bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the Na+/H+ exchanger protein
Authors: Bullis Bonnie L. , Li Xiuju , Rieder Carmen V. , Singh Dyal N. , Berthiaume Luc G. , Fliegel Larry
Published: March 2003

The Na+/H+ exchanger is a ubiquitous membrane protein of bacteria, plants and mammals. The first isoform discovered (NHE1) is present on the mammalian plasma membrane and transports one H+ out of cells in exchange for one extracellular Na+. With solubilization in standard SDS/PAGE buffer, this protein had a high tendency to aggregate when subjected to elevated temperature. The aggregates were stable and did not dissociate in high concentrations of SDS or 2‐mercaptoethanol. We examined the distribution of the Na+/H+ exchanger within membrane subfractions. The Na+/H+ exchanger was found both in caveolin‐containing fractions and, in lesser amounts, in higher density membrane fractions where the bulk of proteins were contained. Treatment with cytochalasin D caused only a minor reduction of the amount of Na+/H+ exchanger present in caveolin‐enriched fractions suggesting an intact cytoskeleton was not important for NHE1 localization to these microdomains. Treatment of cells with methyl β‐cyclodextrin had a small stimulatory effect on Na+/H+ exchanger activity and reduced the amount of Na+/H+ exchanger in low density membrane fractions. Our study demonstrates that SDS cannot maintain the protein in a monomeric state suggesting that strong hydrophobic interactions are responsible for this temperature dependent aggregation behavior. In addition a large proportion of the Na+/H+ exchanger protein is found to be enriched in low density caveolin‐containing fractions.
The analysis of the fine specificity of celiac disease antibodies using tissue transglutaminase fragments
Authors: Sblattero Daniele , Florian Fiorella , Azzoni Elisabetta , Zyla Trevin , Park Min , Baldas Valentina , Not Tarcisio , Ventura Alessandro , Bradbury Andrew , Marzari Roberto
Published: March 2003

Celiac disease is an intestinal malabsorption characterized by an intolerance to cereal proteins accompanied by immunological responses to dietary gliadins and an autoantigen located in the endomysium. The latter has been identified as the enzyme tissue transglutaminase which belongs to a family of enzymes that catalyze protein cross‐linking reactions and is constitutively expressed in many tissues as well as being activated during apoptosis. In a recent paper, we described the selection and characterization of anti‐transglutaminase Igs from phage antibody libraries created from intestinal lymphocytes from celiac disease patients. In this work, using transglutaminase gene fragments, we identify a region of tissue transglutaminase recognized by these antibodies as being conformational and located in the core domain of the enzyme. This is identical to the region recognized by anti‐transglutaminase Igs found in the serum of celiac disease patients.
Domain V of m‐calpain shows the potential to form an oblique‐orientated α‐helix, which may modulate the enzyme's activity via interactions with anionic lipid
Authors: Brandenburg Klaus , Harris Frederick , Dennison Sarah , Seydel Ulrich , Phoenix David
Published: March 2003

The activity of m‐calpain, a heterodimeric, Ca2+‐dependent cysteine protease appears to be modulated by membrane interactions involving oblique‐orientated α‐helix formation by a segment, GTAMRILGGVI, in the protein's smaller subunit. Here, graphical and hydrophobic moment‐based analyses predicted that this segment may form an α‐helix with strong structural resemblance to the influenza virus peptide, HA2, a known oblique‐orientated α‐helix former. Fourier transform infrared spectroscopy showed that a peptide homologue of the GTAMRILGGVI segment, VP1, adopted low levels of α‐helical structure (≈ 20%) in the presence of zwitterionic lipid and induced a minor decrease (3 °C) in the gel to liquid‐crystalline phase transition temperature, TC, of the hydrocarbon chains of zwitterionic membranes, suggesting interaction with the lipid headgroup region. In contrast, VP1 adopted high levels of α‐helical structure (65%) in the presence of anionic lipid, induced a large increase (10 °C) in the TC of anionic membranes, and showed high levels of anionic lipid monolayer penetration (ΔSP = 5.5 mN·m−1), suggesting deep levels of membrane penetration. VP1 showed strong haemolytic ability (LD50 = 1.45 mm), but in the presence of ionic agents, this ability, and that of VP1 to penetrate anionic lipid monolayers, was greatly reduced. In combination, our results suggest that m‐calpain domain V may penetrate membranes via the adoption of an oblique‐orientated α‐helix and electrostatic interactions. We speculate that these interactions may involve snorkelling by an arginine residue located in the polar face of this α‐helix.
The cytoplasmic C‐terminus of the sulfonylurea receptor is important for KATP channel function but is not key for complex assembly or trafficking
Authors: Giblin Jonathan P. , Quinn Kathryn , Tinker Andrew
Published: March 2003

ATP‐sensitive K+ channels are an octameric assembly of two proteins, a sulfonylurea receptor (SUR1) and an ion conducting subunit (Kir 6.0). We have examined the role of the C‐terminus of SUR1 by expressing a series of truncation mutants together with Kir6.2 stably in HEK293 cells. Biochemical analyses using coimmunoprecipitation indicate that SUR1 deletion mutants and Kir6.2 assemble and that a SUR1 deletion mutant binds glibenclamide with high affinity. Electrophysiological recordings indicate that ATP sensitivity is normal but the response of the mutant channel complexes to tolbutamide, MgADP and diazoxide is disturbed. Quantitative immunofluorescence and cell surface biotinylation supports the idea that there is little disturbance in the efficiency of trafficking. Our data show that deletions of the C‐terminal most cytoplasmic domain of SUR1, can result in functional channels at the plasma membrane in mammalian cells that have an abnormal response to physiological and pharmacological agents.
Characterization of the exopolysaccharide produced by Streptococcus thermophilus 8S containing an open chain nononic acid
Authors: Faber Elisabeth J. , van Haaster Daan J. , Kamerling Johannis P. , Vliegenthart Johannes F. G.
Published: March 2003

The exopolysaccharide produced by Streptococcus thermophilus 8S in reconstituted skimmed milk is a heteropolysaccharide containing d‐galactose, d‐glucose, d‐ribose, and N‐acetyl‐d‐galactosamine in a molar ratio of 2 : 1 : 1 : 1. Furthermore, the polysaccharide contains one equivalent of a novel open chain nononic acid constituent, 3,9‐dideoxy‐d‐threo‐d‐altro‐nononic acid, ether‐linked via C‐2 to C‐6 of an additional d‐glucose per repeating unit. Methylation analysis and 1D/2D NMR studies (1H and 13C) performed on the native polysaccharide, and mass spectrometric and NMR analyses of the oligosaccharide obtained from the polysaccharide by de‐N‐acetylation followed by deamination and reduction demonstrated the ‘hepta’saccharide repeating unit to be:

in which Sug is 6‐O‐(3′,9′‐dideoxy‐d‐threo‐d‐altro‐nononic acid‐2′‐yl)‐α‐d‐glucopyranose.
Concerted regulation of free arachidonic acid and hormone‐induced steroid synthesis by acyl‐CoA thioesterases and acyl‐CoA synthetases in adrenal cells
Authors: Maloberti Paula , Lozano Rocío Castilla , Mele Pablo G. , Cano Florencia , Colonna Cecilia , Mendez Carlos F. , Paz Cristina , Podestá Ernesto J.
Published: March 2003

Although the role of arachidonic acid (AA) in the regulation of steroidogenesis is well documented, the mechanism for AA release is not clear. Therefore, the aim of this study was to characterize the role of an acyl‐CoA thioesterase (ARTISt) and an acyl‐CoA synthetase as members of an alternative pathway in the regulation of the intracellular levels of AA in steroidogenesis. Purified recombinant ARTISt releases AA from arachidonoyl‐CoA (AA‐CoA) with a Km of 2 µm. Antibodies raised against recombinant acyl‐CoA thioesterase recognize the endogenous protein in both adrenal tissue and Y1 adrenal tumor cells by immunohistochemistry and immunocytochemistry and Western blot. Stimulation of Y1 cells with ACTH significantly stimulated endogenous mitochondrial thioesterases activity (1.8‐fold). Nordihydroguaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochondrial acyl‐CoA thioesterases. ACTH‐stimulated steroid synthesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA‐CoA synthetase. The apparent IC50 for NDGA was reduced from 50 µm to 25, 7.5 and 4.5 µm in the presence of 0.1, 0.5 and 2 µm triacsin C, respectively. Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells.
Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N‐terminal region of the heavy chain
Authors: Nikolaeva Olga P. , Orlov Victor N. , Bobkov Andrey A. , Levitsky Dmitrii I.
Published: March 2003

The thermal unfolding of myosin subfragment 1 (S1) cleaved by trypsin was studied by differential scanning calorimetry. In the absence of nucleotides, trypsin splits the S1 heavy chain into three fragments (25, 50, and 20 kDa). This cleavage has no appreciable influence on the thermal unfolding of S1 examined in the presence of ADP, in the ternary complexes of S1 with ADP and phosphate analogs, such as orthovanadate (Vi) or beryllium fluoride (BeFx), and in the presence of F‐actin. In the presence of ATP and in the complexes S1·ADP·Vi or S1·ADP·BeFx, trypsin produces two additional cleavages in the S1 heavy chain: a faster cleavage in the N‐terminal region between Arg23 and Ile24, and a slower cleavage at the 50 kDa fragment. It has been shown that the N‐terminal cleavage strongly decreases the thermal stability of S1 by shifting the maximum of its thermal transition by about 7 °C to a lower temperature, from 50 °C to 42.4 °C, whereas the cleavage at both these sites causes dramatic destabilization of the S1 molecule leading to total loss of its thermal transition. Our results show that S1 with ATP‐induced N‐terminal cleavage is able, like uncleaved S1, to undergo global structural changes in forming the stable ternary complexes with ADP and Pi analogs (Vi, BeFx). These changes are reflected in a pronounced increase of S1 thermal stability. However, S1 cleaved by trypsin in the N‐terminal region is unable, unlike S1, to undergo structural changes induced by interaction with F‐actin that are expressed in a 4–5 °C shift of the S1 thermal transition to higher temperature. Thus, the cleavage between Arg23 and Ile24 does not significantly affect nucleotide‐induced structural changes in the S1, but it prevents structural changes that occur when S1 is bound to F‐actin. The results suggest that the N‐terminal region of the S1 heavy chain plays an important role in structural stabilization of the entire motor domain of the myosin head, and a long‐distance communication pathway may exist between this region and the actin‐binding sites.
Characterization of selenoprotein P as a selenium supply protein
Authors: Saito Yoshiro , Takahashi Kazuhiko
Published: March 2003

Selenium (Se) is well known to be essential for cell culture when using a serum‐free medium, but not when a medium containing serum is used. This finding suggests that serum contains some usable form of Se. To identify the Se‐supplier, T‐lymphoma (Jurkat) cells were cultured for 3 days in the presence of human serum immunodepleted of Se‐containing serum protein, selenoprotein P or extracellular glutathione peroxidase. The Se‐dependent enzyme activities (glutathione peroxidases and thioredoxin reductase) and Se content within the cells markedly decreased only when cultured with selenoprotein P‐depleted serum. Compared with other Se‐containing proteins, the addition of purified selenoprotein P to the selenoprotein P‐depleted serum or a serum‐free medium was the most effective for the recovery of cellular glutathione peroxidase activity (index of Se status). These results suggest that selenoprotein P functions as a Se‐supply protein, delivering Se to the cells.
Molecular characterization of MRG19 of Saccharomyces cerevisiae
Authors: Khanday Firdous A. , Saha Maitreyi , Bhat Paike Jayadeva
Published: March 2003

We have reported previously that multiple copies of MRG19 suppress GAL genes in a wild‐type but not in a gal80 strain of Saccharomyces cerevisiae. In this report we show that disruption of MRG19 leads to a decrease in GAL induction when S. cerevisiae is induced with 0.02% but not with 2.0% galactose. Disruption of MRG19 in a gal3 background (this strain shows long‐term adaptation phenotype) further delays the GAL induction, supporting the notion that its function is important only under low inducing signals. As a corollary, disruption of MRG19 in a gal80 strain did not decrease the constitutive expression of GAL genes. These results suggest that MRG19 has a role in GAL regulation only when the induction signal is weak. Unlike the effect on GAL gene expression, disruption of MRG19 leads to de‐repression of CYC1‐driven β‐galactosidase activity. MRG19 disruptant also showed a twofold increase in the rate of oxygen uptake as compared with the wild‐type strain. ADH2, CTA1, DLD1, and CYC7 promoters that are active during nonfermentative growth did not show any de‐repression of β‐galactosidase activity in the MRG19 disruptant. Western blot analysis indicated that MRG19 is a glucose repressible gene and is expressed in galactose and glycerol plus lactate. Experiments using green fluorescent protein fusion constructs indicate that Mrg19p is localized in the nucleus consistent with the presence of a consensus nuclear localization signal sequence. Based on the above results, we propose that Mrg19p is a regulator of galactose and nonfermentable carbon utilization.
Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI‐A down regulates c‐mycgene expression and endothelial cell proliferation
Authors: Pintus Gianfranco , Tadolini Bruna , Posadino Anna Maria , Sanna Bastiano , Debidda Marcella , Bennardini Federico , Sava Gianni , Ventura Carlo
Published: March 2003

Imidazoliumtrans‐imidazoledimethyl sulfoxide‐tetrachlororuthenate (NAMI‐A) is a novel ruthenium‐containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI‐A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down‐regulation of ERK phosphorylation in serum‐cultured cells. In addition, NAMI‐A markedly reduced serum stimulated‐ and completely suppressed phorbol 12‐myristate 13‐acetate (PMA)‐triggered MAPK/ERK kinase activity. NAMI‐A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA‐induced ERK phosphorylation. Finally, NAMI‐A and PD98059 down regulated c‐mycgene expression to the same extent in serum‐cultured cells and dose‐dependently counteracted, and ultimately abolished, the increase in c‐mycgene expression elicited by PMA in serum‐free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI‐A may have an important role in modulating c‐mycgene expression and ECV304 proliferation.
Characterization of a low redox potential laccase from the basidiomycete C30
Authors: Klonowska Agnieszka , Gaudin Christian , Fournel André , Asso Marcel , Le Petit Jean , Giorgi Michel , Tron Thierry
Published: March 2003

A new exocellular laccase was purified from the basidiomycete C30. LAC2 is an acidic protein (pI = 3.2) preferentially produced upon a combined induction by copper and p‐hydroxybenzoate. The spectroscopic signature (UV/visible and EPR) of this isoform is typical of multicopper oxidases, but its enzymatic and physico‐chemical properties proved to be markedly different from those of LAC1, the constitutive laccase previously purified from the same organism. In particular, the LAC2 kcat values observed for the oxidation of the substrates syringaldazine (kcat = 65 600 min−1), ABTS (2,2‐azino‐bis‐[3‐ethylthiazoline‐6‐sulfonate] (kcat = 41 000 min−1) and guaiacol (kcat = 75 680 min−1) are 10–40 times those obtained with LAC1 and the redox potential of its T1 copper is 0.17 V lower than that of LAC1 (E° = 0.73 V). This is the first report on a single organism producing simultaneously both a high and a low redox potential laccase. The cDNA, clac2, was cloned and sequenced. It encodes a protein of 528 amino acids that shares 69% identity (79% similarity) with LAC1 and 81% identity (95% similarity) with Lcc3‐2 from Polyporus ciliatus (AF176321‐1), its nearest neighbor in database. Possible reasons for why this basidiomycete produces, in vivo, enzyme forms with such different behaviors are discussed.
Trehalose‐phosphate synthase of Mycobacterium tuberculosis
Authors: Pan Y. T. , Carroll J. D. , Elbein A. D.
Published: March 2003

The trehalose‐phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of ≈ 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1‐L culture of E. coli (≈ 2.3 g cell paste). The purified recombinant enzyme showed a single band of ≈ 58 kDa on SDS/PAGE, but a molecular mass of ≈ 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP–glucose, CDP–glucose, GDP–glucose, TDP–glucose and UDP–glucose, with ADP–glucose, GDP–glucose and UDP–glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose‐P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP–glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP–glucose as the glucosyl donor was ≈ 18 mm, and that for GDP–glucose was ≈ 16 mm. The enzyme was specific for glucose‐6‐P as the glucosyl acceptor, and the Km for this substrate was ≈ 7 mm when UDP–glucose was the glucosyl donor and ≈ 4 mm with GDP–glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.
Transcriptional activity of interferon regulatory factor (IRF)‐3 depends on multiple protein–protein interactions
Authors: Yang Hongmei , Lin Charles H. , Ma Gang , Orr Melissa , Baffi Michael O. , Wathelet Marc G.
Published: March 2003

Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF‐3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF‐3 is not fully elucidated, yet it involves a conformational change triggered by the virus‐dependent phosphorylation of its C‐terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF‐3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF‐3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF‐3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF‐3.
The tungsten‐containing formate dehydrogenase from Methylobacterium extorquens AM1: Purification and properties
Authors: Laukel Markus , Chistoserdova Ludmila , Lidstrom Mary E. , Vorholt Julia A.
Published: March 2003

NAD‐dependent formate dehydrogenase (FDH1) was isolated from the α‐proteobacterium Methylobacterium extorquens AM1 under oxic conditions. The enzyme was found to be a heterodimer of two subunits (α1β1) of 107 and 61 kDa, respectively. The purified enzyme contained per mol enzyme ≈ 5 mol nonheme iron and acid‐labile sulfur, 0.6 mol noncovalently bound FMN, and ≈ 1.8 mol tungsten. The genes encoding the two subunits of FDH1 were identified on the M. extorquens AM1 chromosome next to each other in the order fdh1B, fdh1A. Sequence comparisons revealed that the α‐subunit harbours putative binding motifs for the molybdopterin cofactor and at least one iron–sulfur cluster. Sequence identity was highest to the catalytic subunits of the tungsten‐ and selenocysteine‐containing formate dehydrogenases characterized from Eubacterium acidaminophilum and Moorella thermoacetica (Clostridium thermoaceticum). The β‐subunit of FDH1 contains putative motifs for binding FMN and NAD, as well as an iron–sulfur cluster binding motif. The β‐subunit appears to be a fusion protein with its N‐terminal domain related to NuoE‐like subunits and its C‐terminal domain related to NuoF‐like subunits of known NADH‐ubiquinone oxidoreductases.
Structural diversity in lipopolysaccharide expression in nontypeable Haemophilus influenzae
Authors: Månsson Martin , Hood Derek W. , Moxon E. Richard , Schweda Elke K. H.
Published: March 2003

Structural elucidation of the lipopolysaccharide (LPS) from three nontypeable Haemophilus influenzae clinical isolates, 1209, 1207 and 1233 was achieved using NMR spectroscopy and ESI‐MS on O‐deacylated LPS and core oligosaccharide (OS) material as well as ESI‐MSn on permethylated dephosphorylated OS. It was found that the organisms expressed a tremendous heterogeneous glycoform mixture resulting from the variable length of the OS chains attached to the common structural element of H. influenzae, l‐α‐d–Hepp‐(1→2)‐[PEtn→6]‐l‐α‐d–Hepp‐(1→3)‐[β‐d‐Glcp‐(1→4)]‐l‐α‐d–Hepp‐(1→5)‐[PPEtn→4]‐α‐Kdop‐(2→6)‐Lipid A. Notably, the O‐6 position of the β‐d‐Glcp residue could either be occupied by PCho or l‐glycero‐d‐manno‐heptose (l,d–Hep), which is a location for l,d–Hep that has not been seen previously in H. influenzae LPS. The outer‐core l,d–Hep residue was further chain elongated at the O‐6 position by the structural element β‐d‐GalpNAc‐(1→3)‐α‐d‐Galp‐(1→4)‐β‐d‐Galp, or sequentially truncated versions thereof. The distal heptose residue in the inner‐core was found to be chain elongated at O‐2 by the globotetraose unit, β‐d‐GalpNAc‐(1→3)‐α‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→4)‐β‐d‐Glcp, or sequentially truncated versions thereof. Investigation of LPS from an lpsA mutant of isolate 1233 and a lic1 mutant of isolate 1209 was also performed, which aside from confirming the functions of the gene products, simplified elucidation of the OS extending from the proximal heptose (the lpsA mutant), and showed that the organism exclusively expresses LPS glycoforms comprising the outer‐core l,d–Hep residue when PCho is not expressed (the lic1 mutant).
Co‐incorporation of Aβ40 and Aβ42 to form mixed pre‐fibrillar aggregates
Authors: Frost David , Gorman Paul M , Yip Christopher M. , Chakrabartty Avijit
Published: March 2003

Senile plaques, the invariable hallmark and likely proximal cause of Alzheimer's disease (AD), are structured depositions of the 40‐ and 42‐residue forms of the Aβ peptide. Conversely, diffuse plaques, which are not associated with neurodegeneration, consist mainly of unstructured Aβ42. We have investigated the interaction between Aβ40 and Aβ42 through an assay, which involves labeling both variants with an environment‐sensitive fluorophore. We have monitored association of Aβ without fibrillar seeds, which allows investigation of molecular species preceding fibrils. Immediately upon mixture, Aβ40 and Aβ42 associate into mixed aggregates, in which the peptides are unstructured and relatively accessible to water. When left to incubate for an extended period, larger, more tightly packed aggregates, which show secondary structure, replace the small, unstructured aggregates formed earlier. Our results show that in vitro the two Aβ variants coassemble early in the fibrillogenesis pathway. The ease of formation for mixed and homogeneous aggregates is similar. A change in the local Aβ variant ratio can therefore have a significant impact on Aβ aggregation; indeed such a change has been reported in some types of familial AD.
Calreticulin–melatonin
Authors: Macías Manuel , Escames Germaine , Leon Josefa , Coto Ana , Sbihi Younes , Osuna Antonio , Acuña‐Castroviejo Darío
Published: March 2003

Increasing evidence suggests that melatonin can exert some effect at nuclear level. Previous experiments using binding techniques clearly showed the existence of specific melatonin binding sites in cell nucleus of rat liver. To further identify these sites, nuclear extracts from rat hepatocytes were treated with different percentages of ammonium sulfate and purified by affinity chromatography. Subsequent ligand blot analysis shows the presence of two polypeptides of ≈ 60 and ≈ 74 kDa that bind specifically to melatonin. N‐Terminal sequence analysis showed that the 60 kDa protein shares a high homology with rat calreticulin, whereas the 74 kDa protein shows no homology with any known protein. The binding of melatonin to calreticulin was further characterized incubating 2‐[125I]melatonin with recombinant calreticulin. Binding kinetics show a Kd = 1.08 ± 0.2 nm and Bmax = 290 ± 34 fmol·mg protein−1, compatible with other binding sites of melatonin in the cell. The presence of calreticulin was further identified by Western blot analysis, and the lack of endoplasmic reticulum contamination in our material was assessed by Western blot and immunostaining with anti‐calnexin Ig. The results suggest that calreticulin may represent a new class of high‐affinity melatonin binding sites involved in some functions of the indoleamine including genomic regulation.
Interaction of the small heat shock protein with molecular mass 25 kDa (hsp25) with actin
Authors: Panasenko Olesya O. , Kim Maria V. , Marston Steven B. , Gusev Nikolai B.
Published: March 2003

The interaction of heat shock protein with molecular mass 25 kDa (HSP25) and its point mutants S77D + S81D (2D mutant) and S15D + S77D + S81D (3D mutant) with intact and thermally denatured actin was analyzed by means of fluorescence spectroscopy and ultracentrifugation. Wild type HSP25 did not affect the polymerization of intact actin. The HSP25 3D mutant decreased the initial rate without affecting the maximal extent of intact actin polymerization. G‐actin heated at 40–45 °C was partially denatured, but retained its ability to polymerize. The wild type HSP25 did not affect polymerization of this partially denatured actin. The 3D mutant of HSP25 increased the initial rate of polymerization of partially denatured actin. Heating at more than 55 °C induced complete denaturation of G‐actin. Completely denatured G‐actin cannot polymerize, but it aggregates at increased ionic strength. HSP25 and especially its 2D and 3D mutants effectively prevent salt‐induced aggregation of completely denatured actin. It is concluded that the interaction of HSP25 with actin depends on the state of both actin and HSP25. HSP25 predominantly acts as a chaperone and preferentially interacts with thermally unfolded actin, preventing the formation of insoluble aggregates.
Interaction of caspase‐3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1)
Authors: Frame Mhairi J. , Tate Rothwelle , Adams David R. , Morgan Keith M. , Houslay M. D. , Vandenabeele Peter , Pyne Nigel J.
Published: March 2003

Here, we show that recombinant bovine PDE5A1 is proteolysed by recombinant caspase‐3 in in vitro and transfected Cos‐7 cells. In addition, the treatment of PDE5A1‐transfected Cos‐7 and PC12 cells with staurosporine, an apoptotic agent that activates endogenous caspase‐3, also induced proteolysis and inactivation of PDE5A1. These findings suggest that there is specificity in the interaction between caspase‐3 and PDE5A1 that requires application of an apoptotic stimulus. The potential proteolysis of the [778]DQGD[781] site in PDE5A1 by caspase‐3 might affect cGMP's hydrolyzing activity as this is within the boundary of the active site. We therefore created a truncated D781 mutant corresponding exactly to the potential cleavage product. This mutant was expressed equally well compared with the wild‐type enzyme in transfected Cos‐7 cells and was inactive. Inactivity of the truncated mutant was not due to potential misfolding of the enzyme as it eluted from gel filtration chromatography in the same fraction as the wild‐type enzyme. Homology model comparison with the catalytic domain of PDE4B2 was used to probe a functional role for the region in PDE5A1 that might be cleaved by caspase‐3. From this, we can predict that a caspase‐3‐mediated cleavage of the [778]DQGD[781] motif would result in removal of the C‐terminal tail containing Q807 and F810, which are potentially important amino acids required for substrate binding.
Characterisation of a binding‐protein‐dependent, active transport system for short‐chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus
Authors: Mills James , Wyborn Neil R. , Greenwood Jacqueline A. , Williams Steven G. , Jones Colin W.
Published: March 2003

Three genes (fmdCAB) encoding an outer‐membrane porin for short‐chain amides and urea, formamidase, and a putative regulatory protein in Methylophilus methylotrophus have previously been cloned and characterised. Three genes have now been identified downstream of fmdB, viz fmdD encoding a hydrophilic protein containing an N‐terminal signal sequence, and fmdEF encoding hydrophobic transmembrane proteins. The derived amino acid sequence of mature FmdD (predicted molecular mass 41 870 Da) was similar to the cytoplasmic, amide‐binding protein (AmiC) from Pseudomonas aeruginosa and to several periplasmic, solute‐binding proteins from other bacteria. Mature FmdD was purified and shown to be a monomer (40−45 kDa) with the predicted N‐terminal amino acid sequence (ADYPTA‐). Equilibrium dialysis showed that the purified protein bound short‐chain amides and urea with high affinity (Kd 7.2 μM for [14C]urea). SDS/PAGE and western blotting using antiserum to mature FmdD showed it was induced by short‐chain amides and urea, and repressed by excess ammonia. The derived amino acid sequences of FmdE (32 822 Da) and FmdF (incomplete; > 25 435 Da) were similar to the transmembrane proteins BraD/LivH and BraE/LivM, respectively, in various leucine/isoleucine/valine transport systems. Uptake of [14C]urea by washed cells was inhibited by the uncoupling agent carbonyl cyanide p‐trifluoromethoxyphenylhydrazone and unlabelled formamide. It is concluded that FmdDEF comprise part of a high‐affinity, binding‐protein‐dependent active‐transport system for short‐chain amides and urea in M. methylotrophus.
Rhodobacter capsulatus HypF is involved in regulation of hydrogenase synthesis through the HupUV proteins
Authors: Colbeau Annette , Elsen Sylvie , Tomiyama Masamitsu , Zorin Nikolaï A. , Dimon Bernard , Vignais Paulette M.
Published: March 2003

The photosynthetic bacterium Rhodobacter capsulatus contains a membrane‐bound [NiFe]hydrogenase encoded by the hupSL genes. We show in this study that hypF mutants are devoid of hydrogenase activity and lack the HupL protein. We also observed that, in contrast to the wild‐type strain B10, transcription of the hupSL genes was not stimulated by H2 in the hypF mutants RS13 and BSE19. Complementation of the hypF mutants with the plasmid borne hypF gene restored hydrogenase activity to wild‐type levels and inducibility by H2. The R. capsulatus hupU and hupV gene products share significant similarities with the small (HupS) and the large (HupL) hydrogenase subunits, respectively. Active HupUV proteins can catalyze the hydrogen‐deuterium exchange reaction. In whole cells, this H‐D exchange is distinguishable from the H‐D exchange catalyzed by the membrane‐bound HupSL proteins by its insensitivity to O2 and to acetylene. By measuring the formation of H2 and HD in exchange with D2 uptake, we demonstrated that the hypF mutants have no active HupUV nor HupSL proteins. H‐D exchange activity, of both HupUV and HupSL, was restored by hypF gene complementation. These data indicate that the HypF protein participates not only in the maturation of HupSL, but also in the maturation of the HupUV proteins and that the latter are involved in the cellular response to H2.
GroEL/ES chaperonins protect interferon‐γ against physicochemical stress
Authors: Vandenbroeck Koen , Martens Eric , Billiau Alfons
Published: March 2003

Interferon‐γ (IFN‐γ) is a structurally labile cytokine that rapidly denatures upon exposure to acid or heat. Here we show that both acid‐denatured (pH 2) and thermally inactivated (50 +C) porcine IFN‐γ can be rescued with the Escherichia coli GroEL/ES chaperonin system and ATP, and reassembled into bio‐active dimers. At 35 +C, spontaneous refolding of acid‐denatured IFN‐γ was found to be dependent on the presence of guanidinium hydrochloride (0.15−0.25 M) or NaCl (0.1−0.2 M). Under non‐permissive reaction conditions for regain of native structure (low‐ionic‐strength buffer at 35 +C), the yield of IFN‐γ refolded with GroEL/ES/ATP increased about 30‐fold above the level of spontaneous refolding. In the absence of GroES, GroEL captured IFN‐γ in a folding‐competent complex. Under these conditions, both ATP and α‐casein induced release of IFN‐γ from GroEL but with the released protein tending to partition into sedimentable aggregates. Only in the presence of GroES, did ATP induce complete discharge of IFN‐γ from GroEL, with the released protein refolded into a conformation that is (a) immunoreactive/bio‐active, (b) resistant to precipitation and (c) in a dimeric configuration. Chicken egg albumin and 90kDa heat‐shock protein were inactive in the exertion of any protective effect against physicochemical stress. The precise amount of protein refolded to the native state at different times of the folding reaction was determined by α‐casein quenching and ELISA. The former is based on the conversion by excess αcasein of any population of unfolded IFN‐γ into one that escapes antibody recognition by subsequent ELISA. Since the native dimers, however, are not affected by α‐casein quenching, immunoreactivity is directly proportional to the yield of correctly refolded protein. The validity of this approach was confirmed by measurement of biological activity. GroEL/ES‐meditated reactivation amounted to > 80 % both by ELISA and antiviral assay.
Contribution of advanced glycosylation to the amyloidogenicity of islet amyloid polypeptide
Authors: Kapurniotu Aphrodite , Bernhagen Jürgen , Greenfield Norma , Al‐Abed Yousef , Teichberg Saul , Frank Rainer W. , Voelter Wolfgang , Bucala Richard
Published: March 2003

The formation of amyloid within the islets of Langerhans is associated with the development of type II diabetes mellitus and occurs by the aggregation and insolubilization of islet amyloid polypeptide (IAPP). Recent in vitro studies suggest that amyloid formation follows a nucleation‐dependent polymerization mechanism, i.e. aggregation is initiated by pre‐formed aggregates or nucleation seeds. Modification of the Alzheimer's disease amyloid peptide by advanced glycosylation end products (AGEs), which form spontaneously by the non‐enzymatic addition of glucose to protein amino groups, has been shown to enhance peptide aggregation in vitro. To explore the possibility that AGEs contribute to islet amyloid formation, we preparedAGE‐modified IAPP (AGE‐IAPP) in vitro and studied its properties by biochemical and biophysical techniques. AGE modification induced the formation of high‐molecular‐mass IAPP aggregates and amyloid formation was demonstrated by Congo red green‐gold birefringence and by the presence of a characteristic fibrillar structure by electron microscopy. AGE‐IAPP also showed an increase in cytotoxicity toward the astroglioma cell line HTB14. When added to soluble IAPP, AGE‐IAPP seeds accelerated IAPP aggregation and abolished the nucleation period required for the polymerization of unseeded IAPP. Circular dichroism spectropolarimetry indicated that AGE‐IAPP seeds may act as a template to stabilize the β‐sheet conformation of IAPP, thereby promoting its aggregation. Our studies demonstrate that AGE modification of IAPP results in high‐molecular mass, fibrillar amyloid structures that nucleate IAPP amyloid formation and suggest a model for intra‐islet amyloid deposition that may occur by the progressive advanced glycosylation of IAPP in vivo.
A stress‐inducible rat liver endoplasmic reticulum protein, ERp29
Authors: Mkrtchian Souren , Fang Che , Hellman Ulf , Ingelman‐Sundberg Magnus
Published: March 2003

We have isolated, cDNA cloned and characterised a 29‐kDa protein (ERp29), containing a C‐terminal endoplasmic reticulum(ER)‐retrieval signal, from the rat liver ER. ERp29 was induced to high levels in the rat hepatoma cells under metabolic stress conditions known to cause an aberrant accumulation of proteins in the ER [(e.g. culture in presence of the Ca2+ ionophore A23187, inhibitors of Ca2+‐ATPase (thapsigargin), intracellular protein transport (brefeldin A), or protein N‐glycosylation (tunicamycin)]. Experimental evidence of its localisation in the luminal compartment of the ER was obtained by topology studies including immunofluorescence microscopy, in vitro translation and proteinase protection assay. ERp29 constitutes about 0.1 % of the rat hepatic microsomal proteins and is constitutively expressed in all rat tissues examined, as evident from northern blot analysis. In rat hepatoma cells ERp29 was found to be associated with the abundant molecular chaperone/stress protein BiP/GRP78 and this interaction was significantly enhanced after treatment with tunicamycin and A23187. Taken together, these results suggest that ERp29 is a member of the stress‐response machinery of the ER.
Amino acid substitutions at the dimer interface of human glucose‐6‐phosphate dehydrogenase that increase thermostability and reduce the stabilising effect of NADP
Authors: Scopes Deborah A. , Bautista José M. , Naylor Claire E. , Adams Margaret J. , Mason Philip J.
Published: March 2003

Over 100 mutations of the G6PD gene have been documented. With the construction of the molecular model of glucose‐6‐phosphate dehydrogenase, based on the structure of the bacterial Leuconostoc mesenteroides glucose‐6‐phosphate dehydrogenase, it has been possible to superimpose these amino acid changes on to the structure of the glucose‐6‐phosphate dehydrogenase molecule. There are a large number of severe disease causing mutations at the dimer interface which usually cause decreased thermostability. We have used this knowledge to predict amino acid changes which would effect an increase in the stability of the dimer. The aspartic acid at residue 421 was chosen as it is a negatively charged residue at the centre of the dimer interface in an area rich in negatively charged residues. This residue was changed to a neutrally charged alanine or asparagine, or a positively charged lysine or arginine. The thermostability of the enzyme was increased when residue 421 was neutral (A or N) and increased further when positive (K or R). NADP is known to exert a concentration dependent stabilising effect on the glucose‐6‐phosphate dehydrogenase dimer. However the concentration‐dependent stabilising effect of NADP was reduced in the residue‐421 substitutions in a manner which was inversly proportional to charge change. These results suggest that changes at the dimer interface can also affect the distant (> 20 Å) NADP‐binding site, and vice versa; an attempt has been made to explain these interactions based on the molecular model of human glucose‐6‐phosphate dehydrogenase.
Functional modules and expression of mouse p40 phox and p67phox, SH3‐domain‐containing proteins involved in the phagocyte NADPH oxidase complex
Authors: Mizuki Kazuhito , Kadomatsu Kenji , Hata Kenichiro , Ito Takashi , Fan Qi‐Wen , Kage Yohko , Fukumaki Yasuyuki , Sakaki Yoshiyuki , Takeshige Koichiro , Sumimoto Hideki
Published: March 2003

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The formation of the active oxidase complex at the membrane requires translocation of the Rac GTPase and two specialized cytosolic proteins that harbor SH3 domains, p67phox and p47phox. Another SH3‐domain‐containing protein p40 phox, which is constitutively associated with p67phox in phagocytes, also enters the complex upon cell stimulation. Here we describe how we cloned mouse cDNAs encoding p40 phox and its partner in phagocytes, p67phox. Both p40 phox and p67phox comprise several protein‐binding modules that are structurally and functionally well conserved between mouse and human, indicating their nature as adaptor proteins. We have also systematically investigated expression of the gene for p40 phox in comparison with those for p67phox and p47phox. Distributions of the mRNAs for the three proteins among tissues are similar, with the most abundant expression in the spleen. The messages are abundant not only in phagocytic cells, but also in B cell lineage. The p40 phox gene, but not the other two, is expressed in some types of cells such as plasma cells and T lymphocytes. Furthermore, in situ hybridization analysis shows that the p40 phox mRNA is distributed in neuronal cells of mouse brain, providing evidence that one of the genes for the specialized oxidase factors is expressed in neurons. These observations raise the possibility that the adaptor protein p40 phox plays a heretofore unsuspected role via interacting with other proteins in the cells that do not express p67phox or p47phox.
Site‐directed mutagenesis of the human β3‐adrenoceptor
Authors: Gros Jérôme , Manning Brian S. , Pietri‐Rouxel France , Guillaume Jean‐Luc , Drumare Marie‐Françoise , Strosberg A. Donny
Published: March 2003

All three subtypes of β‐adrenoceptors are coupled to stimulation of adenylyl cyclase activity via the stimulatory guanine‐nucleotide‐binding protein. Nevertheless, the β3 adrenoceptor (β3‐AR) differs significantly from the other subtypes in terms of pharmacology. Most strikingly, it recognizes as agonists several compounds acting as potent β1‐AR and β2‐AR antagonists. Furthermore, the human β3‐AR is quite different from the animal β3‐AR. Molecular modelling studies followed by site‐directed mutagenesis was used here to identify some of the amino acid residues which may be implicated in ligand binding and signal transduction of the β3‐AR. Three contiguous residues, valine‐leucine‐alanine, which are present in the first transmembrane domain at positions 48−50 of the human receptor but are absent in all known rodent sequences, were thought to be important for species specificity. When these three residues were deleted from the human receptor, no ’rodent‐like' pharmacological profile was obtained in terms of either binding or adenylyl cyclase activation. Glycine at position 53, also in the first transmembrane domain in the human β3‐AR, has been suggested to participate in β2‐/β3‐AR subtype selectivity. Replacement of this glycine residue by phenylalanine, which is the residue present at the homologous position in the human β2‐AR, left the β3‐AR pharmacological profile unaltered in terms of specificity and selectivity. Aspartate residue 117, in the third transmembrane domain, has been found to be essential for ligand binding and consequently adenylyl cyclase activation in several bioamine receptors. When this residue was replaced by a leucine residue in the β3‐AR, ligand binding and signal transduction were suppressed. Finally, replacement of asparagine at position 312 in the sixth transmembrane domain by an alanine residue, led to alterations in the signal‐transduction pathway.
Molecular cloning of the peroxisome proliferator‐induced 46‐kDa cytosolic acyl‐CoA thioesterase from mouse and rat liver
Authors: Lindquist Per J. G. , Svensson L. Thomas , Alexson Stefan E. H.
Published: March 2003

Feeding clofibrate to rats and mice results in a strong induction of acyl‐CoA thioesterase activity in the liver that is mainly due to increases in the enzyme activities in mitochondria and cytosol. The cytosolic acyl‐CoA thioesterase protein of about 40 kDa, referred to as CTE‐I, is strongly induced by the treatment. We report here the molecular cloning of the cDNA corresponding to the rat and mouse enzymes, and the further characterization of the mouse CTE‐I by recombinant expression in bacteria and regulation of expression of the enzyme. The cDNAs corresponding to the rat and mouse enzymes contained open reading frames encoding proteins of 419 amino acids with calculated molecular masses of 45 938 Da and 46 135 Da, respectively. Sequence analysis revealed an active site serine consensus sequence commonly found in lipases and carboxylesterases. Recombinant expression of the mouse CTE‐I cDNA in Escherichia coli resulted in production of immunoreactive protein that was mainly active with long‐chain acyl‐CoAs. Northern blot analysis showed that the full‐length CTE‐I cDNA probe hybridized to two major transcripts corresponding to CTE‐I and MTE‐I (mitochondrial acyl‐CoA thioesterase I), respectively. The expression of both mRNA species was found to be highly regulated. As expected, both CTE‐I and MTE‐I were strongly upregulated (> 50‐fold) by clofibrate treatment. Interestingly, fasting for 48 h resulted in a similar magnitude of induction as two days of clofibrate feeding. In addition, feeding a fat‐free diet resulted in down‐regulation of CTE‐I mRNA. CTE‐I mRNA was strongly expressed in kidney and brown adipose tissue and MTE‐I mRNA was expressed mainly in brown adipose tissue and heart but was also expressed in kidney and white adipose tissue. Dietary regulation and tissue‐specific expression suggest that CTE‐I and MTE‐I play important roles in lipid metabolism.
Can monomers of yeast enolase have enzymatic activity ?
Authors: Kornblatt Mary Judith , Lange Reinhard , Balny Claude
Published: March 2003

Using a combination of ultraviolet spectroscopy under pressure and stopped‐flow kinetics under pressure, we have shown that the monomers of yeast enolase produced by hydrostatic pressure are inactive. Keq, ΔV and ΔV‡ for the dissociation/inactivation produced by hydrostatic pressure have been determined under various conditions. Removing the Mg2+ from enolase, either by adding EDTA or by preparing apoenzyme, displaces the equilibrium towards monomers and decreases both ΔV and ΔV‡. Loss of Mg2+ contributes to the negative ΔV for dissociation; this loss occurs, at least partially, in the transition state for dissociation. Both removal of Mg(II) and dissociation of the enzyme produce major changes in the intensity of the aromatic region of the CD spectrum. We propose that these changes in the CD spectra reflect changes in the conformations of the ’mobile loops' of enolase. The precise conformation of these loops is necessary for binding Mg2+ (and, hence, for activity) and for maintaining subunit interactions.