bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies
Authors: SIGMAN Jeffrey A. , PATWA Tasneem H. , TABLANTE Ana V. , JOSEPH Calleen D. , GLUCKSMAN Marc J. , WOLFSON Adele J. 
Published: May 2005

Thimet oligopeptidase (TOP) is a soluble metalloendopeptidase belonging to a family of enzymes including neurolysin and neprilysin that utilize the HEXXH metal-binding motif. TOP is widely distributed among cell types and is able to cleave a number of structurally unrelated peptides. A recent focus of interest has been on structure–function relationships in substrate selectivity by TOP. The enzyme's structural fold comprises two domains that are linked at the bottom of a deep substrate-binding cleft via several flexible loop structures. In the present study, fluorescence spectroscopy has been used to probe structural changes in TOP induced by the chemical denaturant urea. Fluorescence emission, anisotropy and collisional quenching data support a two-step unfolding process for the enzyme in which complete loss of the tertiary structure occurs in the second step. Complete loss of activity and loss of catalytic Zn(II) from the active site, monitored by absorption changes of the metal chelator 4-(2-pyridylazo)-resorcinol, are also connected with the second step. In contrast, the first unfolding event, which is linked to changes in the non-catalytic domain, leads to a sharp increase in kcat towards a 9-residue substrate and a sharp decrease in kcat for a 5-residue substrate. Thus a conformational change in TOP has been directly correlated with a change in substrate selectivity. These results provide insight into how the enzyme can process the range of structurally unrelated peptides necessary for its many physiological roles.
IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities
Authors: FILLEBEEN Carine , CALTAGIRONE Annie , MARTELLI Alain , MOULIS Jean-Marc , PANTOPOULOS Kostas 
Published: May 2005

In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711→Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1S711E displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1S711E retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1S711E completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1S711E is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.
Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin)
Authors: KELLY Leonard E. , PHILLIPS A. Marie 
Published: May 2005

The stoned locus of Drosophila produces a dicistronic transcript and encodes two proteins, stoned-A (STNA) and stoned-B (STNB). Both proteins are located at synaptic terminals. The STNB protein contains a domain that has homology with the μ-subunit of the AP (adaptor protein) complex, as well as a number of NPF (Asp-Pro-Phe) motifs known to bind EH (Eps15 homology) domains. Mutations at the stoned locus interact synergistically with mutations at the shibire (dynamin) locus and alter synaptic vesicle endocytosis. The STNB protein has also been shown to interact with synaptic vesicles via synaptogamin-I. We initiated an investigation of the possible interaction of DAP-160 (dynamin-associated protein of 160 kDa), a Drosophila member of the intersectin family, with the STNB protein. We show here that both of the viable stoned alleles interacted with a genetic construct that reduces DAP-160 levels to 25% of normal. One of these stoned alleles contains a substitution resulting in a stop codon in the open reading frame encoding STNB. This allele also shows markedly reduced levels of both DAP-160 and dynamin. As anticipated, the NPF motifs in STNB are found to be high-affinity binding motifs for the EH domains of DAP-160. One of the SH3 (Src homology 3) domains of DAP-160 also interacts with STNB. Finally, we show that immunoprecipitation of STNB from fly head extracts co-precipitates with DAP-160, and we conclude that the interaction of the STNB protein with both synaptotagmin I and DAP-160 may regulate synaptic vesicle recycling by recruiting dynamin to a pre-fission complex.
Interactions between Cdc42 and the scaffold protein Scd2: requirement of SH3 domains for GTPase binding
Authors: WHEATLEY Edward , RITTINGER Katrin 
Published: May 2005

The multi-domain protein Scd2 acts as a scaffold upon which the small GTPase Cdc42 (cell division cycle 42), its nucleotide-exchange factor Scd1 and the p21-activated kinase Shk1 assemble to regulate cell polarity and the mating response in fission yeast. In the present study, we show using isothermal titration calorimetry that Scd2 binds two molecules of active GTP-bound Cdc42 simultaneously, but independently of one another. The two binding sites have significantly different affinities, 21 nM and 3 μM, suggesting that they play distinct roles in the Shk1 signalling network. Each of the Cdc42-binding sites includes one of the SH3 (Src homology 3) domains of Scd2. Our data indicate that complex formation does not occur in a conventional manner via the conserved SH3 domain ligand-binding surface. Neither of the isolated SH3 domains is sufficient to interact with the GTPase, and they both require adjacent regions to either stabilize their conformations or contribute to the formation of the Cdc42-binding surface. Furthermore, we show that there is no evidence for an intramolecular PX–SH3 domain interaction, which could interfere with SH3 domain function. This work suggests that SH3 domains might contribute directly to signalling through small GTPases and thereby adds another aspect to the diverse nature of SH3 domains as protein–protein-interaction modules.
Quantification of endogenous retinoic acid in limited biological samples by LC/MS/MS
Authors: KANE Maureen A. , CHEN Na , SPARKS Susan , NAPOLI Joseph L. 
Published: May 2005

We report a sensitive LC (liquid chromatography)/MS/MS assay using selected reaction monitoring to quantify RA (retinoic acid), which is applicable to biological samples of limited size (10–20 mg of tissue wet weight), requires no sample derivatization, provides mass identification and resolves atRA (all-trans-RA) from its geometric isomers. The assay quantifies over a linear range of 20 fmol to 10 pmol, and has a 10 fmol limit of detection at a signal/noise ratio of 3. Coefficients of variation are: instrumental, 0.5–2.9%; intra-assay, 5.4±0.4%; inter-assay 8.9±1.0%. An internal standard (all-trans-4,4-dimethyl-RA) improves accuracy by confirming extraction efficiency and revealing handling-induced isomerization. Tissues of 2–4-month-old C57BL/6 male mice had atRA concentrations of 7–9.6 pmol/g and serum atRA of 1.9±0.6 pmol/ml (±S.E.M.). Tissue 13-cis-RA ranged from 2.9 to 4.2 pmol/g, and serum 13-cis-RA was 1.2±0.3 pmol/ml. CRBP (cellular retinol-binding protein)-null mouse liver had atRA ∼30% lower than wild-type (P&lt;0.05), but kidney, testis, brain and serum atRA were similar to wild-type. atRA in brain areas of 12-month-old female C57BL/6 mice were (±S.E.M.): whole brain, 5.4±0.4 pmol/g; cerebellum, 10.7±0.3 pmol/g; cortex, 2.6±0.4 pmol/g; hippocampus, 8.4±1.2 pmol/g; striatum, 15.3±4.7 pmol/g. These data provide the first analytically robust quantification of atRA in animal brain and in CRBP-null mice. Direct measurements of endogenous RA should have a substantial impact on investigating target tissues of RA, mechanisms of RA action, and the relationship between RA and chronic disease.
Cellular factors required for protection from hyperoxia toxicity in Saccharomyces cerevisiae
Authors: OUTTEN Caryn E. , FALK Robert L. , CULOTTA Valeria C. 
Published: May 2005

Prolonged exposure to hyperoxia represents a serious danger to cells, yet little is known about the specific cellular factors that affect hyperoxia stress. By screening the yeast deletion library, we have identified genes that protect against high-O2 damage. Out of approx. 4800 mutants, 84 were identified as hyperoxia-sensitive, representing genes with diverse cellular functions, including transcription and translation, vacuole function, NADPH production, and superoxide detoxification. Superoxide plays a significant role, since the majority of hyperoxia-sensitive mutants displayed cross-sensitivity to superoxide-generating agents, and mutants with compromised SOD (superoxide dismutase) activity were particularly vulnerable to hyperoxia. By comparison, factors known to guard against H2O2 toxicity were poorly represented amongst hyperoxia-sensitive mutants. Although many cellular components are potential targets, our studies indicate that mitochondrial glutathione is particularly vulnerable to hyperoxia damage. During hyperoxia stress, mitochondrial glutathione is more susceptible to oxidation than cytosolic glutathione. Furthermore, two factors that help maintain mitochondrial GSH in the reduced form, namely the NADH kinase Pos5p and the mitochondrial glutathione reductase (Glr1p), are critical for hyperoxia resistance, whereas their cytosolic counterparts are not. Our findings are consistent with a model in which hyperoxia toxicity is manifested by superoxide-related damage and changes in the mitochondrial redox state.
Muscle endopin 1, a muscle intracellular serpin which strongly inhibits elastase: purification, characterization, cellular localization and tissue distribution
Authors: TASSY Caroline , HERRERA-MENDEZ Carlos H. , SENTANDREU Miguel A. , AUBRY Laurent , BRÉMAUD Laure , PÉLISSIER Patrick , DELOURME Didier , BRILLARD Michèle , GAUTHIER Francis , LEVÉZIEL Hubert , OUALI Ahmed 
Published: May 2005

In the present work, an endopin-like elastase inhibitor was purified for the first time from bovine muscle. A three-step chromatography procedure was developed including successively SP-Sepharose, Q-Sepharose and EMD-DEAE 650. This procedure provides about 300 μg of highly pure inhibitor from 500 g of bovine diaphragm muscle. The N-terminal sequence of the muscle elastase inhibitor, together with the sequence of a trypsin-generated peptide, showed 100% similarity with the cDNA deduced sequence of chromaffin cell endopin 1. Hence, the muscle inhibitor was designated muscle endopin 1 (mEndopin 1). mEndopin 1 had a molecular mass of 70 kDa, as assessed by both gel filtration and SDS/PAGE. According to the association rates determined, mEndopin 1 is a potent inhibitor of elastase (kass=2.41×107 M−1·s−1) and trypsin (kass=3.92×106 M−1·s−1), whereas plasmin (kass=1.78×103 M−1·s−1) and chymotrypsin (kass=1.0×102 M−1·s−1) were only moderately inhibited. By contrast, no inhibition was detected against several other selected serine proteinases, as well as against cysteine proteinases of the papain family. The cellular location of mEndopin in muscle tissue and its tissue distribution were investigated using a highly specific rabbit antiserum. The results obtained demonstrate an intracellular location and a wide distribution in bovine tissues.
Cyclic nucleotide phosphodiesterases in Drosophila melanogaster
Authors: DAY Jonathan P. , DOW Julian A. T. , HOUSLAY Miles D. , DAVIES Shireen-A. 
Published: May 2005

Cyclic nucleotide PDEs (phosphodiesterases) are important enzymes that regulate intracellular levels of cAMP and cGMP. In the present study, we identify and characterize novel PDEs in the genetic model, Drosophila melanogaster. The Drosophila genome encodes five novel PDE genes in addition to dunce. Predicted PDE sequences of Drosophila show highly conserved critical domains when compared with human PDEs. Thus PDE-encoding genes of D. melanogaster are CG14940-PDE1C, CG8279-PDE6β, CG5411-PDE8A, CG32648-PDE9 and CG10231-PDE11. Reverse transcriptase–PCRs of adult tissues reveal widespread expression of PDE genes. Drosophila Malpighian (renal) tubules express all the six PDEs: Drosophila PDE1, dunce (PDE4), PDE6, PDE8, PDE9 and PDE11. Antipeptide antibodies were raised against PDE1, PDE6, PDE9 and PDE11. Verification of antibody specificity by Western blotting of cloned and expressed PDE constructs allowed the immunoprecipitation studies of adult Drosophila lysates. Biochemical characterization of immunoprecipitated endogenous PDEs showed that PDE1 is a dual-specificity PDE (Michaelis constant Km for cGMP: 15.3±1 μM; Km cAMP: 20.5±1.5 μM), PDE6 is a cGMP-specific PDE (Km cGMP: 37±13 μM) and PDE11 is a dual-specificity PDE (Km cGMP: 6±2 μM; Km cAMP: 18.5±5.5 μM). Drosophila PDE1, PDE6 and PDE11 display sensitivity to vertebrate PDE inhibitors, zaprinast (IC50 was 71±39 μM for PDE1, 0.65±0.015 μM for PDE6 and 1.6±0.5 μM for PDE11) and sildenafil (IC50 was 1.3±0.9 μM for PDE1, 0.025±0.005 μM for PDE6 and 0.12±0.06 μM for PDE11). We provide the first characterization of a cGMP-specific PDE and two dual-specificity PDEs in Drosophila, and show a high degree of similarity in structure and function between human and Drosophila PDEs.
Glucose and urea kinetics in cows in early lactation
Authors: Bruckental I. , Oldham J. D. , Sutton J. D. 
Published: May 2005

1. Four mature Friesian cows were used. For 2 weeks after calving they were fed 3 kg hay (147 g crude protein (nitrogen × 6·25; CP)/kg dry matter (DM)) plus 9 kg concentrates (177 g CP/kg DM) per d and thereafter 4 kg hay plus 12 kg concentrates/d.2. At approximately the 2nd, 4th and 9th weeks post-partum each cow was given a single intravenous dose of [14C]urea (1 mCi) and [6-3H]glucose (1·5 mCi). Following this, jugular venous blood samples were withdrawn up to 26 h post-injection.3. The log (specific activity)vtime curves were not linear for either metabolite. Glucose irreversible loss rates (IRL) were calculated by a stochastic procedure. The linear part of the urea log (specific activity)v.time curve gave the same estimate for urea IRL rate as a stochastic method of calculation, but urea pool sizes were overestimated so that stochastic analysis of results was preferred.4. Mean milk yields at the time of the three measurements were 27·1, 30·8 and 27·9 kg milk/d.5. Urea IRL was significantly lower (P< 0·05) in the first 4 weeks of lactation than in the 9th, and blood urea concentration was lower (P< 0·05) in the 4th than in the 2nd and 9th week, but there was no close correlation between urea IRL and concentration.6. Glucose IRL rose (P< 0·05) between the 2nd and 9th weeks post-partum, perhaps due to the increase in feed intake. The results were generally consistent with the relationship between milk yield and glucose IRL established by Paterson & Linzell (1974).7. Urea IRL (y) and milk protein-N (x) production values were used to calculate the efficiency of protein utilization (EPU) asx÷ (x+ 0·35y). This assumes that 0·35 of urea IRL was due to protein catabolism. EPU was found to be 0·59–0·80 (mean 0·69).8. The possible contribution of catabolized amino acids to glucose IRL was calculated by assuming that 0·35 of urea IRL was due to protein catabolism and 0·2 of catabolized protein-C passed through the glucose pool. On this basis the contribution of protein-C to glucose-C was < 25 g/kg glucose IRL.
Growth and the development of dietary obesity in adulthood of rats which have been undernourished during development
Authors: Stephens D. N. 
Published: May 2005

1. The effect of feeding a highly-palatable and varied diet on growth and body composition was assessed in male rats, some of which had undergone a period of undernutrition early in their development.2. Undernutrition during gestation had no effects on weight, length or fat content of offspring in adulthood. Rats underfed during the first 3 weeks of postnatal life were shorter, lighter and more lean as adults than controls which had been well nourished through life.3. Feeding the palatable diet from weaning led to increases in length and fat-free mass, and to comparable extents of obesity in all groups, irrespective of whether they had suffered either period of undernutrition. Access to palatable food for 30 d in adulthood also led to obesity, but to increases in length and fat-free mass in only the groups undernourished during suckling.4. Withdrawal of the palatable diet led to some initial weight loss in all groups, irrespective of whether they had been undernourished during development; and 100 d following the replacement of the palatable with stock diet, there were no longer differences in weight between groups which had received palatable food, and those given stock diet throughout.5. Nevertheless, those rats which had been undernourished during the suckling period and subsequently fed on the palatable diet still showed increased length and fat-free mass relative to their controls fed on the stock diet throughout.6. All groups which had received the palatable diet, whether from weaning or as adults, and irrespective of early nutrition, were significantly less fat 100 d after its withdrawal than were those rats fed on the stock diet throughout life.
The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation
Authors: Wang Steven S.‐S. , Good Theresa A. , Rymer Dawn L. 
Published: May 2005

AbstractCalcitonin, a peptide hormone associated with medullary carcinoma of the thyroid, has the potential to form amyloid fibrils and may be a valuable model for investigating the role of peptide–membrane interactions in β‐sheet and amyloid formation. Via a new model peptide system, bovine calcitonin, we found that the exposure of peptide to phospholipid membranes altered its structure relative to the structures formed in aqueous solutions. Of particular relevance to the amyloidoses, incubation of calcitonin with cholesterol‐rich and ganglioside‐containing membranes resulted in significant enrichment in the β‐sheet and amyloid content of the peptide. The formation of amyloid was also accelerated in these systems. A correlation between the phospholipid‐induced structural alterations and calcitonin binding affinities to phospholipid membranes was evident. Bovine calcitonin has considerably higher binding affinity for the phospholipid systems that enhanced its β‐sheet and amyloid structure. Electrostatic forces were not the governing forces behind the observed behavior, as supported by the fact that the ionic strength did not affect the peptide structures or binding affinities. A Van't Hoff analysis of the temperature‐dependent peptide binding affinities indicated that binding led to an increase in enthalpy and possibly an increase in entropy of the peptide–membrane systems. Experiments with other amyloid‐forming peptides such as β‐amyloid of Alzheimer's disease have also shown similar results and may indicate the need to manipulate peptide–membrane interactions in order to control amyloid formation and its associated disease.
p25α is flexible but natively folded and binds tubulin with oligomeric stoichiometry
Authors: Otzen Daniel E. , Lundvig Ditte M.S. , Wimmer Reinhard , Nielsen Lotte H. , Pedersen Jakob R. , Jensen Poul H. 
Published: May 2005

Abstractp25α is a 219‐residue proteinwhich stimulates aberrant tubulin polymerization and is implicated in a variety of other functions. The protein has unusual secondary structure involving significant amounts of random coil, and binding to microtubules is accompanied by a large structural change, suggesting a high degree of plasticity. p25α has been proposed to be natively unfolded, so that folding is coupled to interaction with its physiological partners. Here we show that recombinant human p25α is folded under physiological conditions, since it has a well structured and solvent‐sequestered aromatic environment and considerable chemical shift dispersion of amide and aliphatic protons. With increasing urea concentrations, p25α undergoes clear spectral changes suggesting significant loss of structure. p25α unfolds cooperatively in urea according to a simple two‐state transition with a stability in water of ∼5 kcal/mol. The protein behaves as a monomer and refolds with a transient on‐pathway folding intermediate. However, high sensitivity to proteolytic attack and abnormal gel filtration migration behavior suggests a relatively extended structure, possibly organized in distinct domains. A deletion mutant of p25α lacking residues 3–43 also unfolds cooperatively and with similar stability, suggesting that the N‐terminal region is largely unstructured. Both proteins undergo significant loss of structure when bound to monomeric tubulin. The stoichiometry of binding is estimated to be 3–4 molecules of tubulin per p25α and is not significantly affected by the deletion of residues 3–43. In conclusion, we dismiss the proposal that p25α is natively unfolded, although the protein is relatively flexible. This flexibility may be linked to its tubulin‐binding properties.
Mass spectrometric analysis of the human 40S ribosomal subunit: Native and HCV IRES‐bound complexes
Authors: Yu Yonghao , Ji Hong , Doudna Jennifer A. , Leary Julie A. 
Published: May 2005

AbstractHepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap‐independent translation initiation. Although IRES‐mediated translation initiation is not subject to many of the regulatory mechanisms that control cap‐dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES‐bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top‐down and bottom‐up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty‐one out of a possible 32 ribosomal proteins were identified by combining top‐down and bottom‐up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES‐bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES‐mediated translation initiation complex.