunofluorescence assays to demonstrate that SC-C/SEA could bind specifically to human lung carcinoma cells(A549), but almost human uterine cervix cells(HeLa). We also used the L-lactate dehydrogenase(LDH) release assay to show that SC-C/SEA elicits a strong A549 tumor-specific cytotoxic T lymphocyte(CTL) response in vitro. The results suggest that SC-C/SEA shows specific activity against CEA-positive cells and has potential application in CEA-targeted cancer immunotherapy.

  5. Adenosine Monophosphate-Activated Protein Kinase (AMPK) as a Diverse Therapeutic Target: A Computational Perspective.

    Ramesh, M; Vepuri, Suresh B; Oosthuizen, Frasia; Soliman, Mahmoud E

    2016-02-01

    Adenosine monophosphate-activated protein kinase (AMPK) is viewed as a privileged therapeutic target for several diseases such as cancer, diabetes, inflammation, obesity, etc. In addition, AMPK has entered the limelight of current drug discovery with its evolution as a key metabolic regulator. AMPK also plays a key role in the maintenance of cellular energy homeostasis. Structurally, AMPK is a heterotrimeric protein, which consists of three protein subunits (α, β, and γ). The crystal structure of AMPK was solved, and several computational studies including homology modeling, molecular docking, molecular dynamics, and QSAR have been reported in order to explore the structure and function of this diverse therapeutic target. In this review, we present a comprehensive up-to-date overview on the computational and molecular modeling approaches that have been carried out on AMPK in order to understand its structure, function, dynamics, and its drug binding landscape. Information provided in this review would be of great interest to a wide pool of researchers involved in the design of new molecules against various diseases where AMPK plays a predominant role. Graphical Abstract ᅟ. PMID:26541160

  6. Improved Species-Specific Lysine Acetylation Site Prediction Based on a Large Variety of Features Set.

    Wuyun, Qiqige; Zheng, Wei; Zhang, Yanping; Ruan, Jishou; Hu, Gang

    2016-01-01

    Lysine acetylation is a major post-translational modification. It plays a vital role in numerous essential biological processes, such as gene expression and metabolism, and is related to some human diseases. To fully understand the regulatory mechanism of acetylation, identification of acetylation sites is first and most important. However, experimental identification of protein acetylation sites is often time consuming and expensive. Therefore, the alternative computational methods are necessary. Here, we developed a novel tool, KA-predictor, to predict species-specific lysine acetylation sites based on support vector machine (SVM) classifier. We incorporated different types of features and employed an efficient feature selection on each type to form the final optimal feature set for model learning. And our predictor was highly competitive for the majority of species when compared with other methods. Feature contribution analysis indicated that HSE features, which were firstly introduced for lysine acetylation prediction, significantly improved the predictive performance. Particularly, we constructed a high-accurate structure dataset of H.sapiens from PDB to analyze the structural properties around lysine acetylation sites. Our datasets and a user-friendly local tool of KA-predictor can be freely available at http://sourceforge.net/p/ka-predictor. PMID:27183223

  7. Exploring the Possible Role of Lysine Acetylation on Entamoeba histolytica Virulence: A Focus on the Dynamics of the Actin Cytoskeleton

    L. López-Contreras

    2013-01-01

    Full Text Available Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

  8. Investigation of acetyl migrations in furanosides

    Migaud ME

    2006-07-01

    Full Text Available Abstract Standard reaction conditions for the desilylation of acetylated furanoside (riboside, arabinoside and xyloside derivatives facilitate acyl migration. Conditions which favour intramolecular and intermolecular mechanisms have been identified with intermolecular transesterifications taking place under mild basic conditions when intramolecular orthoester formations are disfavoured. In acetyl ribosides, acyl migration could be prevented when desilylation was catalysed by cerium ammonium nitrate.

  9. Proteome-wide lysine acetylation in cortical astrocytes and alterations that occur during infection with brain parasite Toxoplasma gondii.

    Anne Bouchut

    Full Text Available Lysine acetylation is a reversible post-translational modification (PTM that has been detected on thousands of proteins in nearly all cellular compartments. The role of this widespread PTM has yet to be fully elucidated, but can impact protein localization, interactions, activity, and stability. Here we present the first proteome-wide survey of lysine acetylation in cortical astrocytes, a subtype of glia that is a component of the blood-brain barrier and a key regulator of neuronal function and plasticity. We identified 529 lysine acetylation sites across 304 proteins found in multiple cellular compartments that largely function in RNA processing/transcription, metabolism, chromatin biology, and translation. Two hundred and seventy-seven of the acetylated lysines we identified on 186 proteins have not been reported previously in any other cell type. We also mapped an acetylome of astrocytes infected with the brain parasite, Toxoplasma gondii. It has been shown that infection with T. gondii modulates host cell gene expression, including several lysine acetyltransferase (KAT and deacetylase (KDAC genes, suggesting that the host acetylome may also be altered during infection. In the T. gondii-infected astrocytes, we identified 34 proteins exhibiting a level of acetylation >2-fold and 24 with a level of acetylation <2-fold relative to uninfected astrocytes. Our study documents the first acetylome map for cortical astrocytes, uncovers novel lysine acetylation sites, and demonstrates that T. gondii infection produces an altered acetylome.

  10. Targeted therapies in cancer - challenges and chances offered by newly developed techniques for protein analysis in clinical tissues

    K Malinowsky, C Wolff, S Gündisch, D Berg, KF Becker

    2011-01-01

    Full Text Available In recent years, new anticancer therapies have accompanied the classical approaches of surgery and radio- and chemotherapy. These new forms of treatment aim to inhibit specific molecular targets namely altered or deregulated proteins, which offer the possibility of individualized therapies.The specificity and efficiency of these new approaches, however, bring about a number of challenges. First of all, it is essential to specifically identify and quantify protein targets in tumor tissues for the reasonable use of such targeted therapies. Additionally, it has become even more obvious in recent years that the presence of a target protein is not always sufficient to predict the outcome of targeted therapies. The deregulation of downstream signaling molecules might also play an important role in the success of such therapeutic approaches. For these reasons, the analysis of tumor-specific protein expression profiles prior to therapy has been suggested as the most effective way to predict possible therapeutic results. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow the rapid, precise, inexpensive and simultaneous analysis of many network components while requiring only a small amount of clinical material.Reverse phase protein microarray (RPPA is a promising technology that meets these requirements while enabling the quantitative measurement of proteins. Together with recently developed protocols for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE tissues, RPPA may provide the means to quantify therapeutic targets and diagnostic markers in the near future and reliably screen for new protein targets.With the possibility to quantitatively analyze DNA, RNA and protein from a single FFPE tissue sample, the methods are available for integrated patient profiling at all levels of gene expression, thus allowing optimal patient stratification

  11. Histone Acetylation in Fungal Pathogens of Plants

    Junhyun Jeon

    2014-03-01

    Full Text Available Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed.

  12. Selective cleavage enhanced by acetylating the side chain of lysine.

    Fu, Leixiaomeng; Chen, Tingting; Xue, Gaiqing; Zu, Lily; Fang, Weihai

    2013-01-01

    Selective cleavage is of great interest in mass spectrometry studies as it can help sequence identification by promoting simple fragmentation pattern of peptides and proteins. In this work, the collision-induced dissociation of peptides containing internal lysine and acetylated lysine residues were studied. The experimental and computational results revealed that multiple fragmentation pathways coexisted when the lysine residue was two amino acid residues away from N-terminal of the peptide. After acetylation of the lysine side-chain, b(n)+ ions were the most abundant primary fragment products and the Lys(Ac)-Gly amide bond became the dominant cleavage site via an oxazolone pathway. Acetylating the side-chain of lysine promoted the selective cleavage of Lys-Xxx amide bond and generated much more information of the peptide backbone sequence. The results re-evaluate the selective cleavage due to the lysine basic side-chain and provide information for studying the post-translational modification of proteins and other bio-molecules containing Lys residues. PMID:23303756

  13. Fabrication of a nanocarrier system through self-assembly of plasma protein and its tumor targeting

    Human serum albumin (HSA) nanoparticles hold great promise as a nanocarrier system for targeted drug delivery. The objective of this study was to explore the possibility of preparing size controllable albumin nanoparticles using the disulfide bond breaking reagent β-mercaptoethanol (β-ME). The results showed that the protein concentration and temperature had positive effects on the sizes of the albumin nanoparticles, while pH had a negative effect on the rate of nanoparticle formation. The addition of β-ME induced changes in HSA secondary structure and exposed the hydrophobic core of HSA, leading to the formation of nanoparticles. Human serum albumin nanoparticles could be internalized by MCF-7 cells and mainly accumulated in cytoplasm. After injection in tumor bearing mice, the HSA nanoparticles accumulated in tumor tissues, demonstrating the targeting ability of the nanoparticles. Therefore, human serum albumin can be fabricated into nanoparticles by breaking the disulfide bonds and these nanoparticles exhibit high tumor targeting ability. Human serum albumin nanoparticles could be ideal for the targeted delivery of pharmacologically active substances.

  14. Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

    Chinnasamy, Dhanalakshmi; Yu, Zhiya; Morgan, Richard A.; Lee, Chyi-Chia Richard; Restifo, Nicholas P.

    2013-01-01

    Fibroblast activation protein (FAP) is a candidate universal target antigen because it has been reported to be selectively expressed in nearly all solid tumors by a subset of immunosuppressive tumor stromal fibroblasts. We verified that 18/18 human tumors of various histologies contained pronounced stromal elements staining strongly for FAP, and hypothesized that targeting tumor stroma with FAP-reactive T cells would inhibit tumor growth in cancer-bearing hosts. T cells genetically engineered with FAP-reactive chimeric antigen receptors (CARs) specifically degranulated and produced effector cytokines upon stimulation with FAP or FAP-expressing cell lines. However, adoptive transfer of FAP-reactive T cells into mice bearing a variety of subcutaneous tumors mediated limited antitumor effects and induced significant cachexia and lethal bone toxicities in two mouse strains. We found that FAP was robustly expressed on PDGFR-α+, Sca-1+ multipotent bone marrow stromal cells (BMSCs) in mice, as well as on well-characterized, clinical-grade multipotent human BMSCs. Accordingly, both mouse and human multipotent BMSCs were recognized by FAP-reactive T cells. The lethal bone toxicity and cachexia observed after cell-based immunotherapy targeting FAP cautions against its use as a universal target. Moreover, the expression of FAP by multipotent BMSCs may point toward the cellular origins of tumor stromal fibroblasts. PMID:23712432

  15. Targeted disruption of fibrinogen like protein-1 accelerates hepatocellular carcinoma development

    Nayeb-Hashemi, Hamed; Desai, Anal; Demchev, Valeriy [Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine. Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Bronson, Roderick T. [Department of Microbiology and Immunology, Harvard Medical School, Boston, MA 02115 (United States); Hornick, Jason L. [Department of Pathology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Cohen, David E. [Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine. Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Ukomadu, Chinweike, E-mail: cukomadu@partners.org [Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine. Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States)

    2015-09-18

    Fibrinogen like protein-1 (Fgl1) is a predominantly liver expressed protein that has been implicated as both a hepatoprotectant and a hepatocyte mitogen. Fgl1 expression is decreased in hepatocellular carcinoma (HCC) and its loss correlates with a poorly differentiated phenotype. To better elucidate the role of Fgl1 in hepatocarcinogenesis, we treated mice wild type or null for Fgl1 with diethyl nitrosamine and monitored for incidence of hepatocellular cancer. We find that mice lacking Fgl1 develop HCC at more than twice the rate of wild type mice. We show that hepatocellular cancers from Fgl1 null mice are molecularly distinct from those of the wild type mice. In tumors from Fgl1 null mice there is enhanced activation of Akt and downstream targets of the mammalian target of rapamycin (mTOR). In addition, there is paradoxical up regulation of putative hepatocellular cancer tumor suppressors; tripartite motif-containing protein 35 (Trim35) and tumor necrosis factor super family 10b (Tnfrsf10b). Taken together, these findings suggest that Fgl1 acts as a tumor suppressor in hepatocellular cancer through an Akt dependent mechanism and supports its role as a potential therapeutic target in HCC. - Highlights: • Fgl1 knockout mice (Fgl1KO) are more prone to carcinogen-induced liver cancer compared to wild type (WT) mates. • Tumors from the Fgl1KO are molecularly distinct with enhanced Akt and mTOR activity in comparison with Fgl1WT tumors. • Tumors from the Fgl1KO have enhanced expression of Trim35 and Tnfrsf10b, putative HCC tumor suppressors.

  16. GW182 proteins cause PABP dissociation from silenced miRNA targets in the absence of deadenylation.

    Zekri, Latifa; Kuzuoğlu-Öztürk, Duygu; Izaurralde, Elisa

    2013-04-01

    GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)-binding protein (PABP) and the CCR4-NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. Here, we demonstrate that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4-NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. Our findings indicate that the recruitment of the CCR4-NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation. PMID:23463101

  17. Amyloid-beta Alzheimer targetsprotein processing, lipid rafts, and amyloid-beta pores

    Arbor, Sage C.; LaFontaine, Mike; Cumbay, Medhane

    2016-01-01

    Amyloid beta (Aβ), the hallmark of Alzheimer’s Disease (AD), now appears to be deleterious in its low number aggregate form as opposed to the macroscopic Aβ fibers historically seen postmortem. While Alzheimer targets, such as the tau protein, amyloid precursor protein (APP) processing, and immune system activation continue to be investigated, the recent discovery that amyloid beta aggregates at lipid rafts and likely forms neurotoxic pores has led to a new paradigm regarding why past therapeutics may have failed and how to design the next round of compounds for clinical trials. An atomic resolution understanding of Aβ aggregates, which appear to exist in multiple conformations, is most desirable for future therapeutic development. The investigative difficulties, structures of these small Aβ aggregates, and current therapeutics are summarized in this review.

  18. Amyloid-beta Alzheimer targets - protein processing, lipid rafts, and amyloid-beta pores.

    Arbor, Sage C; LaFontaine, Mike; Cumbay, Medhane

    2016-03-01

    Amyloid beta (Aβ), the hallmark of Alzheimer's Disease (AD), now appears to be deleterious in its low number aggregate form as opposed to the macroscopic Aβ fibers historically seen postmortem. While Alzheimer targets, such as the tau protein, amyloid precursor protein (APP) processing, and immune system activation continue to be investigated, the recent discovery that amyloid beta aggregates at lipid rafts and likely forms neurotoxic pores has led to a new paradigm regarding why past therapeutics may have failed and how to design the next round of compounds for clinical trials. An atomic resolution understanding of Aβ aggregates, which appear to exist in multiple conformations, is most desirable for future therapeutic development. The investigative difficulties, structures of these small Aβ aggregates, and current therapeutics are summarized in this review. PMID:27505013

  19. Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

    Mahfouz, Magdy M.

    2011-12-14

    Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3. SRDX protein efficiently repressed the transcription of the RD29A

  20. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    Lin Jin; Jong Hyun Ham; Rosemary Hage; Wanying Zhao; Jaricelis Soto-Hernández; Sang Yeol Lee; Seung-Mann Paek; Min Gab Kim; Charles Boone; Coplin, David L.; David Mackey

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, w...

  1. Parasite Mitogen-Activated Protein Kinases as Drug Discovery Targets to Treat Human Protozoan Pathogens

    Michael J. Brumlik

    2011-01-01

    Full Text Available Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known.

  2. Small-Protein-Stabilized Semiconductor Nanoprobe for Targeted Imaging of Cancer Cells.

    Zhao, Ning; Liu, Siyu; Jiang, Qike; Lan, Tian; Cheng, Zhen; Liu, Hongguang

    2016-07-01

    Recently, semiconductor nanoparticles such as quantum dots (QDs) have attracted significant attention for bioimaging. Complex chemical functionalization, surface modification, and bioconjugation chemistry are generally required to tag biomolecules to QDs for imaging of different biomarkers. In this study, we report a simple method for production of QDs stabilized by the small protein, Affibody (AF-QDs) for fluorescent imaging of the human epidermal growth factor receptor type 2 (HER2) in human A549 lung cancer cells. This one-pot synthesis of AF-QDs avoids complex chemical conjugation procedures and demonstrates a promising approach for the preparation of fluorescent nanoprobes for imaging of cancer targets. PMID:27123671

  3. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA

    Kedde, Martijn; Strasser, Markus J; Boldajipour, Bijan;

    2007-01-01

    MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally...... evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the...

  4. Locating proteins in the cell using TargetP, SignalP and related tools

    Emanuelsson, O.; Brunak, Søren; von Heijne, G.;

    2007-01-01

    of methods to predict subcellular localization based on these sorting signals and other sequence properties. We then outline how to use a number of internet-accessible tools to arrive at a reliable subcellular localization prediction for eukaryotic and prokaryotic proteins. In particular, we provide detailed...... step-by-step instructions for the coupled use of the amino-acid sequence-based predictors TargetP, SignalP, ChloroP and TMHMM, which are all hosted at the Center for Biological Sequence Analysis, Technical University of Denmark. In addition, we describe and provide web references to other useful...

  5. 5-Fluorocytosine combined with Fcy-hEGF fusion protein targets EGFR-expressing cancer cells

    Lan, Keng-Hsueh [Division of Radiation Oncology, Department of Oncology, National Taiwan University Hospital, Taipei 100, Taiwan (China); Shih, Yi-Sheng [Cancer Center, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Chang, Cheng Allen [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); School of Biomedical Science and Engineering, National Yang-Ming University, Taipei 112, Taiwan (China); Yen, Sang-Hue [Cancer Center, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Lan, Keng-Li, E-mail: kllan@vghtpe.gov.tw [Cancer Center, Taipei Veterans General Hospital, Taipei 112, Taiwan (China); Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei 112, Taiwan (China)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer EGFR-expressing epithelial cancers account for significant portion of cancer deaths. Black-Right-Pointing-Pointer EGF-EGFR signaling pathway is validated as an important anticancer drug target. Black-Right-Pointing-Pointer EGF and Fcy fusion protein (Fcy-hEGF) can bind to EGFR and convert 5-FC to 5-FU. Black-Right-Pointing-Pointer Fcy-hEGF combined with 5-FC preferentially inhibits EGFR-expressing cells viability. -- Abstract: Human epithelial cancers account for approximately 50% of all cancer deaths. This type of cancer is characterized by excessive activation and expression of the epidermal growth factor receptor (EGFR). The EGFR pathway is critical for cancer cell proliferation, survival, metastasis and angiogenesis. The EGF-EGFR signaling pathway has been validated as an important anticancer drug target. Increasing numbers of targeted therapies against this pathway have been either approved or are currently under development. Here, we adopted a prodrug system that uses 5-fluorocytosine (5-FC) and human EGF (hEGF) fused with yeast cytosine deaminase (Fcy) to target EGFR-overexpressing cancer cells and to convert 5-FC to a significantly more toxic chemotherapeutic, 5-fluorouracil (5-FU). We cloned and purified the Fcy-hEGF fusion protein from Pichia pastoris yeast. This fusion protein specifically binds to EGFR with a similar affinity as hEGF, approximately 10 nM. Fcy-hEGF binds tightly to A431 and MDA-MB-468 cells, which overexpress EGFR, but it binds with a lower affinity to MDA-MB-231 and MCF-7, which express lower levels of EGFR. Similarly, the viability of EGFR-expressing cells was suppressed by Fcy-hEGF in the presence of increasing concentrations of 5-FC, and the IC{sub 50} values for A431 and MDA-MB-468 were approximately 10-fold lower than those of MDA-MB-231 and MCF-7. This novel prodrug system, Fcy-hEGF/5-FC, might represent a promising addition to the available class of inhibitors that specifically target EGFR

  6. 5-Fluorocytosine combined with Fcy–hEGF fusion protein targets EGFR-expressing cancer cells

    Highlights: ► EGFR-expressing epithelial cancers account for significant portion of cancer deaths. ► EGF–EGFR signaling pathway is validated as an important anticancer drug target. ► EGF and Fcy fusion protein (Fcy–hEGF) can bind to EGFR and convert 5-FC to 5-FU. ► Fcy–hEGF combined with 5-FC preferentially inhibits EGFR-expressing cells viability. -- Abstract: Human epithelial cancers account for approximately 50% of all cancer deaths. This type of cancer is characterized by excessive activation and expression of the epidermal growth factor receptor (EGFR). The EGFR pathway is critical for cancer cell proliferation, survival, metastasis and angiogenesis. The EGF–EGFR signaling pathway has been validated as an important anticancer drug target. Increasing numbers of targeted therapies against this pathway have been either approved or are currently under development. Here, we adopted a prodrug system that uses 5-fluorocytosine (5-FC) and human EGF (hEGF) fused with yeast cytosine deaminase (Fcy) to target EGFR-overexpressing cancer cells and to convert 5-FC to a significantly more toxic chemotherapeutic, 5-fluorouracil (5-FU). We cloned and purified the Fcy–hEGF fusion protein from Pichia pastoris yeast. This fusion protein specifically binds to EGFR with a similar affinity as hEGF, approximately 10 nM. Fcy–hEGF binds tightly to A431 and MDA-MB-468 cells, which overexpress EGFR, but it binds with a lower affinity to MDA-MB-231 and MCF-7, which express lower levels of EGFR. Similarly, the viability of EGFR-expressing cells was suppressed by Fcy–hEGF in the presence of increasing concentrations of 5-FC, and the IC50 values for A431 and MDA-MB-468 were approximately 10-fold lower than those of MDA-MB-231 and MCF-7. This novel prodrug system, Fcy–hEGF/5-FC, might represent a promising addition to the available class of inhibitors that specifically target EGFR-expressing cancers.

  7. Density Functional Theory Study on the Histidine-assisted Mechanism of Arylamine N-Acetyltransferase Acetylation

    QIAO Qing-An; GAO Shan-Min; JIN Yue-Qing; CHEN Xin; SUN Xiao-Min; YANG Chuan-Lu

    2008-01-01

    Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) catalyze the N-acetylation of primary arylamines, and play a key role in the biotransformation and metabolism of drugs, carcinogens, etc.In this paper, three possible reaction mechanisms are investigated and the results indicate that if the acetyl group directly transfers from the donor to the acceptor, the high activation energies will make it hard to obtain the target products.When using histidine to mediate the acetylation process, these energies will drop in the 15~45 kJ/mol range.If the histidine residue is protonated, the corresponding energies will be decreased by about 35~87 kJ/mol.The calculations predict an enzymatic acetylation mechanism that undergoes a thiolate-imidazolium pair, which agrees with the experimental results very well.

  8. Selective autophagy of non-ubiquitylated targets in plants: looking for cognate receptor/adaptor proteins

    Vasko eVeljanovski

    2014-06-01

    Full Text Available Cellular homeostasis is essential for the physiology of eukaryotic cells. Eukaryotic cells, including plant cells, utilize two main pathways to adjust the level of cytoplasmic components, namely the proteasomal and the lysosomal/vacuolar pathways. Macroautophagy is a lysosomal/vacuolar pathway which, until recently, was thought to be non-specific and a bulk degradation process. However, selective autophagy which can be activated in the cell under various physiological conditions, involves the specific degradation of defined macromolecules or organelles by a conserved molecular mechanism. For this process to be efficient, the mechanisms underlying the recognition and selection of the cargo to be engulfed by the double-membrane autophagosome are critical, and not yet well understood. Ubiquitin (poly-ubiquitin conjugation to the target appears to be a conserved ligand mechanism in many types of selective autophagy, and defined receptors/adaptors recognizing and regulating the autophagosomal capture of the ubiquitylated target have been characterized. However, non-proteinaceous and non-ubiquitylated cargoes are also selectively degraded by this pathway. This ubiquitin-independent selective autophagic pathway also involves receptor and/or adaptor proteins linking the cargo to the autophagic machinery. Some of these receptor/adaptor proteins including accessory autophagy-related (Atg and non-Atg proteins have been described in yeast and animal cells but not yet in plants. In this review we discuss the ubiquitin-independent cargo selection mechanisms in selective autophagy degradation of organelles and macromolecules and speculate on potential plant receptor/adaptor proteins.

  9. Identification of proteins involved in neural progenitor cell targeting of gliomas

    Honeth Gabriella

    2009-06-01

    Full Text Available Abstract Background Glioblastoma are highly aggressive tumors with an average survival time of 12 months with currently available treatment. We have previously shown that specific embryonic neural progenitor cells (NPC have the potential to target glioma growth in the CNS of rats. The neural progenitor cell treatment can cure approximately 40% of the animals with malignant gliomas with no trace of a tumor burden 6 months after finishing the experiment. Furthermore, the NPCs have been shown to respond to signals from the tumor environment resulting in specific migration towards the tumor. Based on these results we wanted to investigate what factors could influence the growth and progression of gliomas in our rodent model. Methods Using microarrays we screened for candidate genes involved in the functional mechanism of tumor inhibition by comparing glioma cell lines to neural progenitor cells with or without anti-tumor activity. The expression of candidate genes was confirmed at RNA level by quantitative RT-PCR and at the protein level by Western blots and immunocytochemistry. Moreover, we have developed in vitro assays to mimic the antitumor effect seen in vivo. Results We identified several targets involved in glioma growth and migration, specifically CXCL1, CD81, TPT1, Gas6 and AXL proteins. We further showed that follistatin secretion from the NPC has the potential to decrease tumor proliferation. In vitro co-cultures of NPC and tumor cells resulted in the inhibition of tumor growth. The addition of antibodies against proteins selected by gene and protein expression analysis either increased or decreased the proliferation rate of the glioma cell lines in vitro. Conclusion These results suggest that these identified factors might be useful starting points for performing future experiments directed towards a potential therapy against malignant gliomas.

  10. Gene and protein targeting technologies create novel opportunities in plant biotechnology

    An important platform technology that is missing in the field of plant science is gene targeting through homologous recombination. Moreover, for many economically important plant varieties, robust and efficient protocols for vegetative propagation and genetic modification are still lacking. The availability of such technologies is essential for the further development of plant biotechnology. Add2X Biosciences BV is a young biotech spin-off from Leiden University that aims to create novel opportunities in plant biotechnology, by developing innovative platform technologies and products for the directed and efficient delivery of DNA and proteins into plant cells. The Add2X portfolio includes: 1. Efficient gene targeting in plants through suppression of the non-homologous recombination pathway. Proof of concept has been obtained in different yeasts and filamentous fungi. Currently, research is in progress to confirm the applicability of this technology in plant species. 2. Agrobacterium-mediated protein translocation to produce and transiently 'inject' proteins of interest into plant cells in order to exert their function without permanently altering the host cells. 3. Induction of somatic embryogenesis in hitherto recalcitrant crop species. A protein family was identified that, when over-expressed, stimulates the spontaneous formation of somatic embryos from vegetative plant cells. The Add2X technologies stand well on their own, but clearly have the potential to be combined into integrated technologies or products. The technologies are developed in close collaboration with Leiden University. In addition, Add2X has established strategic alliances with other academic and industrial partners to explore novel opportunities and to achieve rapid implementation of the technologies in the biotechnology sector. (author)

  11. Cell to cell spreading of misfolded proteins as a therapeutic target in motor neuron disease.

    Pasquali, Livia; Lenzi, Paola; Biagioni, Francesca; Siciliano, Gabriele; Fornai, Francesco

    2014-01-01

    Despite a number of genetic mutations and molecular mechanisms are recognized to participate in amyotrophic lateral sclerosis (ALS), such a devastating neurological disorder still lacks a substantial cure. The present manuscript rather than a general overview of potential therapeutic approaches focuses on novel research findings detailing novel molecular mechanisms which appear to be promising for developing future ALS therapeutics. A special emphasis is given to the abnormal autophagy status and to those autophagy substrates which aggregate in the form of misfolded proteins. In fact, as reviewed in the first part of the manuscript, altered autophagy pathway is present in most genetic mutations responsible for familial ALS. These mutations impair clearance of autophagy substrates, which determines accumulation of giant altered mitochondria and misfolded proteins. Therefore, a considerable piece of the review is dedicated to unconventional processing of misfolded proteins leading to unconventional protein secretions which may underlie a prionoid cellto- cell spreading of ALS neuropathology. The intimate mechanisms regulating these steps are analyzed in order to comprehend which potential therapeutic targets might be considered in future studies. At the same time, negative findings concerning recent trials are explained in light of novel disease mechanisms. In the final part of the review the replacement therapy with focal stem cells implantation is discussed in relationship with toxic mechanisms operating in the intercellular space of the spinal cord and motor-related areas. PMID:24934358

  12. Therapeutic potential of targeting protein for Xklp2 silencing for pancreatic cancer

    The targeting protein for Xklp2 (TPX2) is a microtubule- and, cell cycle-associated protein who’s overexpression has been reported in various malignancies. In this study, we verified the overexpression of TPX2 in both surgically resected specimens of pancreatic cancer and multiple pancreatic cancer cell lines. Subsequently, we found that TPX2 siRNA effectively suppressed the proliferation of pancreatic cancer cells in culture, and the direct injection of TPX2 siRNA into subcutaneously implanted pancreatic cancer cells in nude mice revealed antiproliferative effects. These results implied a therapeutic potential of TPX2 siRNA in pancreatic cancer. Among 56 angiogenesis-related factors examined using angiogenesis arrays, the average protein levels of insulin-like growth factor-binding protein-3 (IGFBP-3) were significantly higher in TPX2 siRNA-treated tumors than in the Control siRNA-treated tumors. Moreover, we demonstrated that CD34-positive microvessels were significantly reduced in tumors treated with TPX2 siRNA compared to tumors that treated with Control siRNA. The attenuated expression of CD34 in TPX2 siRNA-treated tumors coincided with the overexpression of IGFBP-3. These results indicated that TPX2 has an impact on tumor angiogenesis in pancreatic cancer. The results also implied that the antiangiogenic effect observed in TPX2 siRNA-treated pancreatic cancer cells may be partly explained by the upregulation of IGFBP-3

  13. Protein Folding Activity of the Ribosome (PFAR –– A Target for Antiprion Compounds

    Debapriya Banerjee

    2014-10-01

    Full Text Available Prion diseases are fatal neurodegenerative diseases affecting mammals. Prions are misfolded amyloid aggregates of the prion protein (PrP, which form when the alpha helical, soluble form of PrP converts to an aggregation-prone, beta sheet form. Thus, prions originate as protein folding problems. The discovery of yeast prion(s and the development of a red-/white-colony based assay facilitated safe and high-throughput screening of antiprion compounds. With this assay three antiprion compounds; 6-aminophenanthridine (6AP, guanabenz acetate (GA, and imiquimod (IQ have been identified. Biochemical and genetic studies reveal that these compounds target ribosomal RNA (rRNA and inhibit specifically the protein folding activity of the ribosome (PFAR. The domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active site for PFAR. 6AP and GA inhibit PFAR by competition with the protein substrates for the common binding sites on the domain V rRNA. PFAR inhibition by these antiprion compounds opens up new possibilities for understanding prion formation, propagation and the role of the ribosome therein. In this review, we summarize and analyze the correlation between PFAR and prion processes using the antiprion compounds as tools.

  14. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms

    Maeda, K.; Finnie, Christine; Svensson, Birte

    2004-01-01

    thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The...

  15. A plastid-targeted heat shock cognate 70 kDa protein interacts with the Abutilon mosaic virus movement protein

    The movement protein (MP) of bipartite geminiviruses facilitates cell-to-cell as well as long-distance transport within plants and influences viral pathogenicity. Yeast two-hybrid assays identified a chaperone, the nuclear-encoded and plastid-targeted heat shock cognate 70 kDa protein (cpHSC70-1) of Arabidopsis thaliana, as a potential binding partner for the Abutilon mosaic virus (AbMV) MP. In planta, bimolecular fluorescence complementation (BiFC) analysis showed cpHSC70-1/MP complexes and MP homooligomers at the cell periphery and co-localized with chloroplasts. BiFC revealed cpHSC70-1 oligomers associated with chloroplasts, but also distributed at the cellular margin and in filaments arising from plastids reminiscent of stromules. Silencing the cpHSC70 gene of Nicotiana benthamiana using an AbMV DNA A-derived gene silencing vector induced minute white leaf areas, which indicate an effect on chloroplast stability. Although AbMV DNA accumulated within chlorotic spots, a spatial restriction of these occurred, suggesting a functional relevance of the MP-chaperone interaction for viral transport and symptom induction.

  16. Docking Studies in Target Proteins Involved in Antibacterial Action Mechanisms: Extending the Knowledge on Standard Antibiotics to Antimicrobial Mushroom Compounds

    Maria José Alves

    2014-01-01

    Full Text Available In the present work, the knowledge on target proteins of standard antibiotics was extended to antimicrobial mushroom compounds. Docking studies were performed for 34 compounds in order to evaluate their affinity to bacterial proteins that are known targets for some antibiotics with different mechanism of action: inhibitors of cell wall synthesis, inhibitors of protein synthesis, inhibitors of nucleic acids synthesis and antimetabolites. After validation of the molecular docking approach, virtual screening of all the compounds was performed against penicillin binding protein 1a (PBP1a, alanine racemase (Alr, d-alanyl-d-alanine synthetase (Ddl, isoleucyl-tRNA sinthetase (IARS, DNA gyrase subunit B, topoisomerase IV (TopoIV, dihydropteroate synthetase (DHPS and dihydrofolate reductase (DHFR using AutoDock4. Overall, it seems that for the selected mushroom compounds (namely, enokipodins, ganomycins and austrocortiluteins the main mechanism of the action is the inhibition of cell wall synthesis, being Alr and Ddl probable protein targets.

  17. Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

    The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA

  18. Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation.

    Todd J Cohen

    Full Text Available Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD. Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.

  19. Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification.

    von Rechenberg, Moritz; Blake, Brian Kelly; Ho, Yew-Seng J; Zhen, Yuejun; Chepanoske, Cindy Lou; Richardson, Bonnie E; Xu, Nafei; Kery, Vladimir

    2005-05-01

    The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors. PMID:15761956

  20. Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

    The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P2 and PI(3,4,5)P3. A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein

  1. Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

    Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

    2016-03-01

    Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research.

  2. Targeted delivery of siRNA to cell death proteins in sepsis.

    Brahmamdam, Pavan; Watanabe, Eizo; Unsinger, Jacqueline; Chang, Katherine C; Schierding, William; Hoekzema, Andrew S; Zhou, Tony T; McDonough, Jacquelyn S; Holemon, Heather; Heidel, Jeremy D; Coopersmith, Craig M; McDunn, Jonathan E; Hotchkiss, Richard S

    2009-08-01

    Immune suppression is a major cause of morbidity and mortality in the patients with sepsis. Apoptotic loss of immune effector cells such as CD4 T and B cells is a key component in the loss of immune competence in sepsis. Inhibition of lymphocyte apoptosis has led to improved survival in animal models of sepsis. Using quantitative real-time polymerase chain reaction of isolated splenic CD4 T and B cells, we determined that Bim and PUMA, two key cell death proteins, are markedly upregulated during sepsis. Lymphocytes have been notoriously difficult to transfect with small interfering RNA (siRNA). Consequently a novel, cyclodextrin polymer-based, transferrin receptor-targeted, delivery vehicle was used to coadminister siRNA to Bim and PUMA to mice immediately after cecal ligation and puncture. Antiapoptotic siRNA-based therapy markedly decreased lymphocyte apoptosis and prevented the loss of splenic CD4 T and B cells. Flow cytometry confirmed in vivo delivery of siRNA to CD4 T and B cells and also demonstrated decreases in intracellular Bim and PUMA protein. In conclusion, Bim and PUMA are two critical mediators of immune cell death in sepsis. Use of a novel cyclodextrin polymer-based, transferrin receptor-targeted siRNA delivery vehicle enables effective administration of antiapoptotic siRNAs to lymphocytes and reverses the immune cell depletion that is a hallmark of this highly lethal disorder. PMID:19033888

  3. Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition

    Skelding, Zachary; Queen-Baker, Jennie; Craig, Nancy L

    2003-01-01

    The Tn7 transposon avoids inserting into a target DNA that contains a pre-existing copy of Tn7. This phenomenon, known as ‘target immunity’, is established when TnsB, a Tn7 transposase subunit, binds to Tn7 sequences in the target DNA and mediates displacement of TnsC, a critical transposase activator, from the DNA. Paradoxically, TnsB–TnsC interactions are also required to promote transposon insertion. We have probed Tn7 target immunity by isolating TnsB mutants that mediate more frequent in...

  4. Sensitive targeted multiple protein quantification based on elemental detection of Quantum Dots

    Highlights: • Novel generic platform for multiparametric quantification of proteins. • QDs labeling and ICP-MS detection allow significant analytical signal amplification. • ICP-MS mass balances information provided an internal validation of the immunoassay. • Multiparametric determination of 5 proteins in human serum samples. • ICP-MS reduced matrix effects as compared to other conventional detection techniques. - Abstract: A generic strategy based on the use of CdSe/ZnS Quantum Dots (QDs) as elemental labels for protein quantification, using immunoassays with elemental mass spectrometry (ICP-MS), detection is presented. In this strategy, streptavidin modified QDs (QDs-SA) are bioconjugated to a biotinylated secondary antibody (b-Ab2). After a multi-technique characterization of the synthesized generic platform (QDs-SA-b-Ab2) it was applied to the sequential quantification of five proteins (transferrin, complement C3, apolipoprotein A1, transthyretin and apolipoprotein A4) at different concentration levels in human serum samples. It is shown how this generic strategy does only require the appropriate unlabeled primary antibody for each protein to be detected. Therefore, it introduces a way out to the need for the cumbersome and specific bioconjugation of the QDs to the corresponding specific recognition antibody for every target analyte (protein). Results obtained were validated with those obtained using UV–vis spectrophotometry and commercial ELISA Kits. As expected, ICP-MS offered one order of magnitude lower DL (0.23 fmol absolute for transferrin) than the classical spectrophotometric detection (3.2 fmol absolute). ICP-MS precision and detection limits, however turned out to be compromised by procedural blanks. The full analytical performance of the ICP-MS-based immunoassay proposed was assessed for detection of transferrin (Tf), present at the low ng mL−1 range in a complex “model” synthetic matrix, where the total protein concentration was

  5. Sensitive targeted multiple protein quantification based on elemental detection of Quantum Dots

    Montoro Bustos, Antonio R.; Garcia-Cortes, Marta [Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8, Oviedo 33006 (Spain); González-Iglesias, Hector [Fundación de Investigación Oftalmológica, Instituto Oftalmológico Fernandez-Vega, Avenida Doctores Fernández-Vega, 34, Oviedo 33012 (Spain); Ruiz Encinar, Jorge, E-mail: ruizjorge@uniovi.es [Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8, Oviedo 33006 (Spain); Costa-Fernández, José M. [Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8, Oviedo 33006 (Spain); Coca-Prados, Miguel [Fundación de Investigación Oftalmológica, Instituto Oftalmológico Fernandez-Vega, Avenida Doctores Fernández-Vega, 34, Oviedo 33012 (Spain); Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT 06510 (United States); Sanz-Medel, Alfredo, E-mail: asm@uniovi.es [Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8, Oviedo 33006 (Spain)

    2015-06-16

    Highlights: • Novel generic platform for multiparametric quantification of proteins. • QDs labeling and ICP-MS detection allow significant analytical signal amplification. • ICP-MS mass balances information provided an internal validation of the immunoassay. • Multiparametric determination of 5 proteins in human serum samples. • ICP-MS reduced matrix effects as compared to other conventional detection techniques. - Abstract: A generic strategy based on the use of CdSe/ZnS Quantum Dots (QDs) as elemental labels for protein quantification, using immunoassays with elemental mass spectrometry (ICP-MS), detection is presented. In this strategy, streptavidin modified QDs (QDs-SA) are bioconjugated to a biotinylated secondary antibody (b-Ab{sub 2}). After a multi-technique characterization of the synthesized generic platform (QDs-SA-b-Ab{sub 2}) it was applied to the sequential quantification of five proteins (transferrin, complement C3, apolipoprotein A1, transthyretin and apolipoprotein A4) at different concentration levels in human serum samples. It is shown how this generic strategy does only require the appropriate unlabeled primary antibody for each protein to be detected. Therefore, it introduces a way out to the need for the cumbersome and specific bioconjugation of the QDs to the corresponding specific recognition antibody for every target analyte (protein). Results obtained were validated with those obtained using UV–vis spectrophotometry and commercial ELISA Kits. As expected, ICP-MS offered one order of magnitude lower DL (0.23 fmol absolute for transferrin) than the classical spectrophotometric detection (3.2 fmol absolute). ICP-MS precision and detection limits, however turned out to be compromised by procedural blanks. The full analytical performance of the ICP-MS-based immunoassay proposed was assessed for detection of transferrin (Tf), present at the low ng mL{sup −1} range in a complex “model” synthetic matrix, where the total protein

  6. AMPK/Snf1 signaling regulates histone acetylation: Impact on gene expression and epigenetic functions.

    Salminen, Antero; Kauppinen, Anu; Kaarniranta, Kai

    2016-08-01

    AMP-activated protein kinase (AMPK) and its yeast homolog, Snf1, are critical regulators in the maintenance of energy metabolic balance not only stimulating energy production but also inhibiting energy-consuming processes. The AMPK/Snf1 signaling controls energy metabolism by specific phosphorylation of many metabolic enzymes and transcription factors, enhancing or suppressing their functions. The AMPK/Snf1 complexes can be translocated from cytoplasm into nuclei where they are involved in the regulation of transcription. Recent studies have indicated that AMPK/Snf1 activation can control histone acetylation through different mechanisms affecting not only gene transcription but also many other epigenetic functions. For instance, AMPK/Snf1 enzymes can phosphorylate the histone H3S10 (yeast) and H2BS36 (mammalian) sites which activate specific histone acetyltransferases (HAT), consequently enhancing histone acetylation. Moreover, nuclear AMPK can phosphorylate type 2A histone deacetylases (HDAC), e.g. HDAC4 and HDAC5, triggering their export from nuclei thus promoting histone acetylation reactions. AMPK activation can also increase the level of acetyl CoA, e.g. by inhibiting fatty acid and cholesterol syntheses. Acetyl CoA is a substrate for HATs, thus increasing their capacity for histone acetylation. On the other hand, AMPK can stimulate the activity of nicotinamide phosphoribosyltransferase (NAMPT) which increases the level of NAD(+). NAD(+) is a substrate for nuclear sirtuins, especially for SIRT1 and SIRT6, which deacetylate histones and transcription factors, e.g. those regulating ribosome synthesis and circadian clocks. Histone acetylation is an important epigenetic modification which subsequently can affect chromatin remodeling, e.g. via bromodomain proteins. We will review the signaling mechanisms of AMPK/Snf1 in the control of histone acetylation and subsequently clarify their role in the epigenetic regulation of ribosome synthesis and circadian clocks

  7. Target Identification by Chromatographic Co-elution: Monitoring of Drug-Protein Interactions without Immobilization or Chemical Derivatization*

    Chan, Janet N. Y.; Vuckovic, Dajana; Sleno, Lekha; Olsen, Jonathan B.; Pogoutse, Oxana; Havugimana, Pierre; Hewel, Johannes A.; Bajaj, Navgeet; Wang, Yale; Musteata, Marcel F.; Nislow, Corey; Emili, Andrew

    2012-01-01

    Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely mo...

  8. Targeting the Wolbachia cell division protein FtsZ as a new approach for antifilarial therapy.

    Zhiru Li

    2011-11-01

    Full Text Available The use of antibiotics targeting the obligate bacterial endosymbiont Wolbachia of filarial parasites has been validated as an approach for controlling filarial infection in animals and humans. Availability of genomic sequences for the Wolbachia (wBm present in the human filarial parasite Brugia malayi has enabled genome-wide searching for new potential drug targets. In the present study, we investigated the cell division machinery of wBm and determined that it possesses the essential cell division gene ftsZ which was expressed in all developmental stages of B. malayi examined. FtsZ is a GTPase thereby making the protein an attractive Wolbachia drug target. We described the molecular characterization and catalytic properties of Wolbachia FtsZ. We also demonstrated that the GTPase activity was inhibited by the natural product, berberine, and small molecule inhibitors identified from a high-throughput screen. Furthermore, berberine was also effective in reducing motility and reproduction in B. malayi parasites in vitro. Our results should facilitate the discovery of selective inhibitors of FtsZ as a novel anti-symbiotic approach for controlling filarial infection. NOTE: The nucleotide sequences reported in this paper are available in GenBank™ Data Bank under the accession number wAlB-FtsZ (JN616286.

  9. Protein disulfide isomerase as a novel target for cyclopentenone prostaglandins: implications for hypoxic ischemic injury

    Liu, Hao; Chen, Jie; Li, Wenjin; Rose, Marie E.; Shinde, Sunita N.; Balasubramani, Manimalha; Uechi, Guy T.; Mutus, Bülent; Graham, Steven H.; Hickey, Robert W.

    2016-01-01

    Cyclooxygenase-2 (COX-2) is an important contributor to ischemic brain injury. Identification of the downstream mediators of COX-2 toxicity may allow the development of targeted therapies. Of particular interest is the cyclopentenone family of prostaglandin metabolites. Cyclopentenone prostaglandins (CyPGs) are highly reactive molecules that form covalent bonds with cellular thiols. Protein disulfide isomerase (PDI) is an important molecule for the restoration of denatured proteins following ischemia. Because PDI has several thiols, including thiols within the active thioredoxin-like domain, we hypothesized that PDI is a target of CyPGs and that CyPG binding of PDI is detrimental. CyPG–PDI binding was detected in vitro via immunoprecipitation and MS. CyPG–PDI binding decreased PDI enzymatic activity in recombinant PDI treated with CyPG, and PDI immunoprecipitated from neuronal culture treated with CyPG or anoxia. Toxic effects of binding were demonstrated in experiments showing that: (a) pharmacologic inhibition of PDI increased cell death in anoxic neurons, (b) PDI overexpression protected neurons exposed to anoxia and SH-SY5Y cells exposed to CyPG, and (c) PDI overexpression in SH-SY5Y cells attenuated ubiquitination of proteins and decreased activation of pro-apoptotic caspases. In conclusion, CyPG production and subsequent binding of PDI is a novel and potentially important mechanism of ischemic brain injury. We show that CyPGs bind to PDI, cyclopentenones inhibit PDI activity, and CyPG–PDI binding is associated with increased neuronal susceptibility to anoxia. Additional studies are necessary to determine the relative role of CyPG-dependent inhibition of PDI activity in ischemia and other neurodegenerative disorders. PMID:25754985

  10. Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing.

    Kubota, Kenji; Tsuda, Shinya; Tamai, Atsushi; Meshi, Tetsuo

    2003-10-01

    Posttranscriptional gene silencing (PTGS), a homology-dependent RNA degradation system, has a role in defending against virus infection in plants, but plant viruses encode a suppressor to combat PTGS. Using transgenic tobacco in which the expression of green fluorescent protein (GFP) is posttranscriptionally silenced, we investigated a tomato mosaic virus (ToMV)-encoded PTGS suppressor. Infection with wild-type ToMV (L strain) interrupted GFP silencing in tobacco, coincident with visible symptoms, whereas some attenuated strains of ToMV (L(11) and L(11)A strains) failed to suppress GFP silencing. Analyses of recombinant viruses containing the L and L(11)A strains revealed that a single base change in the replicase gene, which causes an amino acid substitution, is responsible for the symptomless and suppressor-defective phenotypes of the attenuated strains. An agroinfiltration assay indicated that the 130K replication protein acts as a PTGS suppressor. Small interfering RNAs (siRNAs) of 21 to 25 nucleotides accumulated during ToMV infection, suggesting that the major target of the ToMV-encoded suppressor is downstream from the production of siRNAs in the PTGS pathway. Analysis with GFP-tagged recombinant viruses revealed that the suppressor inhibits the establishment of the ToMV-targeted PTGS system in the inoculated leaves but does not detectably suppress the activity of the preexisting, sequence-specific PTGS machinery there. Taken together, these results indicate that it is likely that the ToMV-encoded suppressor, the 130K replication protein, blocks the utilization of silencing-associated small RNAs, so that a homology-dependent RNA degradation machinery is not newly formed. PMID:14512550

  11. Cell morphogenesis proteins are translationally controlled through UTRs by the Ndr/LATS target Ssd1.

    Antony G Wanless

    Full Text Available Eukaryotic cells control their growth and morphogenesis to maintain integrity and viability. Free-living cells are further challenged by their direct interaction with the environment and in many cases maintain a resilient cell wall to stay alive under widely varying conditions. For these organisms, stringent and highly localized control of the cell wall's remodeling and expansion is crucial for cell growth and reproduction. In the budding yeast Saccharomyces cerevisiae the RNA binding protein Ssd1 helps control cell wall remodeling by repressing translation of proteins involved in wall expansion. Ssd1 is itself negatively regulated by the highly conserved Ndr/LATS protein kinase Cbk1. We sought to identify mRNA regions that confer Ssd1-mediated translational control. After validating a GFP reporter system as a readout of Ssd1 activity we found that 3' untranslated regions of the known Ssd1 targets CTS1, SIM1 and UTH1 are sufficient for Cbk1-regulated translational control. The 5' untranslated region of UTH1 also facilitated Ssd1-mediated translational control in a heterologous context. The CTS1 and SIM1 3' untranslated regions confer Ssd1 binding, and the SIM1 3' untranslated region improves Ssd1 immunoprecipitation of the endogenous SIM1 transcript. However, SIM1's 3' untranslated region is not essential for Ssd1-regulated control of the message's translation. We propose that Ssd1 regulates translation of its target message primarily through UTRs and the SIM1 message through multiple potential points of interaction, permitting fine translational control in various contexts.

  12. A computational strategy to select optimized protein targets for drug development toward the control of cancer diseases.

    Carels, Nicolas; Tilli, Tatiana; Tuszynski, Jack A

    2015-01-01

    In this report, we describe a strategy for the optimized selection of protein targets suitable for drug development against neoplastic diseases taking the particular case of breast cancer as an example. We combined human interactome and transcriptome data from malignant and control cell lines because highly connected proteins that are up-regulated in malignant cell lines are expected to be suitable protein targets for chemotherapy with a lower rate of undesirable side effects. We normalized transcriptome data and applied a statistic treatment to objectively extract the sub-networks of down- and up-regulated genes whose proteins effectively interact. We chose the most connected ones that act as protein hubs, most being in the signaling network. We show that the protein targets effectively identified by the combination of protein connectivity and differential expression are known as suitable targets for the successful chemotherapy of breast cancer. Interestingly, we found additional proteins, not generally targeted by drug treatments, which might justify the extension of existing formulation by addition of inhibitors designed against these proteins with the consequence of improving therapeutic outcomes. The molecular alterations observed in breast cancer cell lines represent either driver events and/or driver pathways that are necessary for breast cancer development or progression. However, it is clear that signaling mechanisms of the luminal A, B and triple negative subtypes are different. Furthermore, the up- and down-regulated networks predicted subtype-specific drug targets and possible compensation circuits between up- and down-regulated genes. We believe these results may have significant clinical implications in the personalized treatment of cancer patients allowing an objective approach to the recycling of the arsenal of available drugs to the specific case of each breast cancer given their distinct qualitative and quantitative molecular traits. PMID:25625699

  13. A computational strategy to select optimized protein targets for drug development toward the control of cancer diseases.

    Nicolas Carels

    Full Text Available In this report, we describe a strategy for the optimized selection of protein targets suitable for drug development against neoplastic diseases taking the particular case of breast cancer as an example. We combined human interactome and transcriptome data from malignant and control cell lines because highly connected proteins that are up-regulated in malignant cell lines are expected to be suitable protein targets for chemotherapy with a lower rate of undesirable side effects. We normalized transcriptome data and applied a statistic treatment to objectively extract the sub-networks of down- and up-regulated genes whose proteins effectively interact. We chose the most connected ones that act as protein hubs, most being in the signaling network. We show that the protein targets effectively identified by the combination of protein connectivity and differential expression are known as suitable targets for the successful chemotherapy of breast cancer. Interestingly, we found additional proteins, not generally targeted by drug treatments, which might justify the extension of existing formulation by addition of inhibitors designed against these proteins with the consequence of improving therapeutic outcomes. The molecular alterations observed in breast cancer cell lines represent either driver events and/or driver pathways that are necessary for breast cancer development or progression. However, it is clear that signaling mechanisms of the luminal A, B and triple negative subtypes are different. Furthermore, the up- and down-regulated networks predicted subtype-specific drug targets and possible compensation circuits between up- and down-regulated genes. We believe these results may have significant clinical implications in the personalized treatment of cancer patients allowing an objective approach to the recycling of the arsenal of available drugs to the specific case of each breast cancer given their distinct qualitative and quantitative molecular

  14. Heart failure-inducible gene therapy targeting protein phosphatase 1 prevents progressive left ventricular remodeling.

    Yosuke Miyazaki

    Full Text Available BACKGROUND: The targeting of Ca(2+ cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR Ca(2+ ATPase, or ablation of phospholamban (PLN and associated protein phosphatase 1 (PP1 protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca(2+ uptake in the SR among the three PP1 isoforms, thereby contributing to Ca(2+ downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9 vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice. METHODS: We created an adeno-associated virus 9 (AAV9 vector encoding PP1β short-hairpin RNA (shRNA or negative control (NC shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP promoter conjugated to emerald-green fluorescence protein (EmGFP and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA were injected into the tail vein (2×10(11 GC/mouse of muscle LIM protein deficient mice (MLPKO, followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months. RESULTS: In the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months. CONCLUSION: Heart failure

  15. Targeting of a Tail-anchored Protein to Endoplasmic Reticulum and Mitochondrial Outer Membrane by Independent but Competing Pathways

    Borgese, Nica; Gazzoni, Ilaria; Barberi, Massimo; Colombo, Sara; Pedrazzini, Emanuela

    2001-01-01

    Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b5, a TA protein existing in two, MOM or ER localized, versions. Substi...

  16. Calcium/calmodulin-dependent protein kinase IV: A multifunctional enzyme and potential therapeutic target.

    Naz, Huma; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-05-01

    The calcium/calmodulin-dependent protein kinase IV (CAMKIV) belongs to the serine/threonine protein kinase family, and is primarily involved in transcriptional regulation in lymphocytes, neurons and male germ cells. CAMKIV operates the signaling cascade and regulates activity of several transcription activators by phosphorylation, which in turn plays pivotal roles in immune response, inflammation and memory consolidation. In this review, we tried to focus on different aspects of CAMKIV to understand the significance of this protein in the biological system. This enzyme is associated with varieties of disorders such as cerebral hypoxia, azoospermia, endometrial and ovarian cancer, systemic lupus, etc., and hence it is considered as a potential therapeutic target. Structure of CAMKIV is comprised of five distinct domains in which kinase domain is responsible for enzyme activity. CAMKIV is involved in varieties of cellular functions such as regulation of gene expression, T-cell maturation, regulation of survival phase of dendritic cells, bone growth and metabolism, memory consolidation, sperm motility, regulation of microtubule dynamics, cell-cycle progression and apoptosis. In this review, we performed an extensive analysis on structure, function and regulation of CAMKIV and associated diseases. PMID:26773169

  17. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

    Bailey, Zachary S.; Grinter, Michael B.; VandeVord, Pamela J.

    2016-01-01

    Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p processes. We have shown aberrant histone acetylation patterns involved in blast induced astrogliosis and cognitive impairments. Further understanding of their role in the injury progression may lead to novel therapeutic targets. PMID:27551260

  18. MicroRNA-137 Targets Carboxyl-terminal Binding Protein 1 in Melanoma Cell Lines

    Yu Deng, Hui Deng, Feng Bi, Jing Liu, Lynn T. Bemis, David Norris, Xiao-Jing Wang, Qinghong Zhang

    2011-01-01

    Full Text Available Carboxyl-terminal binding protein 1 (CtBP1 is a transcriptional co-repressor that represses expression of various tumor suppressor genes. In the present study, we identified miR-137 as a potential regulator of CtBP1 expression in melanoma cells. Expression of miR-137 in melanoma cell lines was found to inversely correlate with CtBP1 levels. Target Scan predicted a putative site for miR-137 within the CtBP1 3′ untranslated region (3′UTR at nt 710-716, which is highly conserved across species. To explore the mechanism of miR-137 targeting CtBP1, we performed an Argonaute 2 (Ago2-pull down assay, and miR-137 was identified in complex with CtBP1 mRNA. miR-137 suppressed CtBP1 3' UTR luciferase-reporter activity, and this effect was lost with deletion of the putative 3' UTR target-site. Consistent with the results of the reporter assay, ectopic expression of miR-137 reduced expression levels of CtBP1. Furthermore, expression of miR-137 increased the immediate downstream effectors of CtBP1, such as E-cadherin and Bax. The human miR-137 gene is located at chromosome 1p22, which has previously been determined to be a susceptive region for melanoma. This study suggests miR-137 may act as a tumor suppressor by directly targeting CtBP1 to inhibit epithelial-mesenchymal transition (EMT and inducing apoptosis of melanoma cells, thus illustrating a functional link between miR-137 and CtBP1 in melanoma development.

  19. Efficient acetylation of primary amines and amino acids in environmentally benign brine solution using acetyl chloride

    Kaushik Basu; Suchandra Chakraborty; Achintya Kumar Sarkar; Chandan Saha

    2013-05-01

    Acetyl chloride is one of the most commonly available and cheap acylating agent but its high reactivity and concomitant instability in water precludes its use to carry out acetylation in aqueous medium. The present methodology illustrates the efficient acetylation of primary amines and amino acids in brine solution by means of acetyl chloride under weakly basic condition in the presence of sodium acetate and/or triethyl amine followed by trituration with aqueous saturated bicarbonate solution. This effort represents the first efficient use of this most reactive but cheap acetylating agent to acetylate amines in excellent yields in aqueous medium. This is a potentially useful green chemical transformation where reaction takes place in environment-friendly brine solution leading to easy work-up and isolation of the amide derivatives. Mechanistic rationale of this methodology is also important.

  20. Acetylation modification regulates GRP78 secretion in colon cancer cells.

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  1. Acetylation modification regulates GRP78 secretion in colon cancer cells

    Li, Zongwei; Zhuang, Ming; Zhang, Lichao; Zheng, Xingnan; Yang, Peng; Li, Zhuoyu

    2016-01-01

    High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors. PMID:27460191

  2. Radio-peptides targeting g-protein coupled receptors in cancer: from bench to bed

    Full text of publication follows. In the development of targeted imaging and therapy agents the most important challenge and prerequisite is to identify and validate the molecular targets of any disease. The targets should be specific, relevant, easily accessible and highly expressed. In addition they should have no or at least very low expression in normal tissue. Among the many drug targets is the large family of G-protein coupled receptors (GPCRs). It is the most important family of marketed drugs and the basic accomplishments in the field were recognised by the award of the recent Nobel price in chemistry. GPCRs also play a role in cancer. Several of these receptors are massively over-expressed in different human tumors such as neuroendocrine tumors (over-expression of the somatostatin receptor family), prostate and breast tumors (bombesin receptor family), brain tumors (NK1 receptor) etc.. This allows to develop (nuclear, MRI, optical) probes for imaging and potentially targeted therapy (theragnostics). Natural ligands targeting GPCRs are often peptides. They need to be modified for metabolic stability, modified for labeling with radio-metals (conjugation of bifunctional chelators) or radio-halogens (prosthetic groups). Preserved biological integrity after modification and labeling needs to be assured, long retention times in the tumor is important, conferred by internalisation. Radio-metal labeling in particular needs to be reasonably fast and the radio metal complexes have to show high stability with regard to radio-metal release. These prerequisites will be discussed for somatostatin receptor based radio-peptides in particular. For a successful clinical application preclinical imaging and biodistribution in adequate animal models are mandatory. New tracers for positron emission tomography (PET) and single photon emission computed tomography (SPECT) will be presented for neuroendocrine tumors and prostate cancer. In particular radiolabeled antagonists will

  3. ATP-citrate lyase is required for production of cytosolic acetyl coenzyme A and development in Aspergillus nidulans.

    Hynes, Michael J; Murray, Sandra L

    2010-07-01

    Acetyl coenzyme A (CoA) is a central metabolite in carbon and energy metabolism and in the biosynthesis of cellular molecules. A source of cytoplasmic acetyl-CoA is essential for the production of fatty acids and sterols and for protein acetylation, including histone acetylation in the nucleus. In Saccharomyces cerevisiae and Candida albicans acetyl-CoA is produced from acetate by cytoplasmic acetyl-CoA synthetase, while in plants and animals acetyl-CoA is derived from citrate via ATP-citrate lyase. In the filamentous ascomycete Aspergillus nidulans, tandem divergently transcribed genes (aclA and aclB) encode the subunits of ATP-citrate lyase, and we have deleted these genes. Growth is greatly diminished on carbon sources that do not result in cytoplasmic acetyl-CoA, such as glucose and proline, while growth is not affected on carbon sources that result in the production of cytoplasmic acetyl-CoA, such as acetate and ethanol. Addition of acetate restores growth on glucose or proline, and this is dependent on facA, which encodes cytoplasmic acetyl-CoA synthetase, but not on the regulatory gene facB. Transcription of aclA and aclB is repressed by growth on acetate or ethanol. Loss of ATP-citrate lyase results in severe developmental effects, with the production of asexual spores (conidia) being greatly reduced and a complete absence of sexual development. This is in contrast to Sordaria macrospora, in which fruiting body formation is initiated but maturation is defective in an ATP-citrate lyase mutant. Addition of acetate does not repair these defects, indicating a specific requirement for high levels of cytoplasmic acetyl-CoA during differentiation. Complementation in heterokaryons between aclA and aclB deletions for all phenotypes indicates that the tandem gene arrangement is not essential. PMID:20495057

  4. Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

    Shirataki, H; Kaibuchi, K; Sakoda, T.; Kishida, S; Yamaguchi, T.; Wada, K.; Miyazaki, M.; Takai, Y.

    1993-01-01

    In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino...

  5. Targeted studies on the interaction of nicotine and morin molecules with amyloid β-protein.

    Boopathi, Subramaniam; Kolandaivel, Ponmalai

    2014-03-01

    Alzheimer's disease (AD) is a neurodegenerative disorder that occurs due to progressive deposition of amyloid β-protein (Aβ) in the brain. Stable conformations of solvated Aβ₁₋₄₂ protein were predicted by molecular dynamics (MD) simulation using the OPLSAA force field. The seven residue peptide (Lys-Leu-Val-Phe-Phe-Ala-Glu) Aβ₁₆₋₂₂ associated with AD was studied and reported in this paper. Since effective therapeutic agents have not yet been studied in detail, attention has focused on the use of natural products as effective anti-aggregation compounds, targeting the Aβ₁₋₄₂ protein directly. Experimental and theoretical investigation suggests that some compounds extracted from natural products might be useful, but detailed insights into the mechanism by which they might act remains elusive. The molecules nicotine and morin are found in cigarettes and beverages. Here, we report the results of interaction studies of these compounds at each hydrophobic residue of Aβ₁₆₋₂₂ peptide using the hybrid ONIOM (B3LYP/6-31G**:UFF) method. It was found that interaction with nicotine produced higher deformation in the Aβ₁₆₋₂₂ peptide than interaction with morin. MD simulation studies revealed that interaction of the nicotine molecule with the β-sheet of Aβ₁₆₋₂₂ peptide transforms the β-sheet to an α-helical structure, which helps prohibit the aggregation of Aβ-protein. PMID:24567151

  6. Novel Cycloheximide Derivatives Targeting the Moonlighting Protein Mip Exhibit Specific Antimicrobial Activity Against Legionella pneumophila

    Rasch, Janine; Theuerkorn, Martin; Ünal, Can; Heinsohn, Natascha; Tran, Stefan; Fischer, Gunter; Weiwad, Matthias; Steinert, Michael

    2015-01-01

    Macrophage infectivity potentiator (Mip) and Mip-like proteins are virulence factors in a wide range of pathogens including Legionella pneumophila. These proteins belong to the FK506 binding protein (FKBP) family of peptidyl-prolyl-cis/trans-isomerases (PPIases). In L. pneumophila, the PPIase activity of Mip is required for invasion of macrophages, transmigration through an in vitro lung–epithelial barrier, and full virulence in the guinea pig infection model. Additionally, Mip is a moonlighting protein that binds to collagen IV in the extracellular matrix. Here, we describe the development and synthesis of cycloheximide derivatives with adamantyl moieties as novel FKBP ligands, and analyze their effect on the viability of L. pneumophila and other bacteria. All compounds efficiently inhibited PPIase activity of the prototypic human FKBP12 as well as Mip with IC50-values as low as 180 nM and 1.7 μM, respectively. Five of these derivatives inhibited the growth of L. pneumophila at concentrations of 30–40 μM, but exhibited no effect on other tested bacterial species indicating a specific spectrum of antibacterial activity. The derivatives carrying a 3,5-dimethyladamantan-1-[yl]acetamide substitution (MT_30.32), and a 3-ethyladamantan-1-[yl]acetamide substitution (MT_30.51) had the strongest effects in PPIase- and liquid growth assays. MT_30.32 and MT_30.51 were also inhibitory in macrophage infection studies without being cytotoxic. Accordingly, by applying a combinatorial approach, we were able to generate novel, hybrid inhibitors consisting of cycloheximide and adamantane, two known FKBP inhibitors that interact with different parts of the PPIase domain, respectively. Interestingly, despite the proven Mip-inhibitory activity, the viability of a Mip-deficient strain was affected to the same degree as its wild type. Hence, we also propose that cycloheximide derivatives with adamantyl moieties are potent PPIase inhibitors with multiple targets in L

  7. False-Positive Rate Determination of Protein Target Discovery using a Covalent Modification- and Mass Spectrometry-Based Proteomics Platform

    Strickland, Erin C.; Geer, M. Ariel; Hong, Jiyong; Fitzgerald, Michael C.

    2014-01-01

    Detection and quantitation of protein-ligand binding interactions is important in many areas of biological research. Stability of proteins from rates of oxidation (SPROX) is an energetics-based technique for identifying the proteins targets of ligands in complex biological mixtures. Knowing the false-positive rate of protein target discovery in proteome-wide SPROX experiments is important for the correct interpretation of results. Reported here are the results of a control SPROX experiment in which chemical denaturation data is obtained on the proteins in two samples that originated from the same yeast lysate, as would be done in a typical SPROX experiment except that one sample would be spiked with the test ligand. False-positive rates of 1.2-2.2 % and protein targets of the drug, manassantin A. The impact of ion purity in the tandem mass spectral analyses and of background oxidation on the false-positive rate of protein target discovery using SPROX is also discussed.

  8. PCAF-primed EZH2 acetylation regulates its stability and promotes lung adenocarcinoma progression

    Wan Junhu; Chin Y Eugene; Zhang Hongquan; Zhan Jun; Li Shuai; Ma Ji; Xu Weizhi; Liu Chang; Xue Xiaowei; Xie Yuping; Fang Weigang

    2015-01-01

    Enhancer of zeste homolog 2 ( EZH2 ) is a key epigenetic regulator that catalyzes the trimethyla-tion of H3K27 and is modulated by post-translational modifications (PTMs). However, the precise regulation of EZH2 PTMs remains elusive. We, herein, report that EZH2 is acetylated by acetyltransferase P300/CBP-associat-ed factor (PCAF) and is deacetylated by deacetylase SIRT1. We identified that PCAF interacts with and acetylates EZH2 mainly at lysine 348 (K348). Mechanistically, K348 acetylation decreases EZH2 phosphorylation at T345 and T487 and increases EZH2 stability without disrupting the formation of polycomb repressive complex 2 ( PRC2 ) . Functionally, EZH2 K348 acetylation enhances its capacity in suppression of the target genes and promotes lung cancer cell migration and invasion. Further, elevated EZH2 K348 acetylation in lung adenocarcinoma patients pre-dicts a poor prognosis. Our findings define a new mechanism underlying EZH2 modulation by linking EZH2 acety-lation to its phosphorylation that stabilizes EZH2 and promotes lung adenocarcinoma progression.

  9. Sulphoraphane, a naturally occurring isothiocyanate induces apoptosis in breast cancer cells by targeting heat shock proteins

    Sarkar, Ruma; Mukherjee, Sutapa [Department of Environmental Carcinogenesis and Toxicology, Chittaranjan National Cancer Institute, 37, SP Mukherjee Road, Kolkata 700 026 (India); Biswas, Jaydip [Chittaranjan National Cancer Institute, 37, SP Mukherjee Road, Kolkata 700 026 (India); Roy, Madhumita, E-mail: mitacnci@yahoo.co.in [Department of Environmental Carcinogenesis and Toxicology, Chittaranjan National Cancer Institute, 37, SP Mukherjee Road, Kolkata 700 026 (India)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer HSPs (27, 70 and 90) and HSF1 are overexpressed in MCF-7 and MDA-MB-231 cells. Black-Right-Pointing-Pointer Sulphoraphane, a natural isothiocyanate inhibited HSPs and HSF1 expressions. Black-Right-Pointing-Pointer Inhibition of HSPs and HSF1 lead to regulation of apoptotic proteins. Black-Right-Pointing-Pointer Alteration of apoptotic proteins activate of caspases particularly caspase 3 and 9 leading to induction of apoptosis. Black-Right-Pointing-Pointer Alteration of apoptotic proteins induce caspases leading to induction of apoptosis. -- Abstract: Heat shock proteins (HSPs) are involved in protein folding, aggregation, transport and/or stabilization by acting as a molecular chaperone, leading to inhibition of apoptosis by both caspase dependent and/or independent pathways. HSPs are overexpressed in a wide range of human cancers and are implicated in tumor cell proliferation, differentiation, invasion and metastasis. HSPs particularly 27, 70, 90 and the transcription factor heat shock factor1 (HSF1) play key roles in the etiology of breast cancer and can be considered as potential therapeutic target. The present study was designed to investigate the role of sulphoraphane, a natural isothiocyanate on HSPs (27, 70, 90) and HSF1 in two different breast cancer cell lines MCF-7 and MDA-MB-231 cells expressing wild type and mutated p53 respectively, vis-a-vis in normal breast epithelial cell line MCF-12F. It was furthermore investigated whether modulation of HSPs and HSF1 could induce apoptosis in these cells by altering the expressions of p53, p21 and some apoptotic proteins like Bcl-2, Bax, Bid, Bad, Apaf-1 and AIF. Sulphoraphane was found to down-regulate the expressions of HSP70, 90 and HSF1, though the effect on HSP27 was not pronounced. Consequences of HSP inhibition was upregulation of p21 irrespective of p53 status. Bax, Bad, Apaf-1, AIF were upregulated followed by down-regulation of Bcl-2 and this effect was prominent

  10. Detection of prion protein in urine-derived injectable fertility products by a targeted proteomic approach.

    Alain Van Dorsselaer

    Full Text Available BACKGROUND: Iatrogenic transmission of human prion disease can occur through medical or surgical procedures, including injection of hormones such as gonadotropins extracted from cadaver pituitaries. Annually, more than 300,000 women in the United States and Canada are prescribed urine-derived gonadotropins for infertility. Although menopausal urine donors are screened for symptomatic neurological disease, incubation of Creutzfeldt-Jakob disease (CJD is impossible to exclude by non-invasive testing. Risk of carrier status of variant CJD (vCJD, a disease associated with decades-long peripheral incubation, is estimated to be on the order of 100 per million population in the United Kingdom. Studies showing infectious prions in the urine of experimental animals with and without renal disease suggest that prions could be present in asymptomatic urine donors. Several human fertility products are derived from donated urine; recently prion protein has been detected in preparations of human menopausal gonadotropin (hMG. METHODOLOGY/PRINCIPAL FINDINGS: Using a classical proteomic approach, 33 and 34 non-gonadotropin proteins were identified in urinary human chorionic gonadotropin (u-hCG and highly-purified urinary human menopausal gonadotropin (hMG-HP products, respectively. Prion protein was identified as a major contaminant in u-hCG preparations for the first time. An advanced prion protein targeted proteomic approach was subsequently used to conduct a survey of gonadotropin products; this approach detected human prion protein peptides in urine-derived injectable fertility products containing hCG, hMG and hMG-HP, but not in recombinant products. CONCLUSIONS/SIGNIFICANCE: The presence of protease-sensitive prion protein in urinary-derived injectable fertility products containing hCG, hMG, and hMG-HP suggests that prions may co-purify in these products. Intramuscular injection is a relatively efficient route of transmission of human prion disease, and

  11. Sulphoraphane, a naturally occurring isothiocyanate induces apoptosis in breast cancer cells by targeting heat shock proteins

    Highlights: ► HSPs (27, 70 and 90) and HSF1 are overexpressed in MCF-7 and MDA-MB-231 cells. ► Sulphoraphane, a natural isothiocyanate inhibited HSPs and HSF1 expressions. ► Inhibition of HSPs and HSF1 lead to regulation of apoptotic proteins. ► Alteration of apoptotic proteins activate of caspases particularly caspase 3 and 9 leading to induction of apoptosis. ► Alteration of apoptotic proteins induce caspases leading to induction of apoptosis. -- Abstract: Heat shock proteins (HSPs) are involved in protein folding, aggregation, transport and/or stabilization by acting as a molecular chaperone, leading to inhibition of apoptosis by both caspase dependent and/or independent pathways. HSPs are overexpressed in a wide range of human cancers and are implicated in tumor cell proliferation, differentiation, invasion and metastasis. HSPs particularly 27, 70, 90 and the transcription factor heat shock factor1 (HSF1) play key roles in the etiology of breast cancer and can be considered as potential therapeutic target. The present study was designed to investigate the role of sulphoraphane, a natural isothiocyanate on HSPs (27, 70, 90) and HSF1 in two different breast cancer cell lines MCF-7 and MDA-MB-231 cells expressing wild type and mutated p53 respectively, vis-à-vis in normal breast epithelial cell line MCF-12F. It was furthermore investigated whether modulation of HSPs and HSF1 could induce apoptosis in these cells by altering the expressions of p53, p21 and some apoptotic proteins like Bcl-2, Bax, Bid, Bad, Apaf-1 and AIF. Sulphoraphane was found to down-regulate the expressions of HSP70, 90 and HSF1, though the effect on HSP27 was not pronounced. Consequences of HSP inhibition was upregulation of p21 irrespective of p53 status. Bax, Bad, Apaf-1, AIF were upregulated followed by down-regulation of Bcl-2 and this effect was prominent in MCF-7 than in MDA-MB-231. However, very little change in the expression of Bid was observed. Alteration in Bcl-2 Bax

  12. Levels of histone acetylation in thyroid tumors.

    Puppin, Cinzia; Passon, Nadia; Lavarone, Elisa; Di Loreto, Carla; Frasca, Francesco; Vella, Veronica; Vigneri, Riccardo; Damante, Giuseppe

    2011-08-12

    Histone acetylation is a major mechanism to regulate gene transcription. This post-translational modification is modified in cancer cells. In various tumor types the levels of acetylation at several histone residues are associated to clinical aggressiveness. By using immunohistochemistry we show that acetylated levels of lysines at positions 9-14 of H3 histone (H3K9-K14ac) are significantly higher in follicular adenomas (FA), papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC) and undifferentiated carcinomas (UC) than in normal tissues (NT). Similar data have been obtained when acetylated levels of lysine 18 of H3 histone (H3K18ac) were evaluated. In this case, however, no difference was observed between NT and UC. When acetylated levels of lysine 12 of H4 histone (H4K12ac) were evaluated, only FA showed significantly higher levels in comparison with NT. These data indicate that modification histone acetylation is an early event along thyroid tumor progression and that H3K18 acetylation is switched off in the transition between differentiated and undifferentiated thyroid tumors. By using rat thyroid cell lines that are stably transfected with doxycyclin-inducible oncogenes, we show that the oncoproteins RET-PTC, RAS and BRAF increase levels of H3K9-K14ac and H3K18ac. In the non-tumorigenic rat thyroid cell line FRTL-5, TSH increases levels of H3K18ac. However, this hormone decreases levels of H3K9-K14ac and H4K12ac. In conclusion, our data indicate that neoplastic transformation and hormonal stimulation can modify levels of histone acetylation in thyroid cells. PMID:21763277

  13. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.;

    2015-01-01

    variable regions, where all variants bind HLA. These regions, although variable, can thus be considered stable in terms of HLA binding and represent valuable vaccine targets. Results: We applied this method to predict CD8+ T-cell targets in influenza A H7N9 hemagglutinin and significantly increased the...... number of potential vaccine targets compared to the number of targets discovered using the traditional approach where low-frequency peptides are excluded. Conclusions: We developed a webserver with an intuitive visualization scheme for summarizing the T cell-based antigenic potential of any given protein...

  14. A targeted library screen reveals a new inhibitor scaffold for protein kinase D.

    Manuj Tandon

    Full Text Available Protein kinase D (PKD has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.

  15. A deep learning framework for modeling structural features of RNA-binding protein targets.

    Zhang, Sai; Zhou, Jingtian; Hu, Hailin; Gong, Haipeng; Chen, Ligong; Cheng, Chao; Zeng, Jianyang

    2016-02-29

    RNA-binding proteins (RBPs) play important roles in the post-transcriptional control of RNAs. Identifying RBP binding sites and characterizing RBP binding preferences are key steps toward understanding the basic mechanisms of the post-transcriptional gene regulation. Though numerous computational methods have been developed for modeling RBP binding preferences, discovering a complete structural representation of the RBP targets by integrating their available structural features in all three dimensions is still a challenging task. In this paper, we develop a general and flexible deep learning framework for modeling structural binding preferences and predicting binding sites of RBPs, which takes (predicted) RNA tertiary structural information into account for the first time. Our framework constructs a unified representation that characterizes the structural specificities of RBP targets in all three dimensions, which can be further used to predict novel candidate binding sites and discover potential binding motifs. Through testing on the real CLIP-seq datasets, we have demonstrated that our deep learning framework can automatically extract effective hidden structural features from the encoded raw sequence and structural profiles, and predict accurate RBP binding sites. In addition, we have conducted the first study to show that integrating the additional RNA tertiary structural features can improve the model performance in predicting RBP binding sites, especially for the polypyrimidine tract-binding protein (PTB), which also provides a new evidence to support the view that RBPs may own specific tertiary structural binding preferences. In particular, the tests on the internal ribosome entry site (IRES) segments yield satisfiable results with experimental support from the literature and further demonstrate the necessity of incorporating RNA tertiary structural information into the prediction model. The source code of our approach can be found in https

  16. Comparative proteome analysis of Saccharomyces cerevisiae: A global overview of in vivo targets of the yeast activator protein 1

    Jun He

    2012-06-01

    Full Text Available Abstract Background The activity of the yeast activator protein 1 (Yap1p increases under stress conditions, which leads to enhanced transcription of a number of genes encoding protective enzymes or other proteins. To obtain a global overview of changes in expression of Yap1p-targeted proteins, we compared a Yap1p-overexpressing transformant with a control transformant by triplicate analysis of the proteome using two-dimensional gel electrophoresis (2-DE. Proteins of interest were identified using MALDI-MS or LC-MS/MS. Results The relative quantities of 55 proteins were elevated significantly upon overexpression of Yap1p, and most of these proteins were found to have a Yap1p-binding site upstream of their coding sequences. Interestingly, the main metabolic enzymes in the glycolysis and pyruvate-ethanol pathways showed a significant increase in the Yap1p-overexpressing transformant. Moreover, a comparison of our proteome data with transcriptome data from the literature suggested which proteins were regulated at the level of the proteome, and which proteins were regulated at the level of the transcriptome. Eight proteins involved in stress response, including seven heat-shock and chaperone proteins, were significantly more abundant in the Yap1p-overexpressing transformant. Conclusions We have investigated the general protein composition in Yap1p-overexpressing S. cerevisiae using proteomic techniques, and quantified the changes in the expression of the potential Yap1p-targeted proteins. Identification of the potential Yap1p targets and analysis of their role in cellular processes not only give a global overview of the ubiquitous cellular changes elicited by Yap1p, but also provide the framework for understanding the mechanisms behind Yap1p-regulated stress response in yeast.

  17. A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting

    Wang SM

    2015-04-01

    Full Text Available Shan-Mei Wang,1,* Xian He,2,* Nan Li,1,* Feng Yu,3 Yang Hu,1 Liu-Sheng Wang,1 Peng Zhang,4 Yu-Kui Du,1 Shan-Shan Du,1 Zhao-Fang Yin,1 Ya-Ru Wei,1 Xavier Mulet,5 Greg Coia,6 Dong Weng,1 Jian-Hua He,3 Min Wu,7 Hui-Ping Li1 1Department of Respiratory Medicine, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, 2School of Medicine, Suzhou University, SuZhou, 3Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 4Department of Chest Surgery, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China; 5CSIRO (Commonwealth Scientific and Industrial Research Materials Science and Engineering, Clayton, 6CSIRO Materials Science and Engineering, Parkville, Melbourne, VIC, Australia; 7Department of Basic Sciences, University of North Dakota, Grand Forks, ND, USA *These authors contributed equally to this work Abstract: Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies’ potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high

  18. Tip60-mediated acetylation activates transcription independent apoptotic activity of Abl

    Pandita Tej K

    2011-07-01

    Full Text Available Abstract Background The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR, is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown. Results We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (KXXK--X3-5--SGS is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase. Conclusion Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function.

  19. The periplasmic protein TolB as a potential drug target in Pseudomonas aeruginosa.

    Alessandra Lo Sciuto

    Full Text Available The Gram-negative bacterium Pseudomonas aeruginosa is one of the most dreaded pathogens in the hospital setting, and represents a prototype of multi-drug resistant "superbug" for which effective therapeutic options are very limited. The identification and characterization of new cellular functions that are essential for P. aeruginosa viability and/or virulence could drive the development of anti-Pseudomonas compounds with novel mechanisms of action. In this study we investigated whether TolB, the periplasmic component of the Tol-Pal trans-envelope protein complex of Gram-negative bacteria, represents a potential drug target in P. aeruginosa. By combining conditional mutagenesis with the analysis of specific pathogenicity-related phenotypes, we demonstrated that TolB is essential for P. aeruginosa growth, both in laboratory and clinical strains, and that TolB-depleted P. aeruginosa cells are strongly defective in cell-envelope integrity, resistance to human serum and several antibiotics, as well as in the ability to cause infection and persist in an insect model of P. aeruginosa infection. The essentiality of TolB for P. aeruginosa growth, resistance and pathogenicity highlights the potential of TolB as a novel molecular target for anti-P. aeruginosa drug discovery.

  20. The periplasmic protein TolB as a potential drug target in Pseudomonas aeruginosa.

    Lo Sciuto, Alessandra; Fernández-Piñar, Regina; Bertuccini, Lucia; Iosi, Francesca; Superti, Fabiana; Imperi, Francesco

    2014-01-01

    The Gram-negative bacterium Pseudomonas aeruginosa is one of the most dreaded pathogens in the hospital setting, and represents a prototype of multi-drug resistant "superbug" for which effective therapeutic options are very limited. The identification and characterization of new cellular functions that are essential for P. aeruginosa viability and/or virulence could drive the development of anti-Pseudomonas compounds with novel mechanisms of action. In this study we investigated whether TolB, the periplasmic component of the Tol-Pal trans-envelope protein complex of Gram-negative bacteria, represents a potential drug target in P. aeruginosa. By combining conditional mutagenesis with the analysis of specific pathogenicity-related phenotypes, we demonstrated that TolB is essential for P. aeruginosa growth, both in laboratory and clinical strains, and that TolB-depleted P. aeruginosa cells are strongly defective in cell-envelope integrity, resistance to human serum and several antibiotics, as well as in the ability to cause infection and persist in an insect model of P. aeruginosa infection. The essentiality of TolB for P. aeruginosa growth, resistance and pathogenicity highlights the potential of TolB as a novel molecular target for anti-P. aeruginosa drug discovery. PMID:25093328

  1. The centrosome protein NEDD1 as a potential pharmacological target to induce cell cycle arrest

    Etievant Chantal

    2009-02-01

    Full Text Available Abstract Background NEDD1 is a protein that binds to the gamma-tubulin ring complex, a multiprotein complex at the centrosome and at the mitotic spindle that mediates the nucleation of microtubules. Results We show that NEDD1 is expressed at comparable levels in a variety of tumor-derived cell lines and untransformed cells. We demonstrate that silencing of NEDD1 expression by treatment with siRNA has differential effects on cells, depending on their status of p53 expression: p53-positive cells arrest in G1, whereas p53-negative cells arrest in mitosis with predominantly aberrant monopolar spindles. However, both p53-positive and -negative cells arrest in mitosis if treated with low doses of siRNA against NEDD1 combined with low doses of the inhibitor BI2536 against the mitotic kinase Plk1. Simultaneous reduction of NEDD1 levels and inhibition of Plk1 act in a synergistic manner, by potentiating the anti-mitotic activity of each treatment. Conclusion We propose that NEDD1 may be a promising target for controlling cell proliferation, in particular if targeted in combination with Plk1 inhibitors.

  2. Virtual screening of the inhibitors targeting at the viral protein 40 of Ebola virus

    V.Karthick; N.Nagasundaram; C.George Priya Doss; Chiranjib Chakraborty; R.Siva; Aiping Lu; Ge Zhang

    2016-01-01

    Background:The Ebola virus is highly pathogenic and destructive to humans and other primates.The Ebola virus encodes viral protein 40 (VP40),which is highly expressed and regulates the assembly and release of viral particles in the host cell.Because VP40 plays a prominent role in the life cycle of the Ebola virus,it is considered as a key target for antiviral treatment.However,there is currently no FDA-approved drug for treating Ebola virus infection,resulting in an urgent need to develop effective antiviral inhibitors that display good safety profiles in a short duration.Methods:This study aimed to screen the effective lead candidate against Ebola infection.First,the lead molecules were filtered based on the docking score.Second,Lipinski rule of five and the other drug likeliness properties are predicted to assess the safety profile of the lead candidates.Finally,molecular dynamics simulations was performed to validate the lead compound.Results:Our results revealed that emodin-8-beta-D-glucoside from the Traditional Chinese Medicine Database (TCMD) represents an active lead candidate that targets the Ebola virus by inhibiting the activity of VP40,and displays good pharmacokinetic properties.Conclusion:This report will considerably assist in the development of the competitive and robust antiviral agents against Ebola infection.

  3. Prediction of Effective Drug Combinations by Chemical Interaction, Protein Interaction and Target Enrichment of KEGG Pathways

    Lei Chen

    2013-01-01

    Full Text Available Drug combinatorial therapy could be more effective in treating some complex diseases than single agents due to better efficacy and reduced side effects. Although some drug combinations are being used, their underlying molecular mechanisms are still poorly understood. Therefore, it is of great interest to deduce a novel drug combination by their molecular mechanisms in a robust and rigorous way. This paper attempts to predict effective drug combinations by a combined consideration of: (1 chemical interaction between drugs, (2 protein interactions between drugs’ targets, and (3 target enrichment of KEGG pathways. A benchmark dataset was constructed, consisting of 121 confirmed effective combinations and 605 random combinations. Each drug combination was represented by 465 features derived from the aforementioned three properties. Some feature selection techniques, including Minimum Redundancy Maximum Relevance and Incremental Feature Selection, were adopted to extract the key features. Random forest model was built with its performance evaluated by 5-fold cross-validation. As a result, 55 key features providing the best prediction result were selected. These important features may help to gain insights into the mechanisms of drug combinations, and the proposed prediction model could become a useful tool for screening possible drug combinations.

  4. How nonspecifically DNA-binding proteins search for the target in crowded environments.

    Ma, Yiding; Chen, Yuhao; Yu, Wancheng; Luo, Kaifu

    2016-03-28

    We investigate how a tracer particle searches a target located in DNA modeled by a stiff chain in crowded environments using theoretical analysis and Langevin dynamics simulations. First, we show that the three-dimensional (3D) diffusion coefficient of the tracer only depends on the density of crowders ϕ, while its one-dimensional (1D) diffusion coefficient is affected by not only ϕ but also the nonspecific binding energy ε. With increasing ϕ and ε, no obvious change in the average 3D diffusion time is observed, while the average 1D sliding time apparently increases. We propose theoretically that the 1D sliding of the tracer along the chain could be well captured by the Kramers' law of escaping rather than the Arrhenius law, which is verified directly by the simulations. Finally, the average search time increases monotonously with an increase in ϕ while it has a minimum as a function of ε, which could be understood from the different behaviors of the average number of search rounds with the increasing ϕ or ε. These results provide a deeper understanding of the role of facilitated diffusion in target search of proteins on DNA in vivo. PMID:27036479

  5. Molecular crowding effect on dynamics of DNA-binding proteins search for their targets

    Liu, Lin; Luo, Kaifu

    2014-12-01

    DNA-binding proteins locate and bind their target sequences positioned on DNA in crowded environments, but the molecular crowding effect on this search process is not clear. Using analytical techniques and Langevin dynamics simulations in two dimensions (2D), we find that the essential physics for facilitated diffusion in 2D search and 3D search is the same. We observe that the average search times have minima at the same optimal nonspecific binding energy for the cases with and without the crowding particle. Moreover, the molecular crowding increases the search time by increasing the average search rounds and the one-dimensional (1D) sliding time of a round, but almost not changing the average 2D diffusion time of a round. In addition, the fraction of 1D sliding time out of the total search time increases with increasing the concentration of crowders. For 2D diffusion, the molecular crowding decreases the jumping length and narrows its distribution due to the cage effect from crowders. These results shed light on the role of facilitated diffusion in DNA targeting kinetics in living cells.

  6. Dynamic Regulation of APE1/Ref-1 as a Therapeutic Target Protein

    Choi, Sunga; Joo, Hee Kyoung

    2016-01-01

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that plays a central role in the cellular response to DNA damage and redox regulation against oxidative stress. APE1/Ref-1 functions in the DNA base excision repair pathway, the redox regulation of several transcription factors, and the control of intracellular redox status through the inhibition of reactive oxygen species (ROS) production. APE1/Ref-1 is predominantly localized in the nucleus; however, its subcellular localization is dynamically regulated and it may be found in the mitochondria or elsewhere in the cytoplasm. Studies have identified a nuclear localization signal and a mitochondrial target sequence in APE1/Ref-1, as well as the involvement of the nuclear export system, as determinants of APE1/Ref-1 subcellular distribution. Recently, it was shown that APE1/Ref-1 is secreted in response to hyperacetylation at specific lysine residues. Additionally, post-translational modifications such as phosphorylation, S-nitrosation, and ubiquitination appear to play a role in fine-tuning the activities and subcellular localization of APE1/Ref-1. In this review, we will introduce the multifunctional role of APE1/Ref-1 and its potential usefulness as a therapeutic target in cancer and cardiovascular disease. PMID:27231670

  7. Design, synthesis and evaluation of Novel 1-(Substituted Acetyl-4-(10-Bromo-8-Chloro-5,6-Dihydro-11H-Benzo[5,6]Cyclohepta[1,2-B]Pyridine-11-Ylidenepiperidines as antitumor agents and farnesyl protein transferase inhibitors

    Gatne P

    2010-01-01

    Full Text Available Eight novel 1-(substituted acetyl-4-(10-bromo-8-chloro-5,6-dihydro-11H-benzo[5,6] cyclohepta [1,2-b] pyridine-11-ylidenepiperidines were designed by incorporating zinc binding groups to enhance activity. The designed molecules were synthesized and were evaluated for antitumor activity in vitro in five cell lines and for farnesyl protein transferase inhibition. Test compounds (6a-h exhibited antitumor activity in most of the cell lines but were less potent than adriamycin. Compound 6e was most active with IC 50 values of <15 μM in two cell lines tested. Test compounds also exhibited potent FPT inhibitory activity and 6c was most potent with IC 50 value of <30 μM.

  8. Protein kinase A of Leishmania amazonensis as a potential target for methoxy-amidine.

    Genestra, M; Echevarria, A; Cysne-Finkelstein, L; Leon, L L

    2001-11-01

    Cyclic 3',5'-adenosine monophosphate (cAMP) is one of the most important signaling molecules for cell growth and differentiation in several systems including protozoal parasites such as Trypanosoma cruzi and Leishmania species. The most important event during Leishmania developmental cycle is the differentiation of procyclic into metacyclic promastigotes, which is associated with the appearance of pathogenicity. As previously demonstrated Leishmania amazonensis metacyclogenesis is associated with an increase of a protein kinase A activity, and therefore further studies on the activity of this phosphorylating enzyme as a target for chemotherapy were performed. Among several amidine derivatives tested by the authors against trypanosomatids (T. cruzi, T. evansi and L. amazonensis) the most effective compounds was defined as that with a methoxy group as substituent. In this work the inhibitory effect of this derivative on the phosphorylating activity of cAMP-dependent protein kinase (PKA) of promastigotes (containing high amounts of metacyclic forms) and axenic amastigotes of L. amazonensis is demonstrated. Soluble fractions (SF) and enriched membrane fractions (MF) were submitted to anion exchange chromatography in a DEAE-cellulose column and the collected fractions used to evaluate the phosphorylating activity associated with cAMP, in the presence/absence of methoxy-amidine and pentamidine (CAS 100-33-4), the latter being used as reference drug. PMID:11765595

  9. Preferential binding of growth inhibitory prostaglandins by the target protein of a carcinogen

    Khan, S.H.; Sorof, S. (Fox Chase Cancer Center, Philadelphia, PA (United States))

    1990-12-01

    Liver fatty acid binding protein (L-FABP) is the principal target protein of the hepatic carcinogen N-(2-fluorenyl)acetamide (2-acetylaminofluorene) in rat liver. In addition, the cyclopentenone prostaglandins (PG), PGA, PGJ{sub 2}, and {Delta}{sup 12}-PGJ{sub 2}, inhibit the growth of many cell types in vitro. This report describes the preferential binding of the growth inhibitory prostaglandins by L-FABP and the reversible inhibition of thymidine incorporation into DNA by PGA{sub 2} and {Delta}{sup 12}-PGJ{sub 2} in primary cultures of purified rat hepatocytes. As a model ligand, ({sup 3}H)PGA{sub 1} bound to L-FABP specifically, reversibly, rapidly, and with high affinity. Its dissociation constants were 134 nM (high affinity) and 3.6 {mu}M (low affinity). The high-affinity finding of ({sup 3}H)PGA{sup 1} correlated with their growth inhibitory activities reported previously and here. The in vitro actions of L-FABP are compatible with those of a specific and dissociable carrier of growth inhibitory prostaglandins in rat hepatocytes and suggest that the carcinogen may usurp the cellular machinery of the growth inhibitory prostaglandins.

  10. Proteins mediating the Neospora caninum-host cell interaction as targets for vaccination.

    Hemphill, Andrew; Debache, Karim; Monney, Thierry; Schorer, Michelle; Guionaud, Christophe; Alaeddine, Ferial; Mueller, Norbert; Mueller, Joachim

    2013-01-01

    Neospora caninum is an apicomplexan parasite that is capable of infecting, a wide range of tissues. The fact that Neospora represents an important abortion-causing parasite in cattle has transformed neosporosis research from an earlier, rather esoteric field, to a significant research topic, and considerable investments have been made in the last years to develop an efficacious vaccine or other means of intervention that would prevent infection and abortion due to N. caninum infection in cattle. Antigenic molecules associated with proteins involved in adhesion/invasion or other parasite-host-cell interaction processes can confer protection against Neospora caninum infection, and such proteins represent valuable targets for the development of a vaccine to limit economical losses due to neosporosis. Although not ideal, small laboratory animal models that mimic cerebral infection, acute disease and fetal loss upon infection during pregnancy have been used for the assessment of vaccine candidates, in parallel with studies on experimental infections in cattle. Herein, we review and critically assess these vaccination approaches and discuss potential options for improvements. PMID:23276967

  11. Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

    Hast, Michael A.; Nichols, Connie B.; Armstrong, Stephanie M.; Kelly, Shannon M.; Hellinga, Homme W.; Alspaugh, J. Andrew; Beese, Lorena S. (Duke)

    2012-09-17

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

  12. Heat shock protein 90: a pathophysiological factor and novel treatment target in autoimmune bullous skin diseases.

    Tukaj, Stefan; Zillikens, Detlef; Kasperkiewicz, Michael

    2015-08-01

    The chaperone heat shock protein 90 (Hsp90), a cell stress-inducible molecule that regulates activity of many client proteins responsible for cellular growth, differentiation and apoptosis, has been proposed as an important therapeutic target in patients with malignancies. More recently, its active participation in (auto)immune processes has been recognized as evidenced by amelioration of inflammatory disease pathways through pharmacological inhibition of Hsp90 in rodent models of autoimmune encephalomyelitis, rheumatoid arthritis and systemic lupus erythematosus. Based on own current research results, this viewpoint essay provides important insights that Hsp90 is also involved as a notable pathophysiological factor in autoimmune blistering dermatoses including epidermolysis bullosa acquisita, bullous pemphigoid and possibly dermatitis herpetiformis. The observed in vitro, ex vivo and in vivo efficacy of anti-Hsp90 treatment in experimental models of autoimmune bullous diseases and its underlying multimodal anti-inflammatory mechanisms of interference with key contributors to autoimmune-mediated blister formation supports the introduction of selective non-toxic Hsp90 inhibitors into the clinical setting for the treatment of patients with these disorders. PMID:25980533

  13. Prediction of TF target sites based on atomistic models of protein-DNA complexes

    Collado-Vides Julio

    2008-10-01

    Full Text Available Abstract Background The specific recognition of genomic cis-regulatory elements by transcription factors (TFs plays an essential role in the regulation of coordinated gene expression. Studying the mechanisms determining binding specificity in protein-DNA interactions is thus an important goal. Most current approaches for modeling TF specific recognition rely on the knowledge of large sets of cognate target sites and consider only the information contained in their primary sequence. Results Here we describe a structure-based methodology for predicting sequence motifs starting from the coordinates of a TF-DNA complex. Our algorithm combines information regarding the direct and indirect readout of DNA into an atomistic statistical model, which is used to estimate the interaction potential. We first measure the ability of our method to correctly estimate the binding specificities of eight prokaryotic and eukaryotic TFs that belong to different structural superfamilies. Secondly, the method is applied to two homology models, finding that sampling of interface side-chain rotamers remarkably improves the results. Thirdly, the algorithm is compared with a reference structural method based on contact counts, obtaining comparable predictions for the experimental complexes and more accurate sequence motifs for the homology models. Conclusion Our results demonstrate that atomic-detail structural information can be feasibly used to predict TF binding sites. The computational method presented here is universal and might be applied to other systems involving protein-DNA recognition.

  14. Preferential binding of growth inhibitory prostaglandins by the target protein of a carcinogen

    Liver fatty acid binding protein (L-FABP) is the principal target protein of the hepatic carcinogen N-(2-fluorenyl)acetamide (2-acetylaminofluorene) in rat liver. In addition, the cyclopentenone prostaglandins (PG), PGA, PGJ2, and Δ12-PGJ2, inhibit the growth of many cell types in vitro. This report describes the preferential binding of the growth inhibitory prostaglandins by L-FABP and the reversible inhibition of thymidine incorporation into DNA by PGA2 and Δ12-PGJ2 in primary cultures of purified rat hepatocytes. As a model ligand, [3H]PGA1 bound to L-FABP specifically, reversibly, rapidly, and with high affinity. Its dissociation constants were 134 nM (high affinity) and 3.6 μM (low affinity). The high-affinity finding of [3H]PGA1 correlated with their growth inhibitory activities reported previously and here. The in vitro actions of L-FABP are compatible with those of a specific and dissociable carrier of growth inhibitory prostaglandins in rat hepatocytes and suggest that the carcinogen may usurp the cellular machinery of the growth inhibitory prostaglandins

  15. Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition.

    Azmi, Asfar S; Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Senapedis, William; Baloglu, Erkan; Bollig-Fischer, Aliccia; Dyson, Gregory; Kauffman, Michael; Landesman, Yosef; Shacham, Sharon; Philip, Philip A; Mohammad, Ramzi M

    2015-01-01

    Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (-ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15 mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore. PMID:26536918

  16. Intracellular Acetyl Unit Transport in Fungal Carbon Metabolism

    Strijbis, K.; Distel, B.

    2010-01-01

    Acetyl coenzyme A (acetyl-CoA) is a central metabolite in carbon and energy metabolism. Because of its amphiphilic nature and bulkiness, acetyl-CoA cannot readily traverse biological membranes. In fungi, two systems for acetyl unit transport have been identified: a shuttle dependent on the carrier carnitine and a (peroxisomal) citrate synthase-dependent pathway. In the carnitine-dependent pathway, carnitine acetyltransferases exchange the CoA group of acetyl-CoA for carnitine, thereby forming...

  17. Structure of drug-target proteins determined by both X-ray and neutron diffraction

    Crystallography enables us to obtain accurate atomic positions within proteins. High resolution X-ray crystallography provides information for most of the atoms comprising a protein, with the exception of hydrogens. Neutron diffraction data can provide information of the location of hydrogen atoms to the structural information determined by X-ray crystallography. Here, we show the recent of the structural determination of drug-target proteins, porcine pancreatic elastase (PPE) and human immuno-deficiency virus type-1 protease (HIV-PR) by both X-ray and neutron diffraction. The structure of porcine pancreatic elastase with its potent inhibitor (FR13080) was determined to 0.94A resolution by X-ray diffraction and 1.75 A resolution by neutron diffraction. It was found that there are two characteristic hydrogen bonding interactions in which hydrogen atoms were confirmed. One is located between a catalytic aspartate and histidine, another is involved in the inhibitor recognition site. The structure of HIV-PR with its potent inhibitor (KNI-272) was also determined to 0.93 A resolution by X-ray diffraction and 2.3 A resolution by neutron diffraction. The ionization state of the catalytic residues were clarified to show that Asp125 is protonated and Asp25 is deprotonated. The ionization state and the location of hydrogen atoms of the catalytic residue in HIV-PR were firstly determined by neutron diffraction. Furthermore, collaborative use of both X-ray and neutron to identify the location of ambiguous hydrogen atoms will be shown. (author)

  18. Acetylated α-Tubulin Regulated by N-Acetyl-Seryl-Aspartyl-Lysyl-Proline(Ac-SDKP) Exerts the Anti-fibrotic Effect in Rat Lung Fibrosis Induced by Silica

    Xiaojun, Wang; Yan, Liu; Hong, Xu; Xianghong, Zhang; Shifeng, Li; Dingjie, Xu; Xuemin, Gao; Lijuan, Zhang; Bonan, Zhang; Zhongqiu, Wei; Ruimin, Wang; Brann, Darrell; Fang, Yang

    2016-01-01

    Silicosis is the most serious occupational disease in China. The objective of this study was to screen various proteins related to mechanisms of the pathogenesis of silicosis underlying the anti-fibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) using proteomic profile analysis. We also aimed to explore a potential mechanism of acetylated α-tubulin (α-Ac-Tub) regulation by Ac-SDKP. Two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to assess the different protein expression profiles between control and silicosis rats treated with or without Ac-SDKP. Twenty-nine proteins were identified to be potentially involved in the progression of silicosis and the anti-fibrotic effect of Ac-SDKP. Our current study finds that 1) the lost expression of Ac-Tub-α may be a new mechanism in rat silicosis; 2) treatment of silicotic rats with N-acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) inhibits myofibroblast differentiation and collagen deposition accompanied by stabilizing the expression of α-Ac-Tub in vivo and in vitro, which is related with deacetylase family member 6 (HDAC6) and α-tubulin acetyl transferase (α-TAT1). Our data suggest that α-Ac-Tub regulation by Ac-SDKP may potentially be a new anti-fibrosis mechanism. PMID:27577858

  19. Heat shock protein 90 (Hsp90) chaperone complex. A molecular target for enhancement of thermosensitivity and radiosensitivity

    Akimoto, Tetsuo; Nonaka, Tetsuo; Kitamoto, Yoshizumi; Sakurai, Hideyuki [Gunma Univ., Maebashi (Japan). School of Medicine; Mitsuhashi, Norio [Tokyo Women' s Medical Coll. (Japan)

    2002-09-01

    Heat shock protein 90 (Hsp90) is a highly conserved heat shock protein in animal and plants, and exists abundantly in the cytoplasm in unstressed condition, accounting for 1-2% in cytoplasmic proteins. Main difference of Hsp90 from other Hsps are its substrate that Hsp90 binds to. These substrates include various signal transduction proteins, kinase, steroid receptors and transcription factors, therefore, Hsp90 plays a key role in maintaining cellular signal transduction networks. Many chaperoned proteins (client proteins) of Hsp90 are associated with cellular proliferation or malignant transformation, thus Hsp90 chaperone complex has been focused as targets for cancer therapy. Among the client proteins, there are several molecules that have been defined as targets or factors for determination or enhancement of radiosensitivity or thermosensitivity. Thus, it is easily speculated that Hsp90 chaperone complex inhibitors that disrupt association of Hsp90 and client protein in combination with radiation or/and heat has potential effect on enhancement of radiosensitivity or thermosensitivity. In this paper, possible mechanisms in enhancing radiosensitivity or thermosensitivity according to the client proteins will be summarized. (author)

  20. A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum.

    Stefanie Wolfram

    Full Text Available Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.