scholarly journals Distribution and paralogue specificity of mammalian deSUMOylating enzymes

2010 ◽  
Vol 430 (2) ◽  
pp. 335-344 ◽  
Author(s):  
Nagamalleswari Kolli ◽  
Jowita Mikolajczyk ◽  
Marcin Drag ◽  
Debaditya Mukhopadhyay ◽  
Nela Moffatt ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Catalytic Efficiency ◽  
Covalent Attachment ◽  
Kinetic Properties ◽  
Kinetic Measurements ◽  
Systematic Analysis ◽  
Catalytic Domains ◽  
Irreversible Inhibitors ◽  
Marked Preference ◽  
Relative Selectivity

The covalent attachment of SUMO (small ubiquitin-like protein modifier) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalysed by SENPs (SUMO-specific processing proteases). Mammals contain four SUMO paralogues and six SENP enzymes. In the present paper, we describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal cSENPs (SENP catalytic domains). We first characterized the reaction of each endogenous SENP and cSENPs with HA–SUMO-VS [HA (haemagglutinin)-tagged SUMO-vinyl sulfones], active-site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogues, whereas all other endogeneous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1- or SUMO2-AMC (7-amino-4-methylcoumarin) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by Km and kcat, whereas cSENP7 works only on SUMO2. Compared with cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity, indicating that paralogue specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggest that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.

Molecular forms and thermal and kinetic properties of purified glutathione reductase from two populations of barnyard grass (Echinochloa crus-galli(L.) Beauv.: Poaceae) from contrasting climatic regions in North America

2000 ◽  
Vol 78 (7) ◽  
pp. 969-980 ◽  
Author(s):  
Nadia Hakam ◽  
Jean-Pierre Simon
Keyword(s):  
Glutathione Reductase ◽  
Thermal Adaptation ◽  
Catalytic Efficiency ◽  
Molecular Forms ◽  
Kinetic Properties ◽  
Barnyard Grass ◽  
Climatic Regions ◽  
Two Populations ◽  
Menten Constant

The thermal, kinetic, and electrophoretic properties of purified glutathione reductase (GR; EC 1.6.4.2) were analyzed in plants from two ecotypes of barnyard grass (Echinochloa crus-galli (L.) Beauv.: Poaceae) originating from sites of contrasting climates in Quebec (QUE) and Mississippi (MISS). Crude and purified GR preparations from plants of both ecotypes consisted of one homodimer isomorph with the same electrophoretic mobility in polyacrylamide gels, a similar molecular mass for the native enzyme (98 kDa) and for each subunit of the dimer (44 kDa), and an identical pI of 5.9. The electrophoretic profile of GR purified from cold-acclimated plants at 14°C light (L) : 8°C dark (D) for 10 days was similar to that of GR from plants grown at 26°C L : 20°C D. Specific activities of purified GR from QUE plants were significantly higher than those of MISS plants. In vitro GR activities from QUE and MISS plants were not differentially affected by thermodenaturation at 55 or 65°C or by cold treatments at 2°C. Apparent energies of activation (Ea) of GR purified from QUE and MISS plants were similar with the exception of estimates of Ea(oxidized glutathione) for Q10(15-5°C) for which significantly lower values were obtained for QUE plants. No differences of physiological significance were observed for Km(Michaelis-Menten constant) values of GR purified from QUE and MISS plants. However, both Vmaxand Kcat(turnover numbers) estimates were significantly higher for GR purified from QUE plants over most of the range of assay temperatures, suggesting superior catalytic efficiency for the enzyme of the cold-adapted ecotype from Québec.Key words: barnyard grass, ecotypes, electrophoresis, enzyme kinetics, glutathione reductase, thermal adaptation.

scholarly journals Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium

2001 ◽  
Vol 353 (3) ◽  
pp. 635-644 ◽  
Author(s):  
Hidekazu KUWAYAMA ◽  
Helena SNIPPE ◽  
Mari DERKS ◽  
Jeroen ROELOFS ◽  
Peter J. M. VAN HAASTERT
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Catalytic Domain ◽  
Expressed Sequence Tag ◽  
Sequence Similarity ◽  
Second Messengers ◽  
Kinetic Properties ◽  
Intracellular Camp ◽  
Catalytic Domains ◽  
Dual Specificity

In Dictyostelium cAMP and cGMP have important functions as first and second messengers in chemotaxis and development. Two cyclic-nucleotide phosphodiesterases (DdPDE 1 and 2) have been identified previously, an extracellular dual-specificity enzyme and an intracellular cAMP-specific enzyme (encoded by the psdA and regA genes respectively). Biochemical data suggest the presence of at least one cGMP-specific phosphodiesterase (PDE) that is activated by cGMP. Using bioinformatics we identified a partial sequence in the Dictyostelium expressed sequence tag database that shows a high degree of amino acid sequence identity with mammalian PDE catalytic domains (DdPDE3). The deduced amino acid sequence of a full-length DdPDE3 cDNA isolated in this study predicts a 60kDa protein with a 300-residue C-terminal PDE catalytic domain, which is preceded by approx. 200 residues rich in asparagine and glutamine residues. Expression of the DdPDE3 catalytic domain in Escherichia coli shows that the enzyme has Michaelis–Menten kinetics and a higher affinity for cGMP (Km = 0.22µM) than for cAMP (Km = 145µM); cGMP does not stimulate enzyme activity. The enzyme requires bivalent cations for activity; Mn2+ is preferred to Mg2+, whereas Ca2+ yields no activity. DdPDE3 is inhibited by 3-isobutyl-1-methylxanthine with an IC50 of approx. 60µM. Overexpression of the DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications of its activation by cGMP. The properties of DdPDE3 resemble those of mammalian PDE9, which also shows the highest sequence similarity within the catalytic domains. DdPDE3 is the first cGMP-selective PDE identified in lower eukaryotes.

scholarly journals Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR.

1996 ◽  
Vol 16 (11) ◽  
pp. 6295-6302 ◽  
Author(s):  
D R Taylor ◽  
S B Lee ◽  
P R Romano ◽  
D R Marshak ◽  
A G Hinnebusch ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Initiation Factor ◽  
Double Stranded Rna ◽  
Catalytic Domains ◽  
Dependent Protein ◽  
Mutant Kinase

The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state. In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR. Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro. The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken together with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, and M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation.

scholarly journals The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke

1976 ◽  
Vol 159 (3) ◽  
pp. 585-600 ◽  
Author(s):  
K S Chapman ◽  
J Ingle
Keyword(s):  
Mammalian Cells ◽  
Molecular Size ◽  
Rrna Synthesis ◽  
Kinetic Measurements ◽  
Polyadenylic Acid ◽  
Nuclear Rna ◽  
The Stability ◽  
Time Required ◽  
Total Tissue ◽  
Isopycnic Centrifugation

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 × 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 × 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 × 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 × 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.

scholarly journals Molecular Basis for Rab Prenylation

2000 ◽  
Vol 150 (1) ◽  
pp. 89-104 ◽  
Author(s):  
Christelle Alory ◽  
William E. Balch
Keyword(s):  
Mammalian Cells ◽  
Normal Growth ◽  
Kinetic Properties ◽  
Temperature Sensitive ◽  
Rab Gtpases ◽  
Inherited Disease ◽  
Vesicular Traffic ◽  
Null Strain

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Δ null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Δ null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6ts) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.

scholarly journals Loss of Glyoxalase 1 Induces Compensatory Mechanism to Achieve Dicarbonyl Detoxification in Mammalian Schwann Cells

2016 ◽  
Vol 292 (8) ◽  
pp. 3224-3238 ◽  
Author(s):  
Jakob Morgenstern ◽  
Thomas Fleming ◽  
Dagmar Schumacher ◽  
Volker Eckstein ◽  
Marc Freichel ◽  
...  
Keyword(s):  
Schwann Cells ◽  
Aldose Reductase ◽  
Mammalian Cells ◽  
Clinical Importance ◽  
Catalytic Efficiency ◽  
Compensatory Mechanism ◽  
Glyoxalase System ◽  
Compensatory Mechanisms ◽  
Knock Out ◽  
Glyoxalase 1

The glyoxalase system is a highly specific enzyme system existing in all mammalian cells that is responsible for the detoxification of dicarbonyl species, primarily methylglyoxal (MG). It has been implicated to play an essential role in preventing the increased formation of advanced glycation end products under certain pathological conditions. We have established the first glyoxalase 1 knock-out model (GLO1−/−) in mammalian Schwann cells using the CRISPR/Cas9 technique to investigate compensatory mechanisms. Neither elevated concentrations of MG nor associated protein modifications were observed in GLO1−/− cells. Alternative detoxification of MG in GLO1−/− is achieved by increased catalytic efficiency of aldose reductase toward hemithioacetal (product of glutathione and MG), which is most likely caused by S-nitrosylation of aldose reductase. The hemithioacetal is mainly converted into lactaldehyde, which is paralleled by a loss of reduced glutathione. Inhibition of aldose reductase in GLO1−/− cells is associated with an increased sensitivity against MG, elevated intracellular MG levels, associated modifications, as well as increased oxidative stress. Our data suggest that aldose reductase can compensate for the loss of GLO1. This might be of clinical importance within the context of neuronal diseases caused by an impaired glyoxalase system and elevated levels of dicarbonyl species, such as MG.

scholarly journals Importance of Covalent and Noncovalent SUMO Interactions with the Major Human Cytomegalovirus Transactivator IE2p86 for Viral Infection

2009 ◽  
Vol 83 (24) ◽  
pp. 12881-12894 ◽  
Author(s):  
Anja Berndt ◽  
Heike Hofmann-Winkler ◽  
Nina Tavalai ◽  
Gabriele Hahn ◽  
Thomas Stamminger
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Mammalian Cells ◽  
Laboratory Strain ◽  
Early Gene ◽  
Covalent Attachment ◽  
Protein Accumulation ◽  
Altered Expression ◽  
Efficient Replication

ABSTRACT The major transactivator protein IE2p86 of human cytomegalovirus (HCMV) has previously been shown to undergo posttranslational modification by the covalent attachment of SUMO proteins, termed SUMOylation, which occurs at two lysine residues located at amino acid positions 175 and 180. Mutation of the acceptor lysines resulted in the abrogation of IE2p86 SUMOylation in mammalian cells and a strong reduction of IE2p86-mediated transactivation. In this paper, we identify an additional SUMO interaction motif (SIM) within IE2p86, which mediates noncovalent binding to SUMO, as shown by yeast two-hybrid analyses. Transient-expression experiments revealed that an IE2p86 SIM mutant exhibited significantly reduced SUMOylation, strongly suggesting that noncovalent SUMO interactions affect the efficacy of covalent SUMO coupling. In order to define the relevance of IE2p86 SUMO interactions for viral replication, recombinant viruses originating from two different HCMV strains (AD169 and VR1814) were generated. Analysis of viruses expressing SUMOylation-negative IE2p86 revealed strongly impaired replication due to reduced viral DNA and protein accumulation, as well as diminished initiation of immediate-early gene expression. The additional introduction of the SIM mutation into the viral genome did not further compromise viral replication but resulted in altered expression of viral proteins at late times postinfection. In summary, this paper clearly shows that IE2p86 SUMOylation is necessary for efficient replication of the HCMV laboratory strain AD169 and the clinical isolate VR1814 and thus for the in vivo function of this viral transcription factor.

scholarly journals Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II

2008 ◽  
Vol 28 (4) ◽  
pp. 205-215 ◽  
Author(s):  
Qian Han ◽  
Tao Cai ◽  
Danilo A. Tagle ◽  
Howard Robinson ◽  
Jianyong Li
Keyword(s):  
Substrate Specificity ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Amino Group ◽  
Kynurenine Aminotransferase ◽  
Broad Substrate Specificity ◽  
Systematic Assessment ◽  
The Brain

KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to α-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested α-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with α-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15–33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

scholarly journals Phosphoenolpyruvate Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2001 ◽  
Vol 183 (2) ◽  
pp. 709-715 ◽  
Author(s):  
Andrea M. Hutchins ◽  
James F. Holden ◽  
Michael W. W. Adams
Keyword(s):  
Molecular Mass ◽  
Functional Role ◽  
Pyrococcus Furiosus ◽  
Active Form ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Kinetic Properties ◽  
Additional Function ◽  
Hyperthermophilic Archaeon ◽  
A Subunit

ABSTRACT Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690 ± 20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large (∼1.6 MDa) complex that is not active. The apparentKm values and catalytic efficiencies for the substrates pyruvate and ATP (at 80°C, pH 8.4) were 0.11 mM and 1.43 × 104 mM−1 · s−1 and 0.39 mM and 3.40 × 103mM−1 · s−1, respectively. Maximal activity was measured at pH 9.0 (at 80°C) and at 90°C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k cat/Km = 32 (mM · s)−1]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration (∼5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.

scholarly journals Molecular, functional and structural properties of the prolyl oligopeptidase of Trypanosoma cruzi (POP Tc80), which is required for parasite entry into mammalian cells

2005 ◽  
Vol 388 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Izabela M. D. BASTOS ◽  
Philippe GRELLIER ◽  
Natalia F. MARTINS ◽  
Gloria CADAVID-RESTREPO ◽  
Marian R. de SOUZA-AULT ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Single Copy ◽  
Catalytic Triad ◽  
Host Cells ◽  
Mammalian Host ◽  
Kinetic Properties ◽  
Prolyl Oligopeptidase ◽  
Single Chromosome

We have demonstrated that the 80 kDa POP Tc80 (prolyl oligopeptidase of Trypanosoma cruzi) is involved in the process of cell invasion, since specific inhibitors block parasite entry into non-phagocytic mammalian host cells. In contrast with other POPs, POP Tc80 is capable of hydrolysing large substrates, such as fibronectin and native collagen. In this study, we present the cloning of the POPTc80 gene, whose deduced amino acid sequence shares considerable identity with other members of the POP family, mainly within its C-terminal portion that forms the catalytic domain. Southern-blot analysis indicated that POPTc80 is present as a single copy in the genome of the parasite. These results are consistent with mapping of POPTc80 to a single chromosome. The active recombinant protein (rPOP Tc80) displayed kinetic properties comparable with those of the native enzyme. Novel inhibitors were assayed with rPOP Tc80, and the most efficient ones presented values of inhibition coefficient Ki≤1.52 nM. Infective parasites treated with these specific POP Tc80 inhibitors attached to the surface of mammalian host cells, but were incapable of infecting them. Structural modelling of POP Tc80, based on the crystallized porcine POP, suggested that POP Tc80 is composed of an α/β-hydrolase domain containing the catalytic triad Ser548–Asp631–His667 and a seven-bladed β-propeller non-catalytic domain. Docking analysis suggests that triple-helical collagen access to the catalytic site of POP Tc80 occurs in the vicinity of the interface between the two domains.

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