scholarly journals Role of the β1 Subunit in the Function and Stability of the 20S Proteasome in the Hyperthermophilic Archaeon Pyrococcus furiosus

2006 ◽  
Vol 189 (2) ◽  
pp. 583-590 ◽  
Author(s):  
Lara S. Madding ◽  
Joshua K. Michel ◽  
Keith R. Shockley ◽  
Shannon B. Conners ◽  
Kevin L. Epting ◽  
...  
Keyword(s):  
Thermal Stress ◽  
Pyrococcus Furiosus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Response Shift ◽  
Transcriptional Analysis ◽  
20S Proteasome ◽  
Significant Activity ◽  
Peptide Hydrolysis ◽  
Hyperthermophilic Archaeon

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one α protein (PF1571) and two β proteins (β1-PF1404 and β2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of the P. furiosus dynamic heat shock response (shift from 90 to 105°C) showed that the β1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the β2/β1 ratio varied linearly with temperature from 3.8, when assembled at 80°C, to 0.9 at 105°C. The recombinant α+β1+β2 CP assembled at 105°C was more thermostable than either the α+β1+β2 version assembled at 90°C or the α+β2 version assembled at either 90°C or 105°C, based on melting temperature and the biocatalytic inactivation rate at 115°C. The recombinant CP assembled at 105°C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90°C assembly (measured at 95°C). Combination of the α and β1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the β1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that β subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.

scholarly journals Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

2000 ◽  
Vol 182 (9) ◽  
pp. 2559-2566 ◽  
Author(s):  
Donald E. Ward ◽  
Servé W. M. Kengen ◽  
John van der Oost ◽  
Willem M. de Vos
Keyword(s):  
Molecular Mass ◽  
Alanine Aminotransferase ◽  
Pyrococcus Furiosus ◽  
Sodium Dodecyl ◽  
Northern Analysis ◽  
Transcriptional Level ◽  
Significant Activity ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts

ABSTRACT Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosusby multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in theP. furiosus genome database. The gene was expressed inEscherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. Thek cat/Km values for alanine and pyruvate formation were 41 and 33 s−1mM−1, respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aatgene. In addition, both the aat and gdh(encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdhgenes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

scholarly journals Molecular Characterization and Functional Analysis of PR-1-Like Proteins Identified from the Wheat Head Blight Fungus Fusarium graminearum

2018 ◽  
Vol 108 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Shunwen Lu ◽  
Michael C. Edwards
Keyword(s):  
Functional Analysis ◽  
Fusarium Graminearum ◽  
Expression Patterns ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Transcriptional Analysis ◽  
Fungal Genome ◽  
Dimensional Structure ◽  
Head Blight ◽  
Fungal Virulence

The group 1 pathogenesis-related (PR-1) proteins originally identified from plants and their homologs are also found in other eukaryotic kingdoms. Studies on nonplant PR-1-like (PR-1L) proteins have been pursued widely in humans and animals but rarely in filamentous ascomycetes. Here, we report the characterization of four PR-1L proteins identified from the ascomycete fungus Fusarium graminearum, the primary cause of Fusarium head blight of wheat and barley (designated FgPR-1L). Molecular cloning revealed that the four FgPR-1L proteins are all encoded by small open reading frames (612 to 909 bp) that are often interrupted by introns, in contrast to plant PR-1 genes that lack introns. Sequence analysis indicated that all FgPR-1L proteins contain the PR-1-specific three-dimensional structure, and one of them features a C-terminal transmembrane (TM) domain that has not been reported for any stand-alone PR-1 proteins. Transcriptional analysis revealed that the four FgPR-1L genes are expressed in axenic cultures and in planta with different spatial or temporal expression patterns. Phylogenetic analysis indicated that fungal PR-1L proteins fall into three major groups, one of which harbors FgPR-1L-2-related TM-containing proteins from both phytopathogenic and human-pathogenic ascomycetes. Low-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteolytic assays indicated that the recombinant FgPR-1L-4 protein exists as a monomer and is resistant to subtilisin of the serine protease family. Functional analysis confirmed that deletion of the FgPR-1L-4 gene from the fungal genome results in significantly reduced virulence on susceptible wheat. This study provides the first example that the F. graminearum–wheat interaction involves a pathogen-derived PR-1L protein that affects fungal virulence on the host.

scholarly journals Antileishmanial Activity of Parthenolide, a Sesquiterpene Lactone Isolated from Tanacetum parthenium

2005 ◽  
Vol 49 (1) ◽  
pp. 176-182 ◽  
Author(s):  
Tatiana Shioji Tiuman ◽  
Tânia Ueda-Nakamura ◽  
Diógenes Aparício Garcia Cortez ◽  
Benedito Prado Dias Filho ◽  
José Andrés Morgado-Díaz ◽  
...  
Keyword(s):  
Sesquiterpene Lactone ◽  
Quantum Coherence ◽  
Morphological Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ionization Mass ◽  
Multiple Quantum ◽  
Significant Activity ◽  
Tanacetum Parthenium ◽  
Isolated Compound

ABSTRACT The in vitro activity of parthenolide against Leishmania amazonensis was investigated. Parthenolide is a sesquiterpene lactone purified from the hydroalcoholic extract of aerial parts of Tanacetum parthenium. This isolated compound was identified through spectral analyses by UV, infrared, 1H and 13C nuclear magnetic resonance imaging, DEPT (distortionless enhancement by polarization transfer), COSY (correlated spectroscopy), HMQC (heteronuclear multiple-quantum coherence), and electron spray ionization-mass spectrometry. Parthenolide showed significant activity against the promastigote form of L. amazonensis, with 50% inhibition of cell growth at a concentration of 0.37 μg/ml. For the intracellular amastigote form, parthenolide reduced by 50% the survival index of parasites in macrophages when it was used at 0.81 μg/ml. The purified compound showed no cytotoxic effects against J774G8 macrophages in culture and did not cause lysis in sheep blood when it was used at higher concentrations that inhibited promastigote forms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with gelatin as the substrate showed that the enzymatic activity of the enzyme cysteine protease increased following treatment of the promastigotes with the isolated compound. This finding was correlated with marked morphological changes induced by parthenolide, such as the appearance of structures similar to large lysosomes and intense exocytic activity in the region of the flagellar pocket, as seen by electron microscopy. These results provide new perspectives on the development of novel drugs with leishmanicidal activities obtained from natural products.

scholarly journals Properties of a Thermostable Nitrate Reductase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum

2001 ◽  
Vol 183 (19) ◽  
pp. 5491-5495 ◽  
Author(s):  
Sepideh Afshar ◽  
Eric Johnson ◽  
Simon de Vries ◽  
Imke Schröder
Keyword(s):  
Nitrate Reductase ◽  
Specific Activity ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Specific Activity ◽  
Competitive Inhibitor ◽  
Pyrobaculum Aerophilum ◽  
Benzyl Viologen ◽  
Hyperthermophilic Archaeon

ABSTRACT The nitrate reductase of the hyperthermophilic archaeonPyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (V max with nitrate, 1,162 s−1 (326 U/mg);V max with chlorate, 1,348 s−1 (378 U/mg) [assayed at 75°C]). The Km values for nitrate and chlorate were 58 and 140 μM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.

scholarly journals A completed KLK activome profile: investigation of activation profiles of KLK9, 10, and 15

2009 ◽  
Vol 390 (4) ◽  
Author(s):  
Hyesook Yoon ◽  
Sachiko I. Blaber ◽  
Mekdes Debela ◽  
Peter Goettig ◽  
Isobel A. Scarisbrick ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Bioactive Peptides ◽  
Additional Data ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Significant Activity ◽  
Terminal Sequence ◽  
Protein System ◽  
Hydrolytic Activities ◽  
Activation Pathways

Abstract We previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10 exhibit low hydrolytic activities towards all of the 15 pro-KLK sequences, while KLK15 exhibits significant activity towards both Arg- and Lys-containing KLK pro-sequences. The ability of KLK15 to activate pro-KLK8, 12, and 14 is confirmed using recombinant pro-KLK proteins, and shown to be significant for activation of pro-KLK8 and 14, but not 12. These additional data for KLK9, 10, and 15 now permit a completed KLK activome profile, using a KLK pro-peptide fusion-protein system, to be described. The results suggest that KLK15, once activated, can potentially feed back into additional pro-KLK activation pathways. Conversely, KLK9 and 10, once activated, are unlikely to participate in further pro-KLK activation pathways, although similar to KLK1 they may activate other bioactive peptides.

scholarly journals Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

2001 ◽  
Vol 67 (6) ◽  
pp. 2445-2452 ◽  
Author(s):  
Yuji Kannan ◽  
Yuichi Koga ◽  
Yohei Inoue ◽  
Mitsuru Haruki ◽  
Masahiro Takagi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Heat Inactivation ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon

ABSTRACT The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensissubtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding,T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly−82 to Gly316), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests thatT. kodakaraensis subtilisin exists in a monomeric form.T. kodakaraensis subtilisin hydrolyzed the synthetic substrateN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca2+ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.

scholarly journals Heat Shock Response by the Hyperthermophilic Archaeon Pyrococcus furiosus

2003 ◽  
Vol 69 (4) ◽  
pp. 2365-2371 ◽  
Author(s):  
Keith R. Shockley ◽  
Donald E. Ward ◽  
Swapnil R. Chhabra ◽  
Shannon B. Conners ◽  
Clemente I. Montero ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Pyrococcus Furiosus ◽  
Small Heat Shock Protein ◽  
Compatible Solute ◽  
Transcriptional Analysis ◽  
Hyperthermophilic Archaeon ◽  
Shock Response ◽  
Differential Gene

ABSTRACT Collective transcriptional analysis of heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus was examined by using a targeted cDNA microarray in conjunction with Northern analyses. Differential gene expression suggests that P. furiosus relies on a cooperative strategy of rescue (thermosome [Hsp60], small heat shock protein [Hsp20], and two VAT-related chaperones), proteolysis (proteasome), and stabilization (compatible solute formation) to cope with polypeptide processing during thermal stress.

scholarly journals Purification and Characterization of Serine Racemase from a Hyperthermophilic Archaeon, Pyrobaculum islandicum

2007 ◽  
Vol 190 (4) ◽  
pp. 1359-1365 ◽  
Author(s):  
Masato Ohnishi ◽  
Makoto Saito ◽  
Sadao Wakabayashi ◽  
Morio Ishizuka ◽  
Katsushi Nishimura ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Serine Racemase ◽  
Threonine Dehydratase ◽  
Hyperthermophilic Archaeon ◽  
Pyrobaculum Islandicum ◽  
Dehydratase Activity ◽  
Allosteric Properties

ABSTRACT Pyrobaculum islandicum is an anaerobic hyperthermophilic archaeon that is most active at 100°C. A pyridoxal 5′-phosphate-dependent serine racemase called Srr was purified from the organism. The corresponding srr gene was cloned, and recombinant Srr was purified from Escherichia coli. It showed the highest racemase activity toward l-serine, followed by l-threonine, d-serine, and d-threonine. Like rodent and plant serine racemases, Srr is bifunctional, showing high l-serine/l-threonine dehydratase activity. The sequence of Srr is 87% similar to that of Pyrobaculum aerophilum IlvA (a putative threonine dehydratase) but less than 32% similar to any other serine racemases and threonine dehydratases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses revealed that Srr is a homotrimer of a 44,000-molecular-weight subunit. Both racemase and dehydratase activities were highest at 95°C, while racemization and dehydration were maximum at pH 8.2 and 7.8, respectively. Unlike other, related Ilv enzymes, Srr showed no allosteric properties: neither of these enzymatic activities was affected by either l-amino acids (isoleucine and valine) or most of the metal ions. Only Fe2+ and Cu2+ caused 20 to 30% inhibition and 30 to 40% stimulation of both enzyme activities, respectively. ATP inhibited racemase activity by 10 to 20%. The Km and V max values of the racemase activity of Srr for l-serine were 185 mM and 20.1 μmol/min/mg, respectively, while the corresponding values of the dehydratase activity of l-serine were 2.2 mM and 80.4 μmol/min/mg, respectively.

scholarly journals Cold Shock of a Hyperthermophilic Archaeon: Pyrococcus furiosus Exhibits Multiple Responses to a Suboptimal Growth Temperature with a Key Role for Membrane-Bound Glycoproteins

2005 ◽  
Vol 187 (1) ◽  
pp. 336-348 ◽  
Author(s):  
Michael V. Weinberg ◽  
Gerrit J. Schut ◽  
Scott Brehm ◽  
Susmita Datta ◽  
Michael W. W. Adams
Keyword(s):  
Growth Temperature ◽  
Cold Shock ◽  
Pyrococcus Furiosus ◽  
Sodium Dodecyl ◽  
Optimal Growth ◽  
Cold Shock Protein ◽  
Hyperthermophilic Archaea ◽  
Lag Phase ◽  
Cold Response ◽  
Hyperthermophilic Archaeon

ABSTRACT The hyperthermophilic archaeon, Pyrococcus furiosus, was grown on maltose near its optimal growth temperature, 95°C, and at the lower end of the temperature range for significant growth, 72°C. In addition, cultures were shocked by rapidly dropping the temperature from 95 to 72°C. This resulted in a 5-h lag phase, during which time little growth occurred. Transcriptional analyses using whole-genome DNA microarrays representing 2,065 open reading frames (ORFs) in the P. furiosus genome showed that cells undergo three very different responses at 72°C: an early shock (1 to 2 h), a late shock (5 h), and an adapted response (occurring after many generations at 72°C). Each response involved the up-regulation in the expression of more than 30 ORFs unique to that response. These included proteins involved in translation, solute transport, amino acid biosynthesis, and tungsten and intermediary carbon metabolism, as well as numerous conserved-hypothetical and/or membrane-associated proteins. Two major membrane proteins were evident after one-dimensional sodium dodecyl sulfate-gel analysis of cold-adapted cells, and staining revealed them to be glycoproteins. Their cold-induced expression evident from the DNA microarray analysis was confirmed by quantitative PCR. Termed CipA (PF0190) and CipB (PF1408), both appear to be solute-binding proteins. While the archaea do not contain members of the bacterial cold shock protein (Csp) family, they all contain homologs of CipA and CipB. These proteins are also related phylogenetically to some cold-responsive genes recently identified in certain bacteria. The Cip proteins may represent a general prokaryotic-type cold response mechanism that is present even in hyperthermophilic archaea.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Sign in / Sign up

Export Citation Format

Share Document