Active site-directed protein regulation

Regulation of protein function is vital for the control of cellular processes. Proteins are often regulated by allosteric mechanisms, in which effecters bind to regulatory sites distinct from the active sites and alter protein function. Intrasteric regulation, directed at the active site and thus the counterpart of allosteric control, is now emerging as an important regulatory mechanism.

What the evolution of the amyloid protein precursor supergene family tells us about its function

The Alzheimer's disease amyloid protein precursor (APP) gene is part of a multi-gene super-family from which sixteen homologous amyloid precursor-like proteins (APLP) and APP species homologues have been isolated and characterised. Comparison of exon structure (including the uncharacterised APL-1 gene), construction of phylogenetic trees, and analysis of the protein sequence alignment of known homologues of the APP super-family were performed to reconstruct the evolution of the family and to assess the functional significance of conserved protein sequences between homologues. This analysis supports an adhesion function for all members of the APP super family, with specificity determined by those sequences which are not conserved between APLP lineages, and provides evidence for an increasingly complex APP superfamily during evolution. The analysis also suggests that Drosophila APPL and Caenorhabdotids elegans APL-1 may be a fourth APLP lineage indicating that these proteins, while not functional homologues of human APP, are similarly likely to regulate cell adhesion. Furthermore, the beta A4 sequence is highly conserved only in APP orthologues, strongly suggesting this sequence is of significant functional importance in this lineage. (C) 2000 Elsevier Science Ltd. All rights reserved.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein, We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis, Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15), A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17. (C) 1999 Academic Press.

Sausage: protein threading with flexible force fields

Sausage is a protein sequence threading program, but with remarkable run-time flexibility. Using different scripts, it can calculate protein sequence-structure alignments, search structure libraries, swap force fields, create models form alignments, convert file formats and analyse results. There are several different force fields which might be classed as knowledge-based, although they do not rely on Boltzmann statistics. Different force fields are used for alignment calculations and subsequent ranking of calculated models.

Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates

Objective. Differentiated dendritic cells (DC) and other antigen-presenting cells are characterized by the nuclear location of RelB, a member of the nuclear factor kappa B/Rel family. To characterize and enumerate differentiated DC in rheumatoid arthritis (RA) peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST), the expression and location of RelB were examined. Methods. RelB protein expression and cellular location were determined in RA PB, SF, and ST by flow cytometry and immunohistochemical analysis of purified cells or formalin-fixed tissue. DNA-binding activity of RelB was determined by electrophoretic: mobility shift-Western immunoblotting assays. Results. Circulating RA PBDC resembled normal immature PBDC in that they did not express intracellular RelB protein. In RA ST serial sections, cells containing nuclear RelB (nRelB) were enriched in perivascular regions. A mean +/- SD of 84 +/- 10% of these cells were DC. The remaining nRelB+,HLA-DR+ cells comprised B cells and macrophages. Only 3% of sorted SFDC contained nRelB, However, RelB present in the nucleus of these SFDC was capable of binding DNA, and therefore capable of transcriptional activity. Conclusion. Circulating DC precursors differentiate and express RelB after entry into rheumatoid ST. Differentiated DC can thus be identified by immunohistochemistry in formalin-fixed ST. Signals for DC maturation may differ between RA ST and SF, resulting in nuclear location of RelB predominantly in ST. This is likely to have functional consequences for the DC in these sites.

Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants

The Green Fluorescent Protein (GFP) from Aequorea victor-in has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER Variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor(TM) Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 +/- 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 mu g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 mu g and 2.11 mu g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfPS-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 mu g mGFP5-ER per mg extractable protein.

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: Correlations with fetal growth

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases of noninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFSP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-5 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGK at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P

Role of phosphorylation in the conformation of tau peptides implicated in Alzheimer's disease

A series of peptides corresponding to isolated regions of Tau (tau) protein have been synthesized and their conformations determined by H-1 NMR spectroscopy. Immunodominant peptides corresponding to tau(224-240) and a bisphosphorylated derivative in which a single Thr and a single Ser are phosphorylated at positions 231 and 235 respectively, and which are recognized by an Alzheimer's disease-specific monoclonal antibody, were the main focus of the study. The nonphosphorylated peptide adopts essentially a random coil conformation in aqueous solution, but becomes slightly more ordered into P-type structure as the hydrophobicity of the solvent is increased by adding up to 50% trifluoroethanol (TFE). Similar trends are observed for the bisphosphorylated peptide, with a somewhat stronger tendency to form an extended structure, There is tentative NMR evidence for a small population of species containing a turn at residues 229-231 in the phosphorylated peptide, and this is strongly supported by CD spectroscopy. A proposal that the selection of a bioactive conformation from a disordered solution ensemble may be an important step (in either tubulin binding or in the formation of PHF) is supported by kinetic data on Pro isomerization. A recent study showed that Thr231 phosphorylation affected the rate of prolyl isomerization and abolished tubulin binding. This binding was restored by the action of the prolyl isomerase Pin1. In the current study, we find evidence for the existence of both trans and cis forms of tau peptides in solution but no difference in the equilibrium distribution of cis-trans isomers upon phosphorylation. Increasing hydrophobicity decreases the prevalence of cis forms and increases the major trans conformation of each of the prolines present in these molecules. We also synthesized mutant peptides containing Tyr substitutions preceding the Pro residues and found that phosphorylation of Tyr appears to have an effect on the equilibrium ratio of cis-trans isomerization and decreases the cis content.

Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system

Virus-like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post-translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti-viral response in vaccines are not well established. In this study we have compared the posttranslational modifications of human papillomavirus type-6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV-1 kidney epithelial cells, Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1 showed several post-translationally modified variants of the protein, whereas rVV-expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [H-3]mannose and [H-3]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [H-3]galactose. We conclude that post-translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human-vaccine candidate, the implication of the observed differences in post-translational modifications on immunogenicity of L1 VLPs warrants investigation.

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding activity and protein expression in the rat brain

This study focused on the DNA-binding activity and protein expression of the transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus after chronic or acute ethanol exposure. DNA-binding activity was reduced in both regions after chronic ethanol exposure and was restored to the level of the pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not altered by chronic ethanol exposure but increased 16 h after withdrawal, thus mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not undergo any change. There was no change in the level of either protein in the hippocampus. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not alter the DNA-binding activity or protein levels of the transcription factor Spl. These observations suggest that chronic exposure to ethanol has region-selective effects on the DNA-binding activity and protein expression of Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur after prolonged ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependency and withdrawal. These effects are also transcription factor-selective. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure must have effects on modulatory agents of Egr DNA-binding activity. (C) 2000 Elsevier Science Ltd, All rights reserved.

Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype-phenotype correlations in phenylketonuria

Phenylalanine hydroxylase (PAH) is the enzyme that converts phenylalanine to tyrosine as a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. Over 300 mutations have been identified in the gene encoding PAH that result in a deficient enzyme activity and lead to the disorders hyperphenylalaninaemia and phenylketonuria. The determination of the crystal structure of PAH now allows the determination of the structural basis of mutations resulting in PAH deficiency. We present an analysis of the structural basis of 120 mutations with a 'classified' biochemical phenotype and/or available in vitro expression data. We find that the mutations can be grouped into five structural categories, based on the distinct expected structural and functional effects of the mutations in each category. Missense mutations and small amino acid deletions are found in three categories:'active site mutations', 'dimer interface mutations', and 'domain structure mutations'. Nonsense mutations and splicing mutations form the category of 'proteins with truncations and large deletions'. The final category, 'fusion proteins', is caused by frameshift mutations. We show that the structural information helps formulate some rules that will help predict the likely effects of unclassified and newly discovered mutations: proteins with truncations and large deletions, fusion proteins and active site mutations generally cause severe phenotypes; domain structure mutations and dimer interface mutations spread over a range of phenotypes, but domain structure mutations in the catalytic domain are more likely to be severe than domain structure mutations in the regulatory domain or dimer interface mutations.

Structured modeling of recombinant protein production in batch and fed-batch culture of baculovirus-infected insect cells

The infection of insect cells with baculovirus was described in a mathematical model as a part of the structured dynamic model describing whole animal cell metabolism. The model presented here is capable of simulating cell population dynamics, the concentrations of extracellular and intracellular viral components, and the heterologous product titers. The model describes the whole processes of viral infection and the effect of the infection on the host cell metabolism. Dynamic simulation of the model in batch and fed-batch mode gave good agreement between model predictions and experimental data. Optimum conditions for insect cell culture and viral infection in batch and fed-batch culture were studied using the model.

Feed intake and liveweight responses to nitrogen and/or protein supplements by steers of Bos taurus, Bos indicus and Bos taurus x Bos indicus breed types offered a low quality grass hay

Thirty steers were used in two pen experiments (Expts 1 and 2). and 27 of these in a third (Expt 3), to quantify their responses of hay intake, rumen ammonia nitrogen (RAN) concentrations, and liveweight to inputs of rumen soluble nitrogen (urea) and rumen undegradable protein (formaldehyde-treated casein; F-casein) when added to a basal diet of low quality hays. The hays were made From unimproved native pastures typical of those grazed by cattle in the subtropics of Australia and contained 7.8 g N/kg dry matter (DM) with coefficient of organic matter digestibility of 0.503 in Expts 1 and 2, and 5.2 g N/kg DM with a digestibility range from 0.385 to 0.448 in Expt 3. The steers (15 months old) were either Brahman (B), Hereford (H) or the F-1 Brahman x Hereford (BH) cross. Steers were offered supplementary minerals with the hays in each experiment. In Expt 1 (35 days) urea was sprayed on part of the hay, allowing for daily urea intakes (g/steer) of either 0, 5, 11, 16 or 26. In Expt 2 (42 days), F-casein was offered daily (g/steer) at either 0, 75, 150, 225 or 300 and in Expt 3 (56 days) discrete offerings were made of soluble casein (225 g/day), of urea (18 g/day) + F-casein (225 g/day) or of nil. There were significant linear effects of urea intake upon hay intake and liveweight change of steers. However, B steers had smaller increases in intake and liveweight change than did H steers, and B steers did not have a linear increase in RAN concentrations with increasing urea intake as did H and SH steers. In Expt 2 there were significant linear effects of F-casein supplements on hay intake and liveweight change of steers and a significant improvement in their feed conversion ratio (i.e. DM intake:liveweight change). The B steers did not differ from H and BH steers in liveweight change but had significantly lower hay intakes and non-significantly smaller increases in RAN with increasing F-casein intake. In Expt 3, hay intake of the steers increased with soluble casein (by 16.8 %) and with urea + F-casein (24.5 %). Only steers given urea + F-casein had a high RAN concentration (94 mg/l) and a high liveweight gain. The B steers had a liveweight loss and a lower hay intake than H or BH steers in Expt 3 but a higher RAN concentration. These studies have indicated the importance of the form and quantity of additional N required by cattle of differing breed types to optimize their feed intake and liveweight gain when offered low-N, low-digestible hays.