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Sample records for nende mju kuritegevusele

  1. The Molecular Basis of N-End Rule Recognition

    SciTech Connect

    Wang, K.H.; Roman-Hernandez, G.; Grant, R.A.; Sauer, R.T.; Baker, T.A.

    2009-03-27

    The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 {angstrom}, reveals specific recognition of the peptide {alpha}-amino group via hydrogen bonding and shows that the peptide's N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that pre-exists on the surface of ClpS. The adaptor side chains that contact the peptide's N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery.

  2. The Molecular Basis of N-end Rule Recognition

    PubMed Central

    Wang, Kevin H.; Roman-Hernandez, Giselle; Grant, Robert A.; Sauer, Robert T.; Baker, Tania A.

    2011-01-01

    Summary The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 Å, reveals specific recognition of the peptide α-amino group via hydrogen bonding and shows that the peptide’s N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that preexists on the surface of ClpS. The adaptor side chains that contact the peptide’s N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery. PMID:18995838

  3. The N-end rule pathway and regulation by proteolysis

    PubMed Central

    Varshavsky, Alexander

    2011-01-01

    The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. The main determinant of an N-degron is a destabilizing N-terminal residue of a protein. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N-end rule and the Arg/N-end rule pathways. The Ac/N-end rule pathway targets proteins containing Nα-terminally acetylated (Nt-acetylated) residues. The Arg/N-end rule pathway recognizes unacetylated N-terminal residues and involves N-terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt-acetylated. Thus, most proteins harbor a specific degradation signal, termed AcN-degron, from the moment of their birth. Specific N-end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N-degrons include methionine-aminopeptidases, caspases, calpains, Nt-acetylases, Nt-amidases, arginyl-transferases, and leucyl-transferases. Regulated degradation of specific proteins by the N-end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation, and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights. PMID:21633985

  4. Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

    SciTech Connect

    Román-Hernández, Giselle; Grant, Robert A.; Sauer, Robert T.; Baker, Tania A.

    2009-06-19

    The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids. In each case, the alpha-amino group and side chain of the N-terminal residue are the major determinants of recognition. The binding pocket for the N-end residue is preformed in the free adaptor, and only small adjustments are needed to accommodate N-end rule residues having substantially different sizes and shapes. M53A ClpS is known to mediate degradation of an expanded repertoire of substrates, including those with N-terminal valine or isoleucine. A structure of Met53A ClpS engaged with an N-end rule tryptophan reveals an essentially wild-type mechanism of recognition, indicating that the Met(53) side chain directly enforces specificity by clashing with and excluding beta-branched side chains. Finally, experimental and structural data suggest mechanisms that make proteins with N-terminal methionine bind very poorly to ClpS, explaining why these high-abundance proteins are not degraded via the N-end rule pathway in the cell.

  5. N-end rule pathway inhibition assists colon tumor regression via necroptosis.

    PubMed

    Agarwalla, Pritha; Banerjee, Rajkumar

    2016-01-01

    Recent study has shown that N-end rule pathway, an ubiquitin dependent proteolytic system, counteracts cell death by degrading many antisurvival protein fragments like BCLxL, BRCA1, RIPK1, etc. Inhibition of the N-end rule pathway can lead to metabolic stabilization of proapoptotic protein fragments like RIPK1, thereby sensitizing cells to programmed cell death. Receptor interacting serine-threonine protein kinase-1 (RIPK1) is one of the upstream regulators of programmed necrosis known as necroptosis. Necroptosis is particularly gaining attention of cancer biologists as it provides an alternate therapeutic modality to kill cancer cells, which often evolve multiple strategies to circumvent growth inhibition by apoptosis. Utilizing the over expression of biotin receptor in cancer cells, herein, we report that coadministration of synthetic hetero-bivalent N-end rule inhibitor RFC11 and anticancer drug shikonin solubilized in a stable biotin receptor-targeted liposome exhibited significant synergistic antitumor effect in both subcutaneous and orthotopic mouse colon tumor model through induction of necroptosis with distinctive upregulation of RIPK1. Besides developing a newly targeted formulation for necroptosis induction, this report is the first in vivo evidence demonstrating that potent inhibition of N-end rule pathway can enhance therapeutic efficacy of conventional chemotherapeutics. PMID:27556106

  6. N-end rule pathway inhibition assists colon tumor regression via necroptosis

    PubMed Central

    Agarwalla, Pritha; Banerjee, Rajkumar

    2016-01-01

    Recent study has shown that N-end rule pathway, an ubiquitin dependent proteolytic system, counteracts cell death by degrading many antisurvival protein fragments like BCLxL, BRCA1, RIPK1, etc. Inhibition of the N-end rule pathway can lead to metabolic stabilization of proapoptotic protein fragments like RIPK1, thereby sensitizing cells to programmed cell death. Receptor interacting serine-threonine protein kinase-1 (RIPK1) is one of the upstream regulators of programmed necrosis known as necroptosis. Necroptosis is particularly gaining attention of cancer biologists as it provides an alternate therapeutic modality to kill cancer cells, which often evolve multiple strategies to circumvent growth inhibition by apoptosis. Utilizing the over expression of biotin receptor in cancer cells, herein, we report that coadministration of synthetic hetero-bivalent N-end rule inhibitor RFC11 and anticancer drug shikonin solubilized in a stable biotin receptor-targeted liposome exhibited significant synergistic antitumor effect in both subcutaneous and orthotopic mouse colon tumor model through induction of necroptosis with distinctive upregulation of RIPK1. Besides developing a newly targeted formulation for necroptosis induction, this report is the first in vivo evidence demonstrating that potent inhibition of N-end rule pathway can enhance therapeutic efficacy of conventional chemotherapeutics. PMID:27556106

  7. Degradation of Serotonin N-Acetyltransferase, a Circadian Regulator, by the N-end Rule Pathway.

    PubMed

    Wadas, Brandon; Borjigin, Jimo; Huang, Zheping; Oh, Jang-Hyun; Hwang, Cheol-Sang; Varshavsky, Alexander

    2016-08-12

    Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two "complementary" forms of rat AANAT are targeted for degradation by two "complementary" branches of the N-end rule pathway. Specifically, the N(α)-terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt-acetylated N-terminal Met residue by the Ac/N-end rule pathway, whereas the non-Nt-acetylated AANAT is targeted by the Arg/N-end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine-to-arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer-lived than its rat counterpart and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N-terminal sequences. PMID:27339900

  8. The mouse and human genes encoding the recognition component of the N-end rule pathway

    PubMed Central

    Kwon, Yong Tae; Reiss, Yuval; Fried, Victor A.; Hershko, Avram; Yoon, Jeong Kyo; Gonda, David K.; Sangan, Pitchai; Copeland, Neal G.; Jenkins, Nancy A.; Varshavsky, Alexander

    1998-01-01

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3α. Mouse UBR1p (E3α) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans ≈120 kilobases of genomic DNA and contains ≈50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway. PMID:9653112

  9. The N-end rule pathway regulates pathogen responses in plants.

    PubMed

    de Marchi, Rémi; Sorel, Maud; Mooney, Brian; Fudal, Isabelle; Goslin, Kevin; Kwaśniewska, Kamila; Ryan, Patrick T; Pfalz, Marina; Kroymann, Juergen; Pollmann, Stephan; Feechan, Angela; Wellmer, Frank; Rivas, Susana; Graciet, Emmanuelle

    2016-01-01

    To efficiently counteract pathogens, plants rely on a complex set of immune responses that are tightly regulated to allow the timely activation, appropriate duration and adequate amplitude of defense programs. The coordination of the plant immune response is known to require the activity of the ubiquitin/proteasome system, which controls the stability of proteins in eukaryotes. Here, we demonstrate that the N-end rule pathway, a subset of the ubiquitin/proteasome system, regulates the defense against a wide range of bacterial and fungal pathogens in the model plant Arabidopsis thaliana. We show that this pathway positively regulates the biosynthesis of plant-defense metabolites such as glucosinolates, as well as the biosynthesis and response to the phytohormone jasmonic acid, which plays a key role in plant immunity. Our results also suggest that the arginylation branch of the N-end rule pathway regulates the timing and amplitude of the defense program against the model pathogen Pseudomonas syringae AvrRpm1. PMID:27173012

  10. The N-end rule pathway regulates pathogen responses in plants

    PubMed Central

    de Marchi, Rémi; Sorel, Maud; Mooney, Brian; Fudal, Isabelle; Goslin, Kevin; Kwaśniewska, Kamila; Ryan, Patrick T.; Pfalz, Marina; Kroymann, Juergen; Pollmann, Stephan; Feechan, Angela; Wellmer, Frank; Rivas, Susana; Graciet, Emmanuelle

    2016-01-01

    To efficiently counteract pathogens, plants rely on a complex set of immune responses that are tightly regulated to allow the timely activation, appropriate duration and adequate amplitude of defense programs. The coordination of the plant immune response is known to require the activity of the ubiquitin/proteasome system, which controls the stability of proteins in eukaryotes. Here, we demonstrate that the N-end rule pathway, a subset of the ubiquitin/proteasome system, regulates the defense against a wide range of bacterial and fungal pathogens in the model plant Arabidopsis thaliana. We show that this pathway positively regulates the biosynthesis of plant-defense metabolites such as glucosinolates, as well as the biosynthesis and response to the phytohormone jasmonic acid, which plays a key role in plant immunity. Our results also suggest that the arginylation branch of the N-end rule pathway regulates the timing and amplitude of the defense program against the model pathogen Pseudomonas syringae AvrRpm1. PMID:27173012

  11. N-Terminal Acetylation-Targeted N-End Rule Proteolytic System: The Ac/N-End Rule Pathway

    PubMed Central

    Lee, Kang-Eun; Heo, Ji-Eun; Kim, Jeong-Mok; Hwang, Cheol-Sang

    2016-01-01

    Although Nα-terminal acetylation (Nt-acetylation) is a pervasive protein modification in eukaryotes, its general functions in a majority of proteins are poorly understood. In 2010, it was discovered that Nt-acetylation creates a specific protein degradation signal that is targeted by a new class of the N-end rule proteolytic system, called the Ac/N-end rule pathway. Here, we review recent advances in our understanding of the mechanism and biological functions of the Ac/N-end rule pathway, and its crosstalk with the Arg/N-end rule pathway (the classical N-end rule pathway). PMID:26883906

  12. Anthrax Edema Factor Toxicity Is Strongly Mediated by the N-end Rule

    PubMed Central

    Leysath, Clinton E.; Phillips, Damilola D.; Crown, Devorah; Fattah, Rasem J.; Moayeri, Mahtab; Leppla, Stephen H.

    2013-01-01

    Anthrax edema factor (EF) is a calmodulin-dependent adenylate cyclase that converts adenosine triphosphate (ATP) into 3’–5’-cyclic adenosine monophosphate (cAMP), contributing to the establishment of Bacillus anthracis infections and the resulting pathophysiology. We show that EF adenylate cyclase toxin activity is strongly mediated by the N-end rule, and thus is dependent on the identity of the N-terminal amino acid. EF variants having different N-terminal residues varied by more than 100-fold in potency in cultured cells and mice. EF variants having unfavorable, destabilizing N-terminal residues showed much greater activity in cells when the E1 ubiquitin ligase was inactivated or when proteasome inhibitors were present. Taken together, these results show that EF is uniquely affected by ubiquitination and/or proteasomal degradation. PMID:24015319

  13. UBL1 of Fusarium verticillioides links the N-end rule pathway to extracellular sensing and plant pathogenesis.

    PubMed

    Ridenour, John B; Smith, Jonathon E; Hirsch, Robert L; Horevaj, Peter; Kim, Hun; Sharma, Sandeep; Bluhm, Burton H

    2014-07-01

    Fusarium verticillioides produces fumonisin mycotoxins during colonization of maize. Currently, molecular mechanisms underlying responsiveness of F.verticillioides to extracellular cues during pathogenesis are poorly understood. In this study, insertional mutants were created and screened to identify genes involved in responses to extracellular starch. In one mutant, the restriction enzyme-mediated integration cassette disrupted a gene (UBL1) encoding a UBR-Box/RING domain E3 ubiquitin ligase involved in the N-end rule pathway. Disruption of UBL1 in F.verticillioides (Δubl1) influenced conidiation, hyphal morphology, pigmentation and amylolysis. Disruption of UBL1 also impaired kernel colonization, but the ratio of fumonisin B1 per unit growth was not significantly reduced. The inability of a Δubl1 mutant to recognize an N-end rule degron confirmed involvement of UBL1 in the N-end rule pathway. Additionally, Ubl1 physically interacted with two G protein α subunits of F.verticillioides, thus implicating UBL1 in G protein-mediated sensing of the external environment. Furthermore, deletion of the UBL1 orthologue in F.graminearum reduced virulence on wheat and maize, thus indicating that UBL1 has a broader role in virulence among Fusarium species. This study provides the first linkage between the N-end rule pathway and fungal pathogenesis, and illustrates a new mechanism through which fungi respond to the external environment. PMID:24237664

  14. The Substrate Recognition Domains of the N-end Rule Pathway*

    PubMed Central

    Tasaki, Takafumi; Zakrzewska, Adriana; Dudgeon, Drew D.; Jiang, Yonghua; Lazo, John S.; Kwon, Yong Tae

    2009-01-01

    The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with Kd of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway. PMID:19008229

  15. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Lecker, S. H.; Goldberg, A. L.

    1998-01-01

    In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

  16. Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Baracos, V.; Sarraf, P.; Goldberg, A. L.

    1998-01-01

    The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

  17. Structure of a putative ClpS N-end rule adaptor protein from the malaria pathogen Plasmodium falciparum.

    PubMed

    AhYoung, Andrew P; Koehl, Antoine; Vizcarra, Christina L; Cascio, Duilio; Egea, Pascal F

    2016-03-01

    The N-end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP-dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N-end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N-degrons. However, while the N-degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal-ClpS binds and discriminates peptides mimicking bona fide N-end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal-ClpS localizes to this plastid-like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti-malarial drugs aimed at disrupting parasite-specific protein quality control pathways. PMID:26701219

  18. The molecular basis for the post-translational addition of amino acids by L/F transferase in the N-end rule pathway.

    PubMed

    Fung, Angela Wai S; Fahlman, Richard P

    2015-01-01

    The N-end rule pathway is a conserved targeted proteolytic process observed in organisms ranging from eubacteria to mammals. The N-end rule relates the metabolic stability of a protein to its N-terminal amino acid residue. The identity of the N-terminal amino acid residue is a primary degradation signal, often referred to as an N-degron, which is recognized by the components of the N-end rule when it is a destabilizing N-terminus. N-degrons may be exposed by non-processive proteolytic cleavages or by post-translational modifications. One modification is the post-translational addition of amino acids to the N-termini of proteins, a reaction catalyzed by aminoacyl-tRNA protein transferases. The aminoacyl-tRNA protein transferase in eubacteria like Escherichia coli is L/F transferase. Recent investigations have reported unexpected observations regarding the L/F transferase catalytic mechanism and its mechanisms of substrate recognition. Additionally, recent proteome-wide identification of putative in vivo substrates facilitates hypothesis into the yet elusive biological functions of the prokaryotic N-end rule pathway. Here we summarize the recent findings on the molecular mechanisms of catalysis and substrate recognition by the E. coli L/F transferase in the prokaryotic N-end rule pathway. PMID:25692952

  19. The Greening after Extended Darkness1 Is an N-End Rule Pathway Mutant with High Tolerance to Submergence and Starvation1[OPEN

    PubMed Central

    Riber, Willi; Müller, Jana T.; Visser, Eric J.W.; Sasidharan, Rashmi; Voesenek, Laurentius A.C.J.; Mustroph, Angelika

    2015-01-01

    Plants respond to reductions in internal oxygen concentrations with adaptive mechanisms (for example, modifications of metabolism to cope with reduced supply of ATP). These responses are, at the transcriptional level, mediated by the group VII Ethylene Response Factor transcription factors, which have stability that is regulated by the N-end rule pathway of protein degradation. N-end rule pathway mutants are characterized by a constitutive expression of hypoxia response genes and abscisic acid hypersensitivity. Here, we identify a novel proteolysis6 (prt6) mutant allele, named greening after extended darkness1 (ged1), which was previously discovered in a screen for genomes uncoupled-like mutants and shows the ability to withstand long periods of darkness at the seedling stage. Interestingly, this ethyl methanesulfonate-derived mutant shows unusual chromosomal rearrangement instead of a point mutation. Furthermore, the sensitivity of N-end rule pathway mutants ged1 and prt6-1 to submergence was studied in more detail to understand previously contradicting experiments on this topic. Finally, it was shown that mutants for the N-end rule pathway are generally more tolerant to starvation conditions, such as prolonged darkness or submergence, which was partially associated with carbohydrate conservation. PMID:25667318

  20. The N-end rule in Escherichia coli: cloning and analysis of the leucyl, phenylalanyl-tRNA-protein transferase gene aat.

    PubMed Central

    Shrader, T E; Tobias, J W; Varshavsky, A

    1993-01-01

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Distinct versions of the N-end rule operate in bacteria, fungi, and mammals. We report the cloning and analysis of aat, the Escherichia coli gene that encodes leucyl, phenylalanyl-tRNA-protein transferase (L/F-transferase), a component of the bacterial N-end rule pathway. L/F-transferase is required for the degradation of N-end rule substrates bearing an N-terminal arginine or lysine. The aat gene maps to the 19-min region of the E. coli chromosome and encodes a 234-residue protein whose sequence lacks significant similarities to sequences in data bases. In vitro, L/F-transferase catalyzes the posttranslational conjugation of leucine or phenylalanine to the N termini of proteins that bear an N-terminal arginine or lysine. However, the isolation and sequence analysis of a beta-galactosidase variant engineered to expose an N-terminal arginine in vivo revealed the conjugation of leucine but not of phenylalanine to the N terminus of the beta-galactosidase variant. Thus, the specificity of L/F-transferase in vivo may be greater than that in vitro. The aat gene is located approximately 1 kb from clpA, which encodes a subunit of ATP-dependent protease Clp. Although both aat and clpA are required for the degradation of certain N-end rule substrates, their nearly adjacent genes are convergently transcribed. The aat gene lies downstream of an open reading frame that encodes a homolog of the mammalian multidrug resistance P glycoproteins. PMID:8331068

  1. A d-Amino Acid at the N-Terminus of a Protein Abrogates Its Degradation by the N-End Rule Pathway

    PubMed Central

    2015-01-01

    Eukaryotes have evolved the ubiquitin (Ub)/proteasome system to degrade polypeptides. The Ub/proteasome system is one way that cells regulate cytosolic protein and amino acids levels through the recognition and ubiquitination of a protein’s N-terminus via E1, E2, and E3 enzymes. The process by which the N-terminus stimulates intracellular protein degradation is referred to as the N-end rule. Characterization of the N-end rule has been limited to only the natural l-amino acids. Using a cytosolic delivery platform derived from anthrax lethal toxin, we probed the stability of mixed chirality proteins, containing one d-amino acid on the N-terminus of otherwise all l-proteins. In all cases, we observed that one N-terminal d-amino acid stabilized the cargo protein to proteasomal degradation with respect to the N-end rule. We found that since the mixed chirality proteins were not polyubiquitinated, they evaded N-end-mediated proteasomal degradation. Evidently, a subtle change on the N-terminus of a natural protein can enhance its intracellular lifetime. PMID:26807441

  2. Modification of PATase by L/F-transferase generates a ClpS-dependent N-end rule substrate in Escherichia coli

    PubMed Central

    Ninnis, Robert L; Spall, Sukhdeep K; Talbo, Gert H; Truscott, Kaye N; Dougan, David A

    2009-01-01

    The N-end rule pathway is conserved from bacteria to man and determines the half-life of a protein based on its N-terminal amino acid. In Escherichia coli, model substrates bearing an N-degron are recognised by ClpS and degraded by ClpAP in an ATP-dependent manner. Here, we report the isolation of 23 ClpS-interacting proteins from E. coli. Our data show that at least one of these interacting proteins—putrescine aminotransferase (PATase)—is post-translationally modified to generate a primary N-degron. Remarkably, the N-terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyl-tRNA-protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N-terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N-end rule pathway, through the ClpAPS-mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N-degron, which is required for substrate delivery to ClpA. PMID:19440203

  3. Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

    NASA Technical Reports Server (NTRS)

    Lecker, S. H.; Solomon, V.; Price, S. R.; Kwon, Y. T.; Mitch, W. E.; Goldberg, A. L.

    1999-01-01

    Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

  4. The Deubiquitinase USP47 Stabilizes MAPK by Counteracting the Function of the N-end Rule ligase POE/UBR4 in Drosophila.

    PubMed

    Ashton-Beaucage, Dariel; Lemieux, Caroline; Udell, Christian M; Sahmi, Malha; Rochette, Samuel; Therrien, Marc

    2016-08-01

    RAS-induced MAPK signaling is a central driver of the cell proliferation apparatus. Disruption of this pathway is widely observed in cancer and other pathologies. Consequently, considerable effort has been devoted to understanding the mechanistic aspects of RAS-MAPK signal transmission and regulation. While much information has been garnered on the steps leading up to the activation and inactivation of core pathway components, comparatively little is known on the mechanisms controlling their expression and turnover. We recently identified several factors that dictate Drosophila MAPK levels. Here, we describe the function of one of these, the deubiquitinase (DUB) USP47. We found that USP47 acts post-translationally to counteract a proteasome-mediated event that reduces MAPK half-life and thereby dampens signaling output. Using an RNAi-based genetic interaction screening strategy, we identified UBC6, POE/UBR4, and UFD4, respectively, as E2 and E3 enzymes that oppose USP47 activity. Further characterization of POE-associated factors uncovered KCMF1 as another key component modulating MAPK levels. Together, these results identify a novel protein degradation module that governs MAPK levels. Given the role of UBR4 as an N-recognin ubiquitin ligase, our findings suggest that RAS-MAPK signaling in Drosophila is controlled by the N-end rule pathway and that USP47 counteracts its activity. PMID:27552662

  5. The Deubiquitinase USP47 Stabilizes MAPK by Counteracting the Function of the N-end Rule ligase POE/UBR4 in Drosophila

    PubMed Central

    Udell, Christian M.; Sahmi, Malha; Rochette, Samuel; Therrien, Marc

    2016-01-01

    RAS-induced MAPK signaling is a central driver of the cell proliferation apparatus. Disruption of this pathway is widely observed in cancer and other pathologies. Consequently, considerable effort has been devoted to understanding the mechanistic aspects of RAS-MAPK signal transmission and regulation. While much information has been garnered on the steps leading up to the activation and inactivation of core pathway components, comparatively little is known on the mechanisms controlling their expression and turnover. We recently identified several factors that dictate Drosophila MAPK levels. Here, we describe the function of one of these, the deubiquitinase (DUB) USP47. We found that USP47 acts post-translationally to counteract a proteasome-mediated event that reduces MAPK half-life and thereby dampens signaling output. Using an RNAi-based genetic interaction screening strategy, we identified UBC6, POE/UBR4, and UFD4, respectively, as E2 and E3 enzymes that oppose USP47 activity. Further characterization of POE-associated factors uncovered KCMF1 as another key component modulating MAPK levels. Together, these results identify a novel protein degradation module that governs MAPK levels. Given the role of UBR4 as an N-recognin ubiquitin ligase, our findings suggest that RAS-MAPK signaling in Drosophila is controlled by the N-end rule pathway and that USP47 counteracts its activity. PMID:27552662

  6. Regulation of hedgehog Ligand Expression by the N-End Rule Ubiquitin-Protein Ligase Hyperplastic Discs and the Drosophila GSK3β Homologue, Shaggy

    PubMed Central

    Moncrieff, Sophie; Moncan, Matthieu; Scialpi, Flavia; Ditzel, Mark

    2015-01-01

    Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals. PMID:26334301

  7. Die grünende IT - Wie die Computerindustrie das Energiesparen neu erfand

    NASA Astrophysics Data System (ADS)

    von Greiner, Wilhelm

    Die IT-Branche hat ihr grünes Gewissen entdeckt. In der jetzigen Verbreitung und Intensität ist dieses Phänomen noch recht neu - lange Zeit schien die Informationstechnik in puncto Umweltverträglichkeit und Energieverbrauch eine "weiße Weste" zu haben. Schließlich läuft ein PC mit Strom und nicht mit - sagen wir mal - einem Dieselmotor: Beim Booten eines Computers schießt nicht erst eine dunkelgraue Rauchwolke aus dem Auspuff, die Lärmerzeugung beschränkt sich auf das Surren des Lüfters, zum Tanken fahren muss man mit ihm auch nicht, und die Produktion der Komponenten erfolgt… ja, wo eigentlich? Irgendwo in der "dritten Welt", in Fernost oder in Mexiko. So sind die umweltschädlichen Aspekte der Produktion von Leiterplatten und sonstigen Bauteilen aus den Augen, aus dem Sinn und bestenfalls sporadisch Gegenstand eines kritischen Greenpeace-Berichts1, der im Überangebot der Medienlandschaft untergeht.

  8. 78 FR 32632 - 36(b)(1) Arms Sales Notification

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-31

    ...-36B Infrared Decoy Flares, 30,000 RR-188 w/BBU-35B Training Chaff, 3,750 BDU-33D/B w/lugs/ Mk4 spot... (RNLAF) F-16 Formal Training Unit (FTU), 50,000 MJU-7B w/BBU-36B Infrared Decoy Flares, 30,000 RR-188...

  9. Data link relay design. [space probe with entry at Uranus

    NASA Technical Reports Server (NTRS)

    Parsons, P.

    1974-01-01

    The data link for the Ames baseline probe as applied to the MJU spacecraft specifically with an entry at Uranus is analyzed. A frequency analysis, a trajectory analysis, and a discussion of the effects on the spacecraft design by the data link are presented. The possibilities of a two-way link are considered.

  10. Use of a Conditional Ubr5 Mutant Allele to Investigate the Role of an N-End Rule Ubiquitin-Protein Ligase in Hedgehog Signalling and Embryonic Limb Development

    PubMed Central

    Kinsella, Elaine; Dora, Natalie; Mellis, David; Lettice, Laura; Deveney, Paul

    2016-01-01

    Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling. PMID:27299863

  11. Use of a Conditional Ubr5 Mutant Allele to Investigate the Role of an N-End Rule Ubiquitin-Protein Ligase in Hedgehog Signalling and Embryonic Limb Development.

    PubMed

    Kinsella, Elaine; Dora, Natalie; Mellis, David; Lettice, Laura; Deveney, Paul; Hill, Robert; Ditzel, Mark

    2016-01-01

    Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling. PMID:27299863

  12. [New opioids for general anaesthesia and in- and out-hospital analgesia].

    PubMed

    Dabrowska-Wójciak, Iwona; Piotrowski, Andrzej

    2008-01-01

    Over the last 30 years, three new opioids of the piperidine family have been introduced to anaesthesia clinical practice: sufentanil, alfentanil and remifentanil. Alfentanil is a derivative of fentanyl, with quicker onset than that of fentanyl and with shorter duration and more intense vagomimetic properties than those of fentanyl and sufentanil. It may cause less intense respiratory depression than equianalgesic doses of fentanyl. Clinical trials indicate that alfentanil can be used effectively as an analgesic, as an analgesic supplement to anaesthesia, and as the major component of a general anaesthetic. Its short duration of effect makes it attractive as an analgesic supplement for short ambulatory surgical procedures. Sufentanil is a more potent and more lipophilic analgesic than fentanyl. It would appear to maintain haemodynamic stability during surgery better than other opioids. Epidural sufentanil produces a rapid onset and good quality of analgesia. In addition, low doses administered intravenously via a PCA pump seem to have a potential role for analgesia during labour. Remifentanil is an opioid analgesic that is rapidly metabolized by non-specific blood and tissue esterases. According to its unique pharmacokinetic profile, remifentanil-based anaesthesia combines high-dosage opioid analgesia intraoperatively with a rapid and predictable postoperative awakening, even after long procedures. Its vagomimetic properties are especially pronounced in small children, the elderly and hypovolaemic patients, and in these groups atropine should be always given before remifentanil administration. Remifentanil also minimises the adrenergic response to endotracheal intubation. Three mju agonist-antagonists have been used for pain treatment: nalbuphine, butorphanol and buprenorphine. They can be used in ambulatory settings. Nalbuphine can be used parenterally. It reverses morphine-induced respiratory depression while maintaining adequate analgesic effect. Buprenorphine

  13. Direct inelastic scattering of oriented NO from Ag(111) and Pt(111)

    NASA Astrophysics Data System (ADS)

    Tenner, Manfred G.; Kuipers, Edgar W.; Kleyn, Aart W.; Stolte, Steven

    1991-04-01

    A pulsed supersonic and cold oriented beam of NO molecules is incident upon the (111) face of clean Ag and Pt single crystal surfaces. The steric effect in the scattered density distributions is determined by a quadrupole mass spectrometer. It is found that the steric effect in the peak in the distribution of direct inelastically scattered molecules depends linearly on the reflection angle. In all circumstances O-end collisions lead to scattering angles more inclined towards the surface than N-end collisions. For the Pt(111) surface a much stronger steric effect is measured than for the Ag(111) surface. The steric effect seems to scale with the incident normal velocity. These strong steric effects can be explained by the larger trapping probability for the N-end orientation and a leverage effect due to the high trapping probability.

  14. Total haemoglobin mass and red blood cell profile in endurance-trained and non-endurance-trained adolescent athletes.

    PubMed

    Ulrich, Gert; Bärtsch, Peter; Friedmann-Bette, Birgit

    2011-11-01

    To evaluate differences in total haemoglobin mass (tHb mass) and in red blood cell profile between elite endurance-trained (END) and non-endurance-trained (nEND) male and female adolescent athletes, tHb mass (CO rebreathing) and specific variables of red blood cell profile (haemoglobin concentration, haematocrit, erythrocyte indices) were determined in 59 elite junior athletes (29 END, 30 nEND). We hypothesized that at the age of 15-17 years, regular endurance training might induce a significant increase in tHb mass and changes in red blood cell profile. Therefore, all parameters were again determined after 6, 12 and 18 months in a subset of 27 subjects (17 END, 10 nEND). In END, tHb mass related to body weight was ~15% greater than in nEND (11.2 ± 1.6  vs. 9.7 ± 1.3 g kg(-1), P < 0.001), whereas no significant differences were observed for the red blood cell profile. In both groups, tHb mass related to body weight and the variables of red blood cell profile had not changed significantly after 6, 12 and 18 months of regular training. In conclusion, in elite junior athletes, differences in tHb mass between END and nEND were similar, however, smaller compared with previously in adult athletes reported values. At the age of 15-17 years, 18 months of regular training did not induce significant changes in tHb mass beyond alterations explained by physical growth and also variables of red blood cell profile did not change significantly. PMID:21431423

  15. The N-degradome of Escherichia coli: limited proteolysis in vivo generates a large pool of proteins bearing N-degrons.

    PubMed

    Humbard, Matthew A; Surkov, Serhiy; De Donatis, Gian Marco; Jenkins, Lisa M; Maurizi, Michael R

    2013-10-01

    The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates. PMID:23960079

  16. The N-degradome of Escherichia coli

    PubMed Central

    Humbard, Matthew A.; Surkov, Serhiy; De Donatis, Gian Marco; Jenkins, Lisa M.; Maurizi, Michael R.

    2013-01-01

    The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates. PMID:23960079

  17. Ubiquitination of specific mitochondrial matrix proteins.

    PubMed

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G; Ciechanover, Aaron

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems - at least partially - in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. PMID:27157140

  18. Expression and Biochemical Characterization of the Human Enzyme N-Terminal Asparagine Amidohydrolase (hNTAN1)

    PubMed Central

    Cantor, Jason R.; Stone, Everett M.; Georgiou, George

    2011-01-01

    The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein we report the bacterial expression, purification, and biochemical characterization of the human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn, but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the point mutation Cys75Ala. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating L-Met is important for function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway. PMID:21375249

  19. From start to finish: amino-terminal protein modifications as degradation signals in plants.

    PubMed

    Gibbs, Daniel J; Bailey, Mark; Tedds, Hannah M; Holdsworth, Michael J

    2016-09-01

    Contents 1188 I. 1188 II. 1189 III. 1190 IV. 1191 V. 1192 1192 References 1192 SUMMARY: The amino- (N-) terminus (Nt) of a protein can undergo a diverse array of co- and posttranslational modifications. Many of these create degradation signals (N-degrons) that mediate protein destruction via the N-end rule pathway of ubiquitin-mediated proteolysis. In plants, the N-end rule pathway has emerged as a major system for regulated control of protein stability. Nt-arginylation-dependent degradation regulates multiple growth, development and stress responses, and recently identified functions of Nt-acetylation can also be linked to effects on the in vivo half-lives of Nt-acetylated proteins. There is also increasing evidence that N-termini could act as important protein stability determinants in plastids. Here we review recent advances in our understanding of the relationship between the nature of protein N-termini, Nt-processing events and proteolysis in plants. PMID:27439310

  20. KCMF1 (potassium channel modulatory factor 1) Links RAD6 to UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4) and Lysosome-Mediated Degradation*

    PubMed Central

    Hong, Jenny H.; Kaustov, Lilia; Coyaud, Etienne; Srikumar, Tharan; Wan, Janet; Arrowsmith, Cheryl; Raught, Brian

    2015-01-01

    RAD6 is a ubiquitin E2 protein with roles in a number of different biological processes. Here, using affinity purification coupled with mass spectrometry, we identify a number of new RAD6 binding partners, including the poorly characterized ubiquitin E3 ligases KCMF1 (potassium channel modulatory factor 1) and UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4), a protein that can bind N-end rule substrates, and which was recently linked to lysosome-mediated degradation and autophagy. NMR, combined with in vivo and in vitro interaction mapping, demonstrate that the KCMF1 C terminus binds directly to RAD6, whereas N-terminal domains interact with UBR4 and other intracellular vesicle- and mitochondria-associated proteins. KCMF1 and RAD6 colocalize at late endosomes and lysosomes, and cells disrupted for KCMF1 or RAD6 function display defects in late endosome vesicle dynamics. Notably, we also find that two different RAD6A point mutants (R7W and R11Q) found in X-linked intellectual disability (XLID) patients specifically lose the interaction with KCMF1 and UBR4, but not with other previously identified RAD6 interactors. We propose that RAD6-KCMF1-UBR4 represents a unique new E2-E3 complex that targets unknown N-end rule substrates for lysosome-mediated degradation, and that disruption of this complex via RAD6A mutations could negatively affect neuronal function in XLID patients. PMID:25582440

  1. Expression of yeast deubiquitination enzyme UBP1 analogues in E. coli

    PubMed Central

    Wojtowicz, Anna; Mazurkiewicz-Pisarek, Anna; Plucienniczak, Grazyna; Mikiewicz-Sygula, Diana; Chojnacka, Luiza; Lukasiewicz, Natalia; Plucienniczak, Andrzej

    2005-01-01

    Background It has been shown that proteins fused to ubiquitin undergo greater expression in E. coli and are easier to purify and renaturate than nonhybrid foreign proteins. However, there is no commercial source of large quantities of specific deubiquitinating proteases. This is the reason why hybrid proteins containing ubiquitin at their N-end cannot be used in large scale biotechnological processes. Results and Conclusion We have described the synthesis of the yeast deubiquination enzyme UBP1 muteins in E. coli. We have shown that an efficient overproduction of the enzyme in E. coli may be achieved after the introduction of several changes in the nucleotide sequence encoding UBP1. One of the conditions of an effective synthesis of the UBP1 muteins is the removal of the 5'-end sequence encoding the transmembrane region of the enzyme. The obtained variants of the enzyme may be successfully used for processing large amounts of hybrid proteins comprising ubiquitin or tagged ubiquitin at their N-ends. PMID:15924623

  2. Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases.

    PubMed

    Klecker, Maria; Dissmeyer, Nico

    2016-01-01

    The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide libraries on cellulose membrane supports. The synthesis method is based on conventional Fmoc chemistry building up peptides with free N-terminal amino acids starting at their cellulose-coupled C-termini. Several hundreds of peptide sequences can be assembled with this technique on one membrane comprising a strong binding potential due to high local peptide concentrations. Peptide orientation on SPOT membranes qualifies this array type for assaying substrate specificities of N-recognins, the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Pioneer studies described binding capability of mammalian and yeast enzymes depending on a peptide's N-terminus. SPOT arrays have been successfully used to describe substrate specificity of N-recognins which are the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Here, we describe the implementation of SPOT binding assays with focus on the identification of N-recognin substrates, applicable also for plant NERD enzymes. PMID:27424747

  3. Assaying ATE1 Activity in Yeast by β-Gal Degradation.

    PubMed

    Kashina, Anna S

    2015-01-01

    In 1980s it was found that addition of N-terminal Arg to proteins induces their ubiquitination and degradation by the N-end rule pathway. While this mechanism applies only to the proteins which also have other features of the N-degron (including a closely adjacent Lys that is accessible for ubiquitination), several test substrates have been found to follow this mechanism very efficiently after ATE1-dependent arginylation. Such property enabled researchers to test ATE1 activity in cells indirectly by assaying for the degradation of such arginylation-dependent substrates. The most commonly used substrate for this assay is E. coli beta galactosidase (beta-Gal) because its activity can be easily measured using standardized colorimetric assays. Here we describe this method, which has served as a quick and easy way to characterize ATE1 activity during identification of arginyltransferases in different species. PMID:26285881

  4. Monitoring bird populations with Breeding Bird Survey and atlas data

    USGS Publications Warehouse

    Robbins, C.S.; Droege, S.; Sauer, J.R.

    1989-01-01

    The principal means of monitoring avian species in North America since 1966 is the Breeding Bird Survey (BBS). Nearly 2000 random roadside rouleS of 50 3-minule stops each are run once each summer by experienced observers. Two-year and 10ng-lerm population trends have been calculated for 250 of the more common species. We show how Breeding Bird Atlas dala for lWO periods of time can also be used to calculale population nends, and we compare atlas neRds with those from the BBS. If coverage is inlensive and consistent belWeen time periods, atlas dala may be useful for monitoring species that are not adequalely sampled by the BBS.

  5. First Things First: Vital Protein Marks by N-Terminal Acetyltransferases.

    PubMed

    Aksnes, Henriette; Drazic, Adrian; Marie, Michaël; Arnesen, Thomas

    2016-09-01

    N-terminal (Nt) acetylation is known to be a highly abundant co-translational protein modification, but the recent discovery of Golgi- and chloroplast-resident N-terminal acetyltransferases (NATs) revealed that it can also be added post-translationally. Nt-acetylation may act as a degradation signal in a novel branch of the N-end rule pathway, whose functions include the regulation of human blood pressure. Nt-acetylation also modulates protein interactions, targeting, and folding. In plants, Nt-acetylation plays a role in the control of resistance to drought and in regulation of immune responses. Mutations of specific human NATs that decrease their activity can cause either the lethal Ogden syndrome or severe intellectual disability and cardiovascular defects. In sum, recent advances highlight Nt-acetylation as a key factor in many biological pathways. PMID:27498224

  6. The importance of chain conformational mobility during 5-exo-cyclizations of C-, N- and O-centred radicals.

    PubMed

    Walton, John C

    2014-10-28

    The reaction coordinates of an archetypical set of 5-exo cyclizations of C-, N- and O-centred radicals were investigated by computational methods. G4 theory, and DFT with the um062x functional, were able to rationalise counterintuitive factors such as the 'normal' order of rate constants being: N-centred < C-centred < O-centred radicals. The access angle between the radical centre and the double bond was identified as a key factor. Examination of its evolution during ring closure implied that rigidity at the N-ends of the chains, and the consequent extra energy needed to attain chair-like transition states, might be the reason for slow aminyl cyclizations. A novel linear correlation between cyclization activation energies and the access angles was discovered. The preference for cis-1,2-disubstituted product formation was also accounted for in terms of interaction between the hyperconjugatively delocalized SOMO and the alkene π* orbital. PMID:25179567

  7. Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome*

    PubMed Central

    Yu, Houqing; Singh Gautam, Amit K.; Wilmington, Shameika R.; Wylie, Dennis; Martinez-Fonts, Kirby; Kago, Grace; Warburton, Marie; Chavali, Sreenivas; Inobe, Tomonao; Finkelstein, Ilya J.; Babu, M. Madan

    2016-01-01

    The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness. PMID:27226608

  8. Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae.

    PubMed Central

    Ghislain, M; Dohmen, R J; Levy, F; Varshavsky, A

    1996-01-01

    A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics. Images PMID:8890162

  9. Quantitative proteomics analysis of the Arg/N-end rule pathway of targeted degradation in Arabidopsis roots

    PubMed Central

    Zhang, Hongtao; Gannon, Lucy; Powers, Stephen J; Lilley, Kathryn S; Theodoulou, Frederica L

    2015-01-01

    According to the Arg/N-end rule pathway, proteins with basic N-termini are targeted for degradation by the Arabidopsis thaliana E3 ligase, PROTEOLYSIS6 (PRT6). Proteins can also become PRT6 substrates following post-translational arginylation by arginyltransferases ATE1 and 2. Here, we undertook a quantitative proteomics study of Arg/N-end rule mutants, ate1/2 and prt6, to investigate the impact of this pathway on the root proteome. Tandem mass tag labelling identified a small number of proteins with increased abundance in the mutants, some of which represent downstream targets of transcription factors known to be N-end rule substrates. Isolation of N-terminal peptides using terminal amine isotope labelling of samples (TAILS) combined with triple dimethyl labelling identified 1465 unique N-termini. Stabilising residues were over-represented among the free neo-N-termini, but destabilising residues were not markedly enriched in N-end rule mutants. The majority of free neo-N-termini were revealed following cleavage of organellar targeting signals, thus compartmentation may account in part for the presence of destabilising residues in the wild-type N-terminome. Our data suggest that PRT6 does not have a marked impact on the global proteome of Arabidopsis roots and is likely involved in the controlled degradation of relatively few regulatory proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD001719 (http://proteomecentral.proteomexchange.org/dataset/PXD001719). PMID:25728785

  10. Conditional Proteolysis of the Membrane Protein YfgM by the FtsH Protease Depends on a Novel N-terminal Degron.

    PubMed

    Bittner, Lisa-Marie; Westphal, Kai; Narberhaus, Franz

    2015-07-31

    Regulated proteolysis efficiently and rapidly adapts the bacterial proteome to changing environmental conditions. Many protease substrates contain recognition motifs, so-called degrons, that direct them to the appropriate protease. Here we describe an entirely new degron identified in the cytoplasmic N-terminal end of the membrane-anchored protein YfgM of Escherichia coli. YfgM is stable during exponential growth and degraded in stationary phase by the essential FtsH protease. The alarmone (p)ppGpp, but not the previously described YfgM interactors RcsB and PpiD, influence YfgM degradation. By scanning mutagenesis, we define individual amino acids responsible for turnover of YfgM and find that the degron does not at all comply with the known N-end rule pathway. The YfgM degron is a distinct module that facilitates FtsH-mediated degradation when fused to the N terminus of another monotopic membrane protein but not to that of a cytoplasmic protein. Several lines of evidence suggest that stress-induced degradation of YfgM relieves the response regulator RcsB and thereby permits cellular protection by the Rcs phosphorelay system. On the basis of these and other results in the literature, we propose a model for how the membrane-spanning YfgM protein serves as connector between the stress responses in the periplasm and cytoplasm. PMID:26092727

  11. A Shoot-Specific Hypoxic Response of Arabidopsis Sheds Light on the Role of the Phosphate-Responsive Transcription Factor PHOSPHATE STARVATION RESPONSE11[W

    PubMed Central

    Klecker, Maria; Gasch, Philipp; Peisker, Helga; Dörmann, Peter; Schlicke, Hagen; Grimm, Bernhard; Mustroph, Angelika

    2014-01-01

    Plant responses to biotic and abiotic stresses are often very specific, but signal transduction pathways can partially or completely overlap. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), the transcriptional responses to phosphate starvation and oxygen deficiency stress comprise a set of commonly induced genes. While the phosphate deficiency response is systemic, under oxygen deficiency, most of the commonly induced genes are found only in illuminated shoots. This jointly induced response to the two stresses is under control of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), but not of the oxygen-sensing N-end rule pathway, and includes genes encoding proteins for the synthesis of galactolipids, which replace phospholipids in plant membranes under phosphate starvation. Despite the induction of galactolipid synthesis genes, total galactolipid content and plant survival are not severely affected by the up-regulation of galactolipid gene expression in illuminated leaves during hypoxia. However, changes in galactolipid molecular species composition point to an adaptation of lipid fluxes through the endoplasmic reticulum and chloroplast pathways during hypoxia. PHR1-mediated signaling of phosphate deprivation was also light dependent. Because a photoreceptor-mediated PHR1 activation was not detectable under hypoxia, our data suggest that a chloroplast-derived retrograde signal, potentially arising from metabolic changes, regulates PHR1 activity under both oxygen and phosphate deficiency. PMID:24753539

  12. [Isolation and certain properties of mutant alkaline phosphatase of Escherichia coli].

    PubMed

    Nesmeianova, M A; Krupianko, V I; Kalinin, A E; Kadyrova, L Iu

    1996-01-01

    Natural and mutant alkaline phosphatases with amino acid substitutions in the processing site and N-terminal domain of the mature polypeptide chain Val for Ala(-1), Gln for Glu (+4) and simultaneously Gln for Glu (+4) and Ala for Arg (+1) have been isolated from the periplasm and cultural fluid of E. coli. It has been found that these substitutions have little effect on the dependence of the enzyme activity on pH, ionic strength and temperature but influence its isoenzymic spectrum and decrease (almost twofold) the maximal rate of the enzyme-catalyzed reaction. Extracellular enzymes display, in contrast with periplasmic ones, other catalytic properties (Vmax) and binding activity (Km). After translocation through the outer membrane all the enzymes display decreased Vmax and increased Km. These changes are especially well-pronounced in case of the mutant protein PhoA46 which contains an uncleaved signal peptide due to the impossibility of processing resulting from the substitution of Val for Ala(-1). The Vmax for this protein is decreased 20 times, while the Km is increased 4-fold. The protein also shows a higher (in comparison with other proteins) sensitivity towards proteolytic enzymes and is less resistant upon storage. The experimental data suggest that the changes in the N-end of alkaline phosphatase located at a long distance from its active center influence the enzyme function. PMID:8679783

  13. Cytosolic PINK1 promotes the targeting of ubiquitinated proteins to the aggresome-autophagy pathway during proteasomal stress.

    PubMed

    Gao, Ju; Li, Mengen; Qin, Siyue; Zhang, Ting; Jiang, Sicong; Hu, Yuan; Deng, Yongkang; Zhang, Chenliang; You, Dujuan; Li, Hongchang; Mu, Dezhi; Zhang, Zhuohua; Jiang, Changan

    2016-04-01

    During proteasomal stress, cells can alleviate the accumulation of polyubiquitinated proteins by targeting them to perinuclear aggresomes for autophagic degradation, but the mechanism underlying the activation of this compensatory pathway remains unclear. Here we report that PINK1-s, a short form of Parkinson disease (PD)-related protein kinase PINK1 (PTEN induced putative kinase 1), is a major regulator of aggresome formation. PINK1-s is extremely unstable due to its recognition by the N-end rule pathway, and tends to accumulate in the cytosol during proteasomal stress. Overexpression of PINK1-s induces aggresome formation in cells with normal proteasomal activities, while loss of PINK1-s function leads to a significant decrease in the efficiency of aggresome formation induced by proteasomal inhibition. PINK1-s exerts its effect through phosphorylation of the ubiquitin-binding protein SQSTM1 (sequestosome 1) and increasing its ability to sequester polyubiquitinated proteins into aggresomes. These findings pinpoint PINK1-s as a sensor of proteasomal activities that transduces the proteasomal impairment signal to the aggresome formation machinery. PMID:27050454

  14. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

    NASA Technical Reports Server (NTRS)

    Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

    2003-01-01

    PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

  15. Synthesis and biological activity of a lysine-containing cyclic analog of (Leu/sup 5/)enkephalin

    SciTech Connect

    Bobrova, I.V.; Abissova, N.A.; Rozental', G.F.; Nikiforovich, G.V.; Chipens, G.I.

    1986-09-01

    A cyclic analog of enkephalin - cyclo(Lys-Tyr-Gly-Gly-Phe-Leu) -- and two corresponding linear hexapeptides containing a residue of the amino acid lysine at the beginning and the end of the molecule - Lys-Tyr-Gly-Gly-Phe-Leu and Tyr-Gly-Gly-Phe-Leu-Lys - have been synthesized by the classical methods of peptide chemistry. The addition of a lysine residue to the N-end of the enkephalin molecule or the cyclization of this hexapeptide decreased the action of the analogs on the central and peripheral opiate receptors. The addition of lysine through the epsilon-amino group to the C-end of the enkephalin molecule scarcely changed the interaction of the analog with the ..mu..-type of opiate receptor but lowered its affinity for the delta-type of receptor approximately 10-fold. All three analogs that were synthesized possessed an analgesic activity comparable in magnitude with the activity of (Leu/sup 5/)enkephalin determined by the tail pinch method on intracisternal administration to mice.

  16. Three domains comprised in thermostable molecular weight 54,000 pullulanase of type I from Bacillus flavocaldarius KP1228.

    PubMed

    Kashiwabara, S; Ogawa, S; Miyoshi, N; Oda, M; Suzuki, Y

    1999-10-01

    The gene that coded for a cellular pullulanase of type I (alpha-dextrin 6-glucanohydrolase, EC 3.2.1.41) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51 to 82 degrees C was expressed in Escherichia coli MV1184. The enzyme had a half-life of 10 min at 107 degrees C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 475 residues with a molecular weight of 53,856 deduced from the gene was 15-21% and 43% identical to the corresponding C-terminal regions in the sequences of 2 plant and 6 bacterial pullulanases of type I, and of Bacillus stearothermophilus TRS40 neoplullulanase, respectively. Sequence analysis showed that B. flavocaldarius pullulanase comprised 3 domains, i.e., one catalytic (beta/alpha)8-barrel domain, one domain made of the region protruding from the barrel between the third beta-strand and the third alpha-helix, and one beta-stranded domain attached to the C-end of the barrel domain, but that the pullulanase lacked the beta-stranded domain commonly found in addition to the 3 domains in the neopullulanase and all other pullulanases, and attached to the N-end of the barrel domain. PMID:10586502

  17. Annotating N Termini for the Human Proteome Project: N Termini and Nα-Acetylation Status Differentiate Stable Cleaved Protein Species from Degradation Remnants in the Human Erythrocyte Proteome

    PubMed Central

    2014-01-01

    A goal of the Chromosome-centric Human Proteome Project is to identify all human protein species. With 3844 proteins annotated as “missing”, this is challenging. Moreover, proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions, and location. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content and wide dynamic range of protein concentrations that impedes protein identification. Using the N-terminomics procedure TAILS, we identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited improved mass spectrometric identification properties versus the intact tryptic peptide enabling identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome. With an improved bioinformatics workflow, we developed a new classification system and the Terminus Cluster Score. Thereby we described a new stabilizing N-end rule for processed protein termini, which discriminates novel protein species from degradation remnants, and identified protein domain hot spots susceptible to cleavage. Strikingly, 68% of the N-termini were within genome-encoded protein sequences, revealing alternative translation initiation sites, pervasive endoproteolytic processing, and stabilization of protein fragments in vivo. The mass spectrometry proteomics data have been deposited to ProteomeXchange with the data set identifier . PMID:24555563

  18. Mutations in the human UBR1 gene and the associated phenotypic spectrum.

    PubMed

    Sukalo, Maja; Fiedler, Ariane; Guzmán, Celina; Spranger, Stephanie; Addor, Marie-Claude; McHeik, Jiad N; Oltra Benavent, Manuel; Cobben, Jan M; Gillis, Lynette A; Shealy, Amy G; Deshpande, Charu; Bozorgmehr, Bita; Everman, David B; Stattin, Eva-Lena; Liebelt, Jan; Keller, Klaus-Michael; Bertola, Débora Romeo; van Karnebeek, Clara D M; Bergmann, Carsten; Liu, Zhifeng; Düker, Gesche; Rezaei, Nima; Alkuraya, Fowzan S; Oğur, Gönül; Alrajoudi, Abdullah; Venegas-Vega, Carlos A; Verbeek, Nienke E; Richmond, Erick J; Kirbiyik, Ozgür; Ranganath, Prajnya; Singh, Ankur; Godbole, Koumudi; Ali, Fouad A M; Alves, Crésio; Mayerle, Julia; Lerch, Markus M; Witt, Heiko; Zenker, Martin

    2014-05-01

    Johanson-Blizzard syndrome (JBS) is a rare, autosomal recessive disorder characterized by exocrine pancreatic insufficiency, typical facial features, dental anomalies, hypothyroidism, sensorineural hearing loss, scalp defects, urogenital and anorectal anomalies, short stature, and cognitive impairment of variable degree. This syndrome is caused by a defect of the E3 ubiquitin ligase UBR1, which is part of the proteolytic N-end rule pathway. Herein, we review previously reported (n = 29) and a total of 31 novel UBR1 mutations in relation to the associated phenotype in patients from 50 unrelated families. Mutation types include nonsense, frameshift, splice site, missense, and small in-frame deletions consistent with the hypothesis that loss of UBR1 protein function is the molecular basis of JBS. There is an association of missense mutations and small in-frame deletions with milder physical abnormalities and a normal intellectual capacity, thus suggesting that at least some of these may represent hypomorphic UBR1 alleles. The review of clinical data of a large number of molecularly confirmed JBS cases allows us to define minimal clinical criteria for the diagnosis of JBS. For all previously reported and novel UBR1 mutations together with their clinical data, a mutation database has been established at LOVD. PMID:24599544

  19. Physicochemical properties and amino acid composition of highly purified preparation of distinctive estrogen-binding protein from rat liver

    SciTech Connect

    Shchelkunova, T.A.; Smirnov, A.N.; Rozen, V.B.

    1986-12-10

    The structure and properties of a distinctive estrogen-binding protein (DEBP) from the livers of male rats, purified with the aid of an affinity sorbent, was investigated. A high degree of purification of the DEBP obtained (> 99%), associated with the pronounced microheterogeneity, was found. Apparently, this microheterogeneity is the result of partial proteolysis of the protein from the N-end during isolation. Purified DEBP molecules have the following parameters: molecular weight 31,000 (according to the data of electrophoresis in polyacrylamide gel in the presence of SDS), sedimentation coefficient 3.765, Stokes' radius 25.6 A, frictional ratio 1.11. Absorption maximum of the protein in the ultraviolet region 276 nm; extinction coefficient 26; content of ..cap alpha..-helical segments 25-30%. The equilibrium constant of the association with estradiol is 5 x 10/sup 7/ M/sup -1/. Estriol (> 100%) and, to a lesser degree, estrone and testosterone (approx. 10%) compete for the protein-binding sites on (/sup 3/H) estradiol, whereas androsterone has practically no competitive effect. A study of the amino acid composition of the DEBP showed that the protein contains a large number of residues with hydrophobic side groups (34.4%), it has more acidic than basic amino acids, and possesses a low content of cysteine, threonine, and histidine.

  20. Density functional theory study on the interactions of l-cysteine with graphene: adsorption stability and magnetism

    NASA Astrophysics Data System (ADS)

    Luo, Huijuan; Li, Hejun; Fu, Qiangang; Chu, Yanhui; Cao, Xiaoyu; Sun, Can; Yuan, Xiaoyan; Liu, Lei

    2013-12-01

    Understanding the interactions between graphene and biomolecules is of fundamental relevance to the area of nanobiotechnology. Herein, we take l-cysteine as the probe biomolecule and investigate its adsorption on pristine graphene and B-, N-, Al-, Ni-, Ga-, Pd-doped graphene using density functional theory calculations. Three kinds of upright adsorption configurations, via unprotonated functional groups (-SH, -NH2, -COOH), are considered. The calculations reveal pristine graphene physically adsorbs l-cysteine. N-doped graphene shows physisorption towards the S-end and N-end l-cysteine, and chemisorption towards the O-end radical. Strong chemisorption, with site-specific preference, occurs on Al-, Ni-, Ga- and Pd-doped graphene, accompanied by severe structural changes. Spin polarization with an unusual mirror symmetry on Ni- and Pd-doped graphene is induced by chemisorption of unprotonated l-cysteine, except for O-end adsorption on Pd-doped graphene. The magnetization arises mainly from spin polarization of the C 2pz orbital, with a minor magnetism located on Ni or Pd. The influence of van der Waals forces is also evaluated. A thorough analysis of the adsorption stability and magnetism of these systems would be beneficial to facilitate applications in graphene-based biosensing, biomolecule immobilization, magnetic bio-separation and other fields in bionanotechnology.

  1. N-terminal protein processing: A comparative proteogenomic analysis

    SciTech Connect

    Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

    2013-01-01

    N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

  2. What do we really know about the ubiquitin-proteasome pathway in muscle atrophy?

    NASA Technical Reports Server (NTRS)

    Jagoe, R. T.; Goldberg, A. L.

    2001-01-01

    Studies of many different rodent models of muscle wasting have indicated that accelerated proteolysis via the ubiquitin-proteasome pathway is the principal cause of muscle atrophy induced by fasting, cancer cachexia, metabolic acidosis, denervation, disuse, diabetes, sepsis, burns, hyperthyroidism and excess glucocorticoids. However, our understanding about how muscle proteins are degraded, and how the ubiquitin-proteasome pathway is activated in muscle under these conditions, is still very limited. The identities of the important ubiquitin-protein ligases in skeletal muscle, and the ways in which they recognize substrates are still largely unknown. Recent in-vitro studies have suggested that one set of ubquitination enzymes, E2(14K) and E3(alpha), which are responsible for the 'N-end rule' system of ubiquitination, plays an important role in muscle, especially in catabolic states. However, their functional significance in degrading different muscle proteins is still unclear. This review focuses on the many gaps in our understanding of the functioning of the ubiquitin-proteasome pathway in muscle atrophy, and highlights the strengths and limitations of the different experimental approaches used in such studies.

  3. A novel N(alpha)-acetyl alanine aminopeptidase from Allomyces arbuscula.

    PubMed

    Beti, Raniera; Cattaneo, Arlette; Gabriel, Jean Marc; Ojha, Mukti

    2002-04-01

    An N(alpha)-acetyl alanine aminopeptidase has been purified from the aquatic fungus Allomyces arbuscula. The apparent molecular mass of the enzyme was estimated to be 280 kDa by gel filtration through calibrated Sephacryl S300 column. In SDS-PAGE, the purified enzyme appeared as a single band of M(r) 80 kDa. Catalytic activity of the enzyme was inhibited by specific serine protease inhibitors, 3,4-DCI and APMSF, as well as SH reacting compounds, HgCl(2) and iodoacetate, indicating that the enzyme is a serine protease with some functional SH group(s) involved in the catalytic reaction. 3H-DFP was used to label the reactive serine of the enzyme. When the labeled protein was analyzed in SDS-PAGE, most of the label appeared in the M(r) 80 kDa band, however, a few additional faster migrating minor bands were also seen, probably representing a minor degradation product of the enzyme. The enzyme cleaved mainly N(alpha)-acetlylated alanine, although a small but negligible activity was also obtained with acetylated leucine and phenylalanine. The role of the enzyme in N-end rule proteolysis is discussed. PMID:12106909

  4. Redox regulation in shoot growth, SAM maintenance and flowering.

    PubMed

    Schippers, Jos Hm; Foyer, Christine H; van Dongen, Joost T

    2016-02-01

    Reactive oxygen species (ROS) and associated reduction/oxidation (redox) controls involving glutathione, glutaredoxins and thioredoxins play key roles in the regulation of plant growth and development. While many questions remain concerning redox functions in the shoot apical meristem (SAM), accumulating evidence suggests that redox master switches integrate major hormone signals and transcriptional networks in the SAM, and so regulate organ growth, polarity and floral development. Auxin-induced activation of plasma-membrane located NADPH-oxidases and mitochondrial respiratory bioenergetics are likely regulators of the ROS bursts that drive the cell cycle in proliferating regions, with other hormones such as jasmonic acid playing propagating or antagonistic roles in gene regulation. Moreover, the activation of oxygen production by photosynthesis and oxygen-dependent N-end rule controls are linked to the transition from cell proliferation to cell expansion and differentiation. While much remains to be understood, the nexus of available redox controls provides a key underpinning mechanism linking hormonal controls, energy metabolism and bioenergetics to plant growth and development. PMID:26799134

  5. Quorum sensing controls hyphal initiation in Candida albicans through Ubr1-mediated protein degradation

    PubMed Central

    Lu, Yang; Su, Chang; Unoje, Ohimai; Liu, Haoping

    2014-01-01

    Candida albicans is the most common cause of invasive fungal infections in humans. Its ability to undergo the morphological transition from yeast to hyphal growth forms is critical for its pathogenesis. Hyphal initiation requires the activation of the cAMP-PKA pathway, which down-regulates the expression of NRG1, the major repressor of hyphal development. Hyphal initiation also requires inoculation of a small amount of C. albicans cells from overnight culture to fresh medium. This inoculation releases the inhibition from farnesol, a quorum-sensing molecule of C. albicans, that accumulated in the spent medium. Here, we show that farnesol inhibits hyphal initiation mainly through blocking the protein degradation of Nrg1. Through screening a kinase mutant library, we identified Sok1 as the kinase required for Nrg1 degradation during inoculation. SOK1 expression is transiently activated on inoculation during hyphal initiation, and overexpression of SOK1 overcomes the farnesol-mediated inhibition of hyphal initiation. Screening a collection of transcription factor mutants, the homeodomain-containing transcription repressor Cup9 is found to be responsible for the repression of SOK1 expression in response to farnesol inhibition. Interestingly, farnesol inhibits Cup9 degradation mediated by the N-end rule E3 ubiquitin ligase, Ubr1. Therefore, hyphal initiation requires both the cAMP-PKA pathway-dependent transcriptional down-regulation of NRG1 and Sok1-mediated degradation of Nrg1 protein. The latter is triggered by the release from farnesol inhibition of Cup9 degradation and consequently, derepression of SOK1 transcription. Neither pathway alone is sufficient for hyphal initiation. PMID:24449897

  6. Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase

    PubMed Central

    Amm, Ingo; Norell, Derrick; Wolf, Dieter H.

    2015-01-01

    The Saccharomyces cerevisiae heat shock proteins Hsp31, Hsp32, Hsp33 and Hsp34 belong to the DJ-1/ThiJ/PfpI superfamily which includes the human protein DJ-1 (PARK7) as the most prominent member. Mutations in the DJ-1 gene are directly linked to autosomal recessive, early-onset Parkinson’s disease. DJ-1 acts as an oxidative stress-induced chaperone preventing aggregation and fibrillation of α-synuclein, a critical factor in the development of the disease. In vivo assays in Saccharomyces cerevisiae using the model substrate ΔssCPY*Leu2myc (ΔssCL*myc) as an aggregation-prone misfolded cytoplasmic protein revealed an influence of the Hsp31 chaperone family on the steady state level of this substrate. In contrast to the ubiquitin ligase of the N-end rule pathway Ubr1, which is known to be prominently involved in the degradation process of misfolded cytoplasmic proteins, the absence of the Hsp31 chaperone family does not impair the degradation of newly synthesized misfolded substrate. Also degradation of substrates with strong affinity to Ubr1 like those containing the type 1 N-degron arginine is not affected by the absence of the Hsp31 chaperone family. Epistasis analysis indicates that one function of the Hsp31 chaperone family resides in a pathway overlapping with the Ubr1-dependent degradation of misfolded cytoplasmic proteins. This pathway gains relevance in late growth phase under conditions of nutrient limitation. Additionally, the Hsp31 chaperones seem to be important for maintaining the cellular Ssa Hsp70 activity which is important for Ubr1-dependent degradation. PMID:26466368

  7. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability1[OPEN

    PubMed Central

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H.; van Wijk, Klaas J.

    2015-01-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed. PMID:26371235

  8. The region adjacent to the C-end of the inner gate in transient receptor potential melastatin 8 (TRPM8) channels plays a central role in allosteric channel activation.

    PubMed

    Taberner, Francisco José; López-Córdoba, Ainara; Fernández-Ballester, Gregorio; Korchev, Yuri; Ferrer-Montiel, Antonio

    2014-10-10

    The ability of transient receptor potential (TRP) channels to sense and respond to environmental and endogenous cues is crucial in animal sensory physiology. The molecular mechanism of channel gating is yet elusive. The TRP box, a conserved region in the N-end of the C terminus domain, has been signaled as pivotal for allosteric activation in TRP channels. Here, we have examined the role of the linker region between the TRPM8 inner gate and the TRP box (referred to as the S6-TRP box linker) to identify structural determinants of channel gating. Stepwise substitutions of segments in the S6-TRP box linker of TRPM8 channel with the cognate TRPV1 channel sequences produced functional chimeric channels, and identified Tyr(981) as a central molecular determinant of channel function. Additionally, mutations in the 986-990 region had a profound impact on channel gating by voltage and menthol, as evidenced by the modulation of the conductance-to-voltage (G-V) relationships. Simulation of G-V curves using an allosteric model for channel activation revealed that these mutations altered the allosteric constants that couple stimuli sensing to pore opening. A molecular model of TRPM8, based on the recently reported TRPV1 structural model, showed that Tyr(981) may lie in a hydrophobic pocket at the end of the S6 transmembrane segment and is involved in inter-subunit interactions with residues from neighbor subunits. The 986-990 region holds intrasubunit interactions between the TRP domain and the S4-S5 linker. These findings substantiate a gating mechanism whereby the TRP domain acts as a coupling domain for efficient channel opening. Furthermore, they imply that protein-protein interactions of the TRP domain may be targets for channel modulation and drug intervention. PMID:25157108

  9. N-Alpha-Acetyltransferases and Regulation of CFTR Expression

    PubMed Central

    Patrick, Anna E.; Hudson, Henry; Thomas, Philip J.

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein. PMID:27182737

  10. [Design of the novel dipeptide neuropsychotropic drug preparations].

    PubMed

    Gudasheva, T A; Skoldinov, A P

    2003-01-01

    The paper considers a new strategy in the field of neuropsychotropic dipeptide drug design, the main points being as follows: (i) determination of the structural elements of dipeptides, such as fragments of amino acid side radicals and peptide bonds, in nonpeptide drugs; (ii) design of peptide analogs topologically close to the drug; (iii) synthesis and activity testing of these analogs; (iv) determination of the corresponding endogenous neuropeptide among the known neuropeptides or identification of the new neuropeptides in the brain of experimental animals. Using this approach, new pyroglutamyl- and prolyl-containing dipeptides were obtained based on the structure of the well-known classical nootropic drug piracetam. The new drugs exhibit nootropic activity in doses 100-10,000 times lower than those of piracetam. The structure of most active pyroglutamyl dipeptide pGlu-Asn-NH2 coincides with that of the N-end fragment of the endogenous memory peptide AVP(4-9). Noopept (N-phenylacetylprolylglycine ethyl ester), patented in Russia and USA as a new nootropic drug, is currently under stage 2 of successful clinical trials. The main metabolite of noopept, cyclo-Pro-Gly, is identical to the endogenous dipeptide designed in this work and is most close analog of piracetam with respect to pharmacological activity. The universal character of the proposed strategy is demonstrated by the design of active dipeptide analogs of an atypical neuroleptic drug sulpiride. As a result, a potential dipeptide neuroleptic dilept was obtained, which has been patented in Russia and now passes broad preclinical trials. PMID:12962042

  11. Rotationally inelastic scattering of NO(A{sup 2}Σ{sup +}) + Ar: Differential cross sections and rotational angular momentum polarization

    SciTech Connect

    Sharples, Thomas R.; Luxford, Thomas F. M.; McKendrick, Kenneth G.; Costen, Matthew L.; Townsend, Dave

    2015-11-28

    We present the implementation of a new crossed-molecular beam, velocity-map ion-imaging apparatus, optimized for collisions of electronically excited molecules. We have applied this apparatus to rotational energy transfer in NO(A{sup 2}Σ{sup +}, v = 0, N = 0, j = 0.5) + Ar collisions, at an average energy of 525 cm{sup −1}. We report differential cross sections for scattering into NO(A{sup 2}Σ{sup +}, v = 0, N′ = 3, 5, 6, 7, 8, and 9), together with quantum scattering calculations of the differential cross sections and angle dependent rotational alignment. The differential cross sections show dramatic forward scattered peaks, together with oscillatory behavior at larger scattering angles, while the rotational alignment moments are also found to oscillate as a function of scattering angle. In general, the quantum scattering calculations are found to agree well with experiment, reproducing the forward scattering and oscillatory behavior at larger scattering angles. Analysis of the quantum scattering calculations as a function of total rotational angular momentum indicates that the forward scattering peak originates from the attractive minimum in the potential energy surface at the N-end of the NO. Deviations in the quantum scattering predictions from the experimental results, for scattering at angles greater than 10°, are observed to be more significant for scattering to odd final N′. We suggest that this represents inaccuracies in the potential energy surface, and in particular in its representation of the difference between the N- and O-ends of the molecule, as given by the odd-order Legendre moments of the surface.

  12. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    PubMed

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein. PMID:27182737

  13. Liat1, an arginyltransferase-binding protein whose evolution among primates involved changes in the numbers of its 10-residue repeats.

    PubMed

    Brower, Christopher S; Rosen, Connor E; Jones, Richard H; Wadas, Brandon C; Piatkov, Konstantin I; Varshavsky, Alexander

    2014-11-18

    The arginyltransferase Ate1 is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. At least six isoforms of mouse Ate1 are produced through alternative splicing of Ate1 pre-mRNA. We identified a previously uncharacterized mouse protein, termed Liat1 (ligand of Ate1), that interacts with Ate1 but does not appear to be its arginylation substrate. Liat1 has a higher affinity for the isoforms Ate1(1A7A) and Ate1(1B7A). Liat1 stimulated the in vitro N-terminal arginylation of a model substrate by Ate1. All examined vertebrate and some invertebrate genomes encode proteins sequelogous (similar in sequence) to mouse Liat1. Sequelogs of Liat1 share a highly conserved ∼30-residue region that is shown here to be required for the binding of Liat1 to Ate1. We also identified non-Ate1 proteins that interact with Liat1. In contrast to Liat1 genes of nonprimate mammals, Liat1 genes of primates are subtelomeric, a location that tends to confer evolutionary instability on a gene. Remarkably, Liat1 proteins of some primates, from macaques to humans, contain tandem repeats of a 10-residue sequence, whereas Liat1 proteins of other mammals contain a single copy of this motif. Quantities of these repeats are, in general, different in Liat1 of different primates. For example, there are 1, 4, 13, 13, 17, and 17 repeats in the gibbon, gorilla, orangutan, bonobo, neanderthal, and human Liat1, respectively, suggesting that repeat number changes in this previously uncharacterized protein may contribute to evolution of primates. PMID:25369936

  14. Structure of the genetic code suggested by the hydropathy correlation between anticodons and amino acid residues

    NASA Astrophysics Data System (ADS)

    Farias, Sávio Torres De; Moreira, Carlos Henrique Costa; Guimarães, Romeu Cardoso

    2007-02-01

    The correlation between hydropathies of anticodons and amino acids, detected by other authors utilizing scales of amino acid molecules in solution, was improved with the utilization of scales of amino acid residues in proteins. Three partitions were discerned in the correlation plot with the principal dinucleotides of anticodons (pDiN, excluding the wobble position). (a) The set of outliers of the correlation: Gly-CC, Pro-GG, Ser-GA and Ser-CU. The amino acids are consistently small, hydro-apathetic, stabilizers of protein N-ends, preferred in aperiodic protein conformations and belong to synthetases class II. The pDiN sequences are representative of the homogeneous sector (triplets N RR and N YY), distinguished from the mixed sector (triplets N RY and N YR), that depict a 70% correspondence to the synthetases class II and I, respectively. The triplet pairs proposed to be responsible for the coherence in the set of outliers are of the palindromic kind, where the lateral bases are the same, C CC: G GG and A GA: U CU. This suggests that U CU previously belonged to Ser, adding to other indications that the attribution of Arg to Y CU was due to an expansion of the Arg- tRNA synthetase specificity. The other attributions produced two correlation sets. (b) One corresponds to the remaining pDiN of the homogeneous sector, containing both synthetase classes; its regression line overlapped the one formed by the remaining attributions to class II. (c) The other contains the pDiN of the mixed sector and produced steeper slopes, especially with the class I attributions. It is suggested that the correlation was established when the amino acid composition of the protein synthetases became progressively enriched and that the set of outliers were the earliest to have been fixed.

  15. CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

    PubMed Central

    Yokota, K; Kagawa, S; Shimizu, Y; Akioka, H; Tsurumi, C; Noda, C; Fujimuro, M; Yokosawa, H; Fujiwara, T; Takahashi, E; Ohba, M; Yamasaki, M; DeMartino, G N; Slaughter, C A; Toh-e, A; Tanaka, K

    1996-01-01

    The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome. Images PMID:8816993

  16. Parity-dependent rotational rainbows in D2-NO and He-NO differential collision cross sections.

    PubMed

    Gijsbertsen, Arjan; Linnartz, Harold; Stolte, Steven

    2006-10-01

    The (j', Omega', epsilon') dependent differential collision cross sections of D2 with fully state selected (j = 12, Omega = 12, epsilon = -1) NO have been determined at a collision energy of about 550 cm(-1). The collisionally excited NO molecules are detected by (1+1') resonance enhanced multiphoton ionization combined using velocity-mapped ion-imaging. The results are compared to He-NO scattering results and tend to be more forward scattered for the same final rotational state. Both for collisions of the atomic He and the molecular D2 with NO, scattering into pairs of rotational states with the same value of n = j' - epsilon epsilon'2 yields the same angular dependence of the cross section. This "parity propensity rule" remains present both for spin-orbit conserving and spin-orbit changing transitions. The maxima in the differential cross sections-that reflect rotational rainbows-have been extracted from the D2-NO and the He-NO differential cross sections. These maxima are found to be distinct for odd and even parity pair number n. Rainbow positions of parity changing transitions (n is odd) occur at larger scattering angles than those of parity conserving transitions (n is even). Parity conserving transitions exhibit-from a classical point of view-a larger effective eccentricity of the shell. No rainbow doubling due to collisions onto either the N-end or the O-end was observed. From a classical point of view the presence of a double rainbow is expected. Rotational excitation of the D2 molecules has not been observed. PMID:17029438

  17. Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12.

    PubMed

    Link, A J; Robison, K; Church, G M

    1997-08-01

    Mining the emerging abundance of microbial genome sequences for hypotheses is an exciting prospect of "functional genomics". At the forefront of this effort, we compared the predictions of the complete Escherichia coli genomic sequence with the observed gene products by assessing 381 proteins for their mature N-termini, in vivo abundances, isoelectric points, molecular masses, and cellular locations. Two-dimensional gel electrophoresis (2-DE) and Edman sequencing were combined to sequence Coomassie-stained 2-DE spots representing the abundant proteins of wild-type E. coli K-12 strains. Greater than 90% of the abundant proteins in the E. coli proteome lie in a small isoelectric point and molecular mass window of 4-7 and 10-100 kDa, respectively. We identified several highly abundant proteins, YjbJ, YjbP, YggX, HdeA, and AhpC, which would not have been predicted from the genomic sequence alone. Of the 223 uniquely identified loci, 60% of the encoded proteins are proteolytically processed. As previously reported, the initiator methionine was efficiently cleaved when the penultimate amino acid was serine or alanine. In contrast, when the penultimate amino acid was threonine, glycine, or proline, cleavage was variable, and valine did not signal cleavage. Although signal peptide cleavage sites tended to follow predicted rules, the length of the putative signal sequence was occassionally greater than the consensus. For proteins predicted to be in the cytoplasm or inner membrane, the N-terminal amino acids were highly constrained compared to proteins localized to the periplasm or outer membrane. Although cytoplasmic proteins follow the N-end rule for protein stability, proteins in the periplasm or outer membrane do not follow this rule; several have N-terminal amino acids predicted to destabilize the proteins. Surprisingly, 18% of the identified 2-DE spots represent isoforms in which protein products of the same gene have different observed pI and M(r), suggesting they are

  18. Pharmacological Inhibitors of the Proteosome in Atrophying Muscles

    NASA Technical Reports Server (NTRS)

    Goldberg, Alfred

    1999-01-01

    depth extracts from normal and atrophying muscles to compare the activities of the Ub-activating enzyme (El), the various LTh-carrier proteins (E2s), and Ub-protein ligases (E3s). Recent studies of other types of muscle wasting -suggest a very important role in muscle proteolysis of certain ubiquitination enzymes, E214k and E3-alpha(i.e. components of the "N-end pathway"). Future studies will focus in understanding their role and test whether they are in fact critical for muscle atrophy in vivo. Since weightlessness leads to a specific loss of contractile proteins and to a switching of myosin isotypes, Dr. Goldberg's group will attempt to identify the ubiquitination enzymes specifically involved in myosin degradation both in normal muscle and after hind-limb suspension.

  19. The February 6, 2013 Mw 8.0 Santa Cruz Islands earthquake and tsunami

    NASA Astrophysics Data System (ADS)

    Lay, Thorne; Ye, Lingling; Kanamori, Hiroo; Yamazaki, Yoshiki; Cheung, Kwok Fai; Ammon, Charles J.

    2013-11-01

    The Santa Cruz Islands region has high seismicity near a 90° bend in the boundary between the Pacific and Australian plates. Southward, along the Vanuatu island arc, the Australian plate under-thrusts the Pacific plate in a well-defined subduction zone. Westward, a transpressional, predominantly transform boundary extends to the southern Solomon Islands subduction zone. The Santa Cruz Islands region has upper plate strike-slip and normal faulting, plate boundary under-thrusting, outer rise extensional faulting, and intraplate compressional faulting. On February 6, 2013 the largest under-thrusting earthquake (Mw 8.0) that has been instrumentally recorded in the region ruptured the megathrust. The epicenter (10.738°S, 165.138°E) is about 1° north of epicenters of prior large shallow under-thrusting events in Vanuatu in 1934 (M ~ 7.8) and 1966 (MS 7.9), and there is overlap of all three events' aftershock zones, but not their large-slip regions. At least 10 lives were lost, with 6 more missing, due to tsunami ~ 1.5 m high striking the town of Lata and several villages on the main Santa Cruz Island of Nendö (Ndeni) and a nearby small island Nibanga. Inundation of 500 m flooded the Lata airport. The tsunami was well-recorded by DART buoys spanning an unusually wide three-quadrant azimuthal aperture. Iterative modeling of teleseismic broadband P waves and the deep-water tsunami recordings resolves the slip distribution. There are two large-slip patches with a southeastward rupture expansion at about 1.5 km/s, with the second patch appearing to have ruptured with large slip at the trench. The event has relatively low short-period seismic wave energy release, but a typical overall moment-scaled total energy. The shallow rupture depth may be associated with the low level of short-period energy, and a near-total stress drop may account for a lack of underthrusting aftershocks among a highly productive aftershock sequence that included three events with Mw ≥ 7.0.

  20. A Trihelix DNA Binding Protein Counterbalances Hypoxia-Responsive Transcriptional Activation in Arabidopsis

    PubMed Central

    Licausi, Francesco; Kosmacz, Monika; Oosumi, Teruko; van Dongen, Joost T.; Bailey-Serres, Julia; Perata, Pierdomenico

    2014-01-01

    Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule–insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein–protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen

  1. A trihelix DNA binding protein counterbalances hypoxia-responsive transcriptional activation in Arabidopsis.

    PubMed

    Giuntoli, Beatrice; Lee, Seung Cho; Licausi, Francesco; Kosmacz, Monika; Oosumi, Teruko; van Dongen, Joost T; Bailey-Serres, Julia; Perata, Pierdomenico

    2014-09-01

    Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule-insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein-protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen