Defining the statistical distribution of vesicle diameters facilitates quantitative assessment of spectral narrowing from small vesicles in protein/lipid interaction studies by 2H-NMR

The diameters of vesicles comprising complexes of an integral membrane protein and 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the choline gamma-methyl groups, exhibited a lognormal distribution defined by the geometric mean (GM) and the logarithmic standard deviation (LSD). The range of vesicle sizes within each complex was very large (e.g., 99% of vesicles measured between 60 nm and 4,000 nm in one complex). This complicated assessment of spectral narrowing artefacts from small vesicles in broad line 2H-NMR results. Artefacts (e.g., decreases in quadrupole splitting and generation of two component spectra) resembled results attributed to membrane proteins in other 2H-NMR studies for which spectral narrowing was dismissed by demonstration of some vesicles > 400 nm in diameter. This was inadequate since 2H-NMR spectra from complexes with GM < 350 nm exhibited spectral narrowing artefacts despite the presence of some vesicles 1,200 nm in diameter. The nature and degree of artefact were determined by both GM and LSD. Quantifying vesicle sizes by GM and LSD accommodated the large size variation within a complex and facilitated quantitative assessment of artefacts through comparison of different complexes.     

Two-dimensional gel protein database of Saccharomyces cerevisiae

With the systematic sequencing of the yeast genome, yeast biology has entered a new era where novel challenges have to be faced. One challenge is the identification of the function of the several hundred novel genes discovered by genome sequencing. Another is to understand how all yeast genes act in concert to ensure and maintain cell organization. Two-dimensional (2-D) gel electrophoresis is the technique of choice to take up these challenges because it provides the opportunity of obtaining an overall view of genome expression. In prospect of these studies we have undertaken the construction of a yeast 2-D gel protein database that contains information on polypeptides of the yeast protein map. In this paper we report the information presently contained in this database. The reported information includes the identification of 250 protein spots and the characterization of polypeptides corresponding to N-terminal acetylated proteins, mitochondrial proteins, glucose-repressed proteins, heat shock induced proteins and proteins encoded by intron-containing genes. In all, 600 spots are annotated. These data can be accessed on the Yeast Protein Map server through the World Wide Web network.     

The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins

Proteins can be denatured by pressures of a few hundred MPa. This finding apparently contradicts the most widely used model of protein stability, where the formation of a hydrophobic core drives protein folding. The pressure denaturation puzzle is resolved by focusing on the pressure-dependent transfer of water into the protein interior, in contrast to the transfer of nonpolar residues into water, the approach commonly taken in models of protein unfolding. Pressure denaturation of proteins can then be explained by the pressure destabilization of hydrophobic aggregates by using an information theory model of hydrophobic interactions. Pressure-denatured proteins, unlike heat-denatured proteins, retain a compact structure with water molecules penetrating their core. Activation volumes for hydrophobic contributions to protein folding and unfolding kinetics are positive. Clathrate hydrates are predicted to form by virtually the same mechanism that drives pressure denaturation of proteins.     

Histone Sequence Database: new histone fold family members

Searches of the major public protein databases with core and linker chicken and human histone sequences have resulted in the compilation of an annotated set of histone protein sequences. In addition, new database searches with two distinct motif search algorithms have identified several members of the histone fold family, including human DRAP1 and yeast CSE4. Database resources include information on conflicts between similar sequence entries in different source databases, multiple sequence alignments, links to the Entrez integrated information retrieval system, structures for histone and histone fold proteins, and the ability to visualize structural data through Cn3D. The database currently contains >1000 protein sequences, which are searchable by protein type, accession number, organism name, or any other free text appearing in the definition line of the entry. All sequences and alignments in this database are available through the World Wide Web at http://www.nhgri.nih. gov/DIR/GTB/HISTONES or http://www.ncbi.nlm.nih. gov/Baxevani/HISTONES     

Separation used for purification of recombinant proteins

The purification of molecules from recombinant cells may be strongly influenced by the molecular biology of gene isolation and expression. At the beginning of the process there may be a demand for information on the minute amounts of proteins and thus for ever increasingly sensitive techniques. Purification of recombinant proteins can differ from conventional purifications in several ways, depending on the solubility of the protein, occurrence in inclusion bodies, creation of fusion proteins with tags that enable simpler purification. Sometimes a (re)naturation step is required to get a bioactive protein. On the other hand, the techniques used in separation are essentially the same as for purification from the natural source and environment.     

Membrane traffic in polarized neurons

The plasma membrane of neurons can be divided into two domains, the soma-dendritic and the axonal. These domains perform different functions: the dendritic surface receives and processes information while the axonal surface is specialized for the rapid transmission of electrical impulses. This functional specialization is generated by sorting and anchoring mechanisms that guarantee the correct delivery and retention of specific membrane proteins. Our understanding of neuronal membrane protein sorting is primarily based on studies of protein overexpression in cultured neurons. These studies revealed that newly synthesized membrane proteins are segregated in the Golgi apparatus in the cell body from where they are transported to the axonal or dendritic surface. Such segregation presumably depends on sorting motifs in the proteins' primary structure. They appear to be located in the cytoplasmic tail for dendritic proteins and in the transmembrane-ectodomain for axonal proteins. Recent studies on neurotransmitter segregation suggest that anchoring in the correct subdomain of the plasma membrane also requires cytoplasmic tail information for binding to the cytoskeleton either directly or by linker proteins. Both mechanisms, sorting and retention, gradually mature during neural development. Young neurons appear to develop initial polarity by other mechanisms, presumably analogous to the mechanisms used by migrating cells.     

Characterization of UT2 cells. The induction of peroxisomal 3-hydroxy-3-methylglutaryl-coenzyme a reductase

In the liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum but also in the peroxisomes. However, to date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and that is localized exclusively to peroxisomes. This cell line was obtained by growing UT2 cells (which lack the endoplasmic reticulum HMG-CoA reductase) in the absence of mevalonate. The cells exhibited a marked increase in a 90-kDa HMG-CoA reductase that was localized exclusively to peroxisomes. The wild type Chinese hamster ovary cells contain two HMG-CoA reductase proteins, the well characterized 97-kDa protein, localized in the endoplasmic reticulum, and a 90-kDa protein localized in peroxisomes. The UT2 cells grown in the absence of mevalonate containing the up-regulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study.     

Functional role of chymase in angiotensin II formation in human vascular tissue

This paper analyzes proteins expressed in a mouse muscle precursor cell line (C2 myoblasts) and compares them with those observed in differentiated myotubes from the same cell line. We observed hundreds of proteins in myoblasts using IPG two-dimensional gel electrophoresis but this number is greatly reduced using Mini-Leak (divinylsulfone-activated agarose) affinity chromatography. Two kinds of affinity columns were prepared. One contained a chemically modified monomeric actin bound to the affinity matrix. The second matrix contained a high-affinity actin-binding protein (DNase I) which was bound to the actin Mini-Leak column to block specific sites on actin. Actin-binding proteins in homogenates of myoblasts or myotubes were passed through the affinity columns and eluted under high salt conditions. The Mini-Leak affinity medium itself appeared to have little ability to bind proteins. Our two-dimensional (2-D) gels identified a small number of proteins and we are currently focusing our attention on a particular protein spot which could correspond to cofilin. Comparison of myoblast and myotube proteins using affinity chromatography shows no qualitative, clearly identifiable differences but the analysis is still in progress. These findings are discussed in relation to reports in which the myoblast-myotube transformation was associated with the up-regulation or de novo synthesis of more than ten proteins.     

Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay time-of-flight mass spectrometry

The potential of matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay (PSD) time-of-flight mass spectrometry for the characterization of peptides and proteins is discussed. Recent instrumental developments provide for levels of sensitivity and accuracy that make these techniques major analytical tools for proteome analysis. New software developments employing protein database searches have greatly enhanced the fields of application of MALDI-PSD. Peptides and proteins can be easily identified even if only a partial sequence information is determined. Derivatization procedures have been optimized for MALDI-PSD to increase the structural information and to obtain a complete peptide sequence even in critical cases. They are fast, simple and can be performed on target. MALDI-PSD is also a very powerful tool to characterize or elucidate post-translational or chemically induced modifications. In association with database searches, proteins issued from electrophoretic gels can be identified after specific enzymatic cleavages and peptide mapping.     

Cell-specific proteins synthesized by Salmonella typhimurium

Studies of the proteins synthesized by Salmonella typhimurium during growth within tissue culture cells have previously focused on a single cell type. In the present study we examine the different protein patterns exhibited by S. typhimurium during growth within three different cell types relevant to those it would encounter throughout the course of a natural infection, including intestinal epithelial cells (Intestine-407), macrophages (J774.A, rat bone marrow-derived macrophages, and mouse bone marrow-derived macrophages), and liver cells (NMuLi). Side-by-side comparisons reveal that S. typhimurium responds to these different cellular environments with specific patterns of protein synthesis unique to each cell type. The numbers of proteins detected in each cell line are as follows: 142 proteins in Intestine-407, of which 58 appear to be unique to growth within this cell line; 413 proteins in J774.A, of which 157 appear to be unique; 260 proteins in rat bone marrow-derived macrophages, of which 40 appear to be unique; 336 proteins in mouse bone marrow-derived macrophages, of which 113 appear to be unique; and 183 proteins in NMuLi, of which 91 appear to be unique.     

Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling

Wingless (Wg) treatment of Drosophila wing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of Drosophila Schneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Dfrizzled-2. However, we found that a line of S2 cells obtained from another source express Dfrizzled-2 and Dfrizzled-1. Thus, we designated this cell line as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and DE-cadherin protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induced the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells was phosphorylated as a consequence of expression of Dfrizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells but can grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling.     

Different functional domains of GLUT2 glucose transporter are required for glucose affinity and substrate specificity

GLUT2 is the major glucose transporter in pancreatic beta-cells and hepatocytes. It plays an important role in insulin secretion from beta-cells and glucose metabolism in hepatocytes. To better understand the molecular determinants for GLUT2's distinctive glucose affinity and its ability to transport fructose, we constructed a series of chimeric GLUT2/GLUT3 proteins and analyzed them in both Xenopus oocytes and mammalian cells. The results showed the following. 1) GLUT3/GLUT2 chimera containing a region from transmembrane segment 9 to part of the COOH-terminus of GLUT2 had Km values for 3-O-methylglucose similar to those of wild-type GLUT2. Further narrowing of the GLUT2 component in the chimeric GLUTs lowered the Km values to those of wild-type GLUT3. 2) GLUT3/GLUT2 chimera containing a region from transmembrane segment 7 to part of the COOH-terminus of GLUT2 retained the ability to transport fructose. Further narrowing of this region in the chimeric GLUTs resulted in a complete loss of the fructose transport ability. 3) Chimeric GLUTs with the NH2-terminal portion of GLUT2 were unable to express glucose transporter proteins in either Xenopus oocytes or mammalian RIN 1046-38 cells. These results indicate that amino acid sequences in transmembrane segments 9-12 are primarily responsible for GLUT2's distinctive glucose affinity, whereas amino acid sequences in transmembrane segments 7-8 enable GLUT2 to transport fructose. In addition, certain region(s) of the amino-terminus of GLUT2 impose strict structural requirements on the carboxy-terminus of the glucose transporter protein. Interactions between these regions and the carboxy-terminus of GLUT2 are essential for GLUT2 expression.     

Toxins produced by arthropod parasites: salivary gland proteins of human body lice and venom proteins of chelonine wasps

A review is presented of our ongoing research projects on the protein components of the saliva of human body lice and of the non-paralyzing venom of wasps in the subfamily Cheloninae. Sodium dodecyl sulfate-polyacryamide gel electrophoretic analysis of lice salivary gland proteins showed a predominance of high and intermediate mol. wt proteins. Immunoblotting with a low titer polyclonal antiserum to lice salivary proteins indicated that some, but not all, of the predominant high mol. wt salivary gland proteins are injected into the host during feeding. The venom of a Chelonus sp. wasp contains a chitinase, and a 33,000 mol. wt protein with a primary structure composed mostly of a series of 12 tandem repeats of a 14-residue sequence. The N-terminus of this protein and its homologs in a related species of Ascogaster share a conserved adjacent pair of acidic residues. Epitope mapping/immunoprecipitation experiments now in progress will provide information on which linear motifs are on the surface of the protein, and will thereby provide information on the tertiary structure of the protein.     

Identification of cellular proteins that bind the central conserved region of p53

The bacterial fusion protein between glutathione S-transferase and the central conserved region of human p53(GST-p53) was purified and fixed on the beads and then used in the binding assay with radiolabeled cell extract from human hepatocarcinoma cell line, Hep3B. The binding assay disclosed the presence of cellular proteins that interact with GST-p53 but not with GST. SV40 large T antigen abrogated the bindings of two cellular proteins with molecular weights of 50 kda and 40 kda. The binding of the proteins to p53 was observed in a cell cycle-dependent manner. These two proteins are candidate cellular proteins which regulate the function of p53.     

Description of a human papillary thyroid carcinoma cell line. Morphologic study and expression of tumoral markers

BACKGROUND: The establishment of cell lines from thyroid carcinomas can provide an in vitro model of oncogenesis. B-CPAP is a new cell line that has been obtained from a differentiated papillary thyroid carcinoma. The data presented give a broader characterization and expression of tumoral markers of this cell line and identify the differentiated functions that are preserved. METHODS: An ultrastructural study was performed to confirm the thyroid nature of the new cell line. The cellular markers (thyroglobulin, S100, neuron-specific enolase [NSE]) and the oncogenes (mutated p53, H-ras, c-myc, PTC, trk) were studied by immunohistochemistry, Southern blot, or in situ hybridization. RESULTS: The cells were of a differentiated ultrastructural thyroid type. All of the cells proved immunoreactive with antibodies specific to thyroglobulin, S100 proteins, NSE, and mutant p53 protein. Mutations of H-ras, PTC, and trk were not observed. The c-myc gene was not amplified. CONCLUSIONS: The cell line described in these data provides a suitable model for the study of thyroid carcinogenesis, given that the cells present thyroid characteristics, and metabolic disorders not previously found in such cell lines. In addition, the coexpression of S100 proteins and mutant p53 proteins in the cells should permit the study of the interaction between these two proteins.     

Self-organization of genetic coding

The self-organization of genetic coding is studied in a simple model system which uses the products of translation as catalysts for the process. The system studied contains protein molecules chosen from a sequence space of high dimension. Catalysts which assign amino acids to codons are chosen in such a way that a random selection of proteins synthesizes further proteins randomly. Under some circumstances, dictated by the genetic information supplied to the system and the manner in which protein function depends on protein sequence, the state of random synthesis is unstable. The system then evolves spontaneously to a new state in which proteins synthesize further proteins in an ordered fashion, typically executing the rules of a simple code for the assignment of amino acids to codons. For some embeddings of protein functions in the protein sequence space, the domains of stability of the ordered and disordered states are calculated. Computer simulation verify that coding self-organization occurs in a variety of systems of the sort studied. Coding self-organization among catalysts which recognize genetic information is a high order co-operative selection process which provides the link between genotype and phenotype needed for the Darwinian evolution of complex biochemical systems.     

Affinity analysis of idiotype-positive and idiotype-negative Ars-binding hybridoma proteins and Ars-immune sera

The possibility that idiotype dominance may be associated with increased affinity for hapten was investigated in the murine A/J anti-p-azophenylarsonate (Ars) response. Fluorescence quenching of 14 Ars-binding hybridoma proteins by Ars-tyrosine was measured and Ka calculated using computer-assisted curve fitting. There was a 200-fold range in Ka for idiotype-positive hybridoma proteins, with 2 IgM hybridoma proteins being near the median. No clear difference in Ka was apparent between idiotype-positive (Id+) and idiotype-negative (Id-) hybridoma proteins. Ka was measured by fluorescence quenching on affinity-purified anti-Ars antibodies from 6 conventional antisera; there was no difference between Id+ and Id- (idiotype suppressed) sera. The affinities of the hybridoma proteins were correlated with the ratio of binding to Ars36-BSA and Ars10-BSA by direct radioimmunoassay. With this calibration, functional affinities of Ars-immune sera could be determined from relative binding ratios without the need for prior affinity purification. This was done for 18 Ars-immune sera, and again there was no clear difference between Id+ and Id- sera. Studies from this laboratory have identified the amino acid sequence of a hybridoma protein which corresponds to the germ line DNA sequence for the cross-reactive idiotype family. The present study shows that the protein directly encoded by the germ line gene has low affinity for hapten suggesting that somatic diversification operating on the germ line sequence can produce antibodies with increased affinity for hapten within the cross-reactive idiotype family. The present study also suggests that affinity is not the driving force behind idiotype dominance of the Ars-immune response.