Showing papers on "Peptide sequence published in 1998"

COI1: An Arabidopsis Gene Required for Jasmonate-Regulated Defense and Fertility

Abstract: The coi1 mutation defines an Arabidopsis gene required for response to jasmonates, which regulate defense against insects and pathogens, wound healing, and pollen fertility. The wild-type allele, COI1, was mapped to a 90-kilobase genomic fragment and located by complementation of coi1-1 mutants. The predicted amino acid sequence of the COI1 protein contains 16 leucine-rich repeats and an F-box motif. It has similarity to the F-box proteins Arabidopsis TIR1, human Skp2, and yeast Grr1, which appear to function by targeting repressor proteins for removal by ubiquitination.

XVI. International Union of Pharmacology Recommendations for the Nomenclature of Neuropeptide Y, Peptide YY, and Pancreatic Polypeptide Receptors

Abstract: Based on structural and evolutionary criteria, neuropeptide Y (NPY)b, peptide YY (PYY) and pancreatic polypetide (PP) are closely related polypeptides ([Larhammar, 1996a][1]). They are composed of 36 amino acids each and share considerable amino acid homology, amidated C-terminal ends, and the

Cloning and characterization of human protease-activated receptor 4

Abstract: Protease-activated receptors 1–3 (PAR1, PAR2, and PAR3) are members of a unique G protein-coupled receptor family. They are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. A partial cDNA sequence of a fourth member of this family (PAR4) was identified in an expressed sequence tag database, and the full-length cDNA clone has been isolated from a lymphoma Daudi cell cDNA library. The ORF codes for a seven transmembrane domain protein of 385 amino acids with 33% amino acid sequence identity with PAR1, PAR2, and PAR3. A putative protease cleavage site (Arg-47/Gly-48) was identified within the extracellular amino terminus. COS cells transiently transfected with PAR4 resulted in the formation of intracellular inositol triphosphate when treated with either thrombin or trypsin. A PAR4 mutant in which the Arg-47 was replaced with Ala did not respond to thrombin or trypsin. A hexapeptide (GYPGQV) representing the newly exposed tethered ligand from the amino terminus of PAR4 after proteolysis by thrombin activated COS cells transfected with either wild-type or the mutant PAR4. Northern blot showed that PAR4 mRNA was expressed in a number of human tissues, with high levels being present in lung, pancreas, thyroid, testis, and small intestine. By fluorescence in situ hybridization, the human PAR4 gene was mapped to chromosome 19p12.

The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions

Abstract: The tetratricopeptide repeat (TPR) is a degenerate 34 amino acid sequence identified in a wide variety of proteins, present in tandem arrays of 3-16 motifs, which form scaffolds to mediate protein-protein interactions and often the assembly of multiprotein complexes. TPR-containing proteins include the anaphase promoting complex (APC) subunits cdc16, cdc23 and cdc27, the NADPH oxidase subunit p67 phox, hsp90-binding immunophilins, transcription factors, the PKR protein kinase inhibitor, and peroxisomal and mitochondrial import proteins. Here, we report the crystal structure of the TPR domain of a protein phosphatase, PP5. Each of the three TPR motifs of this domain consist of a pair of antiparallel alpha-helices of equivalent length. Adjacent TPR motifs are packed together in a parallel arrangement such that a tandem TPR motif structure is composed of a regular series of antiparallel alpha-helices. The uniform angular and spatial arrangement of neighbouring alpha-helices defines a helical structure and creates an amphipathic groove. Multiple-TPR motif proteins would fold into a right-handed super-helical structure with a continuous helical groove suitable for the recognition of target proteins, hence defining a novel mechanism for protein recognition. The spatial arrangement of alpha-helices in the PP5-TPR domain is similar to those within 14-3-3 proteins.

Twenty proteins containing a C-terminal SOCS box form five structural classes

Abstract: The four members of the recently identified suppressor of cytokines signaling family (SOCS-1, SOCS-2, SOCS-3, and CIS, where CIS is cytokine-inducible SH2-containing protein) appear, by various means, to negatively regulate cytokine signal transduction. Structurally, the SOCS proteins are composed of an N-terminal region of variable length and amino acid composition, a central SH2 domain, and a previously unrecognized C-terminal motif that we have called the SOCS box. By using the SOCS box amino acid sequence consensus, we have searched DNA databases and have identified a further 16 proteins that contain this motif. These proteins fall into five classes based on the protein motifs found N-terminal of the SOCS box. In addition to four new SOCS proteins (SOCS-4 to SOCS-7) containing an SH2 domain and a SOCS box, we describe three new families of proteins that contain either WD-40 repeats (WSB-1 and -2), SPRY domains (SSB-1 to -3) or ankyrin repeats (ASB-1 to -3) N-terminal of the SOCS box. In addition, we show that a class of small GTPases also contains a SOCS box. The expression of representative members of each class of proteins differs markedly, as does the regulation of expression by cytokines. The function of the WSB, SSB, and ASB protein families remains to be determined.

Human beta-defensin-1: an antimicrobial peptide of urogenital tissues.

Abstract: Antimicrobial peptides are widely distributed mediators of innate host defense in animals and plants. A 36 amino acid antimicrobial peptide belonging to the defensin family, and named human beta-defensin-1 (HBD-1), was purified recently from hemodialysate fluid, but its tissue sources were not identified. By Northern blotting, we found the highest concentrations of HBD-1 mRNA in the kidney and the female reproductive tract. In situ hybridization localized the HBD-1 mRNA in the epithelial layers of the loops of Henle, distal tubules, and the collecting ducts of the kidney and the epithelial layers of the vagina, ectocervix, endocervix, uterus, and fallopian tubes in the female reproductive tract. Using a novel technique designed to detect cationic peptides in urine, we recovered several forms of HBD-1 ranging in length from 36 to 47 amino acid (aa) residues and differing from each other by amino terminal truncation. The total concentration of HBD-1 forms in voided urine was estimated at 10-100 microg/liter, with individual variations in the total amount of HBD-1 peptides and the relative proportion of HBD-1 forms. Multiple forms of HBD-1 (size 36-47 aa) were also found in the blood plasma, bound to carrier macromolecules that released the peptide under acid conditions, and in vaginal mucosal secretions (39, 40, and 44 aa). By immunostaining, HBD-1 was located in the kidney within the lumen of the loops of Henle, but no intracellular storage sites were identified in renal or female reproductive tissues. Recombinant HBD-1 forms (36, 39, and 42 aa) and natural HBD-1 forms were antimicrobial to laboratory and clinical strains of Escherichia coli at micromolar concentrations. HBD-1 activity was not changed appreciably by low pH, but was inhibited by high salt conditions. Some of the HBD-1 peptides retained their activity against E. coli in unconcentrated (low conductance) urine, and the 36 aa form was microbicidal even in normal (high conductance) urine. Production of HBD-1 in the urogenital tract could contribute to local antimicrobial defense.

Cell penetration by transportan

Abstract: Transportan is a 27 amino acid-long peptide containing 12 functional amino acids from the amino terminus of the neuropeptide galanin and mastoparan in the carboxyl terminus, connected via a lysine Transportan is a cell-penetrating peptide as judged by indirect immunofluorescence using Ne13-biotinyl-transportan The internalization of biotinyl-transportan is energy independent and takes place efficiently at 37°, 4°, and 0°C Cellular uptake of transportan is probably not mediated by endocytosis, since it cannot be blocked by treating the cells with phenylarsine oxide or hyperosmolar sucrose solution and is nonsaturable The kinetics of internalization was studied with the aid of the 125I-labeled peptide At 37°C, the maximal intracellular concentration is reached in about 20 min The internalized transportan is protected from trypsin The cell-penetrating ability of transportan is not restricted by cell type, but seems to be a general feature of this peptide In Bowes' melanoma cells, transportan first lo

Several Common HLA-DR Types Share Largely Overlapping Peptide Binding Repertoires

Abstract: The peptide binding specificities of HLA-DRB1*0401, DRB1*0101, and DRB1*0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1*0101, DRB1*0401, and DRB1*0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1*0101, DRB1*0401, DRB1*0701, DRB5*0101, DRB1*1501, DRB1*0901, and DRB1*1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.

Multimodular Penicillin-Binding Proteins: An Enigmatic Family of Orthologs and Paralogs

Abstract: The monofunctional penicillin-binding DD-peptidases and penicillin-hydrolyzing serine beta-lactamases diverged from a common ancestor by the acquisition of structural changes in the polypeptide chain while retaining the same folding, three-motif amino acid sequence signature, serine-assisted catalytic mechanism, and active-site topology. Fusion events gave rise to multimodular penicillin-binding proteins (PBPs). The acyl serine transferase penicillin-binding (PB) module possesses the three active-site defining motifs of the superfamily; it is linked to the carboxy end of a non-penicillin-binding (n-PB) module through a conserved fusion site; the two modules form a single polypeptide chain which folds on the exterior of the plasma membrane and is anchored by a transmembrane spanner; and the full-size PBPs cluster into two classes, A and B. In the class A PBPs, the n-PB modules are a continuum of diverging sequences; they possess a five-motif amino acid sequence signature, and conserved dicarboxylic amino acid residues are probably elements of the glycosyl transferase catalytic center. The PB modules fall into five subclasses: A1 and A2 in gram-negative bacteria and A3, A4, and A5 in gram-positive bacteria. The full-size class A PBPs combine the required enzymatic activities for peptidoglycan assembly from lipid-transported disaccharide-peptide units and almost certainly prescribe different, PB-module specific traits in peptidoglycan cross-linking. In the class B PBPs, the PB and n-PB modules cluster in a concerted manner. A PB module of subclass B2 or B3 is linked to an n-PB module of subclass B2 or B3 in gram-negative bacteria, and a PB module of subclass B1, B4, or B5 is linked to an n-PB module of subclass B1, B4, or B5 in gram-positive bacteria. Class B PBPs are involved in cell morphogenesis. The three motifs borne by the n-PB modules are probably sites for module-module interaction and the polypeptide stretches which extend between motifs 1 and 2 are sites for protein-protein interaction. The full-size class B PBPs are an assortment of orthologs and paralogs, which prescribe traits as complex as wall expansion and septum formation. PBPs of subclass B1 are unique to gram-positive bacteria. They are not essential, but they represent an important mechanism of resistance to penicillin among the enterococci and staphylococci. Natural evolution and PBP- and beta-lactamase-mediated resistance show that the ability of the catalytic centers to adapt their properties to new situations is limitless. Studies of the reaction pathways by using the methods of quantum chemistry suggest that resistance to penicillin is a road of no return.

Identification of a new human immunodeficiency virus type 1 distinct from group M and group O

Abstract: A highly divergent HIV-1 isolate, designated YBF 30, was obtained in 1995 from a 40-year-old Cameroonian woman with AIDS. Depending on the genes studied, phylogenetic analysis showed that YBF30 branched either with SIVcpz-gab or between SIVcpz-gab and HIV-1 group M. The structural genes and tat, vpr, and nef of YBF30 are approximately equidistant from those of HIV-1 group M and SIVcpz-gab. In contrast, vif and rev are closer to HIV-1 group M, and vpu is highly divergent. Using a YBF30 V3 loop peptide enzyme immunoassay, we screened 700 HIV-1-positive sera collected in Cameroon; three reacted strongly with the YBF30 peptides and one was confirmed as being related to YBF30 by genetic analysis of a pol fragment. YBF30 is as distinct from SIVcpz-gab as it is from HIV-1 group M and can thus be considered as the prototype strain of a new human immunodeficiency virus group.

A Plant Homolog of the Neutrophil NADPH Oxidase gp91phox Subunit Gene Encodes a Plasma Membrane Protein with Ca2+ Binding Motifs

Abstract: Rapid generation of O2- and H2O2, which is reminiscent of the oxidative burst in neutrophils, is a central component of the resistance response of plants to pathogen challenge. Here, we report that the Arabidopsis rbohA (for respiratory burst oxidase homolog A) gene encodes a putative 108-kD protein, with a C-terminal region that shows pronounced similarity to the 69-kD apoprotein of the gp91phox subunit of the neutrophil respiratory burst NADPH oxidase. The RbohA protein has a large hydrophilic N-terminal domain that is not present in gp91phox. This domain contains two Ca2+ binding EF hand motifs and has extended similarity to the human RanGTPase-activating protein 1. rbohA, which is a member of a divergent gene family, generates transcripts of 3.6 and 4.0 kb that differ only in their polyadenylation sites. rbohA transcripts are most abundant in roots, with weaker expression in aerial organs and seedlings. Antibodies raised against a peptide near the RbohA C terminus detected a 105-kD protein that, unlike gp91phox, does not appear to be highly glycosylated. Cell fractionation, two-phase partitioning, and detergent extraction indicate that RbohA is an intrinsic plasma membrane protein. We propose that plants have a plasma membrane enzyme similar to the neutrophil NADPH oxidase but with novel potential regulatory mechanisms for Ca2+ and G protein stimulation of O2- and H2O2 production at the cell surface.

ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

Abstract: A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila , and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ∼130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.

Ezrin/Radixin/Moesin (ERM) Proteins Bind to a Positively Charged Amino Acid Cluster in the Juxta-Membrane Cytoplasmic Domain of CD44, CD43, and ICAM-2

Abstract: . CD44 has been identified as a membrane-binding partner for ezrin/radixin/moesin (ERM) proteins, plasma membrane/actin filament cross-linkers. ERM proteins, however, are not necessarily colocalized with CD44 in tissues, but with CD43 and ICAM-2 in some types of cells. We found that glutathione-S-transferase fusion proteins with the cytoplasmic domain of CD43 and ICAM-2, as well as CD44, bound to moesin in vitro. The regions responsible for the in vitro binding of CD43 and CD44 to moesin were narrowed down to their juxta-membrane 20–30–amino acid sequences in the cytoplasmic domain. These sequences and the cytoplasmic domain of ICAM-2 (28 amino acids) were all characterized by the positively charged amino acid clusters. When E-cadherin chimeric molecules bearing these positively charged amino acid clusters of CD44, CD43, or ICAM-2 were expressed in mouse L fibroblasts, they were co-concentrated with ERM proteins at microvilli, whereas those lacking these clusters were diffusely distributed on the cell surface. The specific binding of ERM proteins to the juxta-membrane positively charged amino acid clusters of CD44, CD43, and ICAM-2 was confirmed by immunoprecipitation and site-directed mutagenesis. From these findings, we conclude that ERM proteins bind to integral membrane proteins bearing a positively charged amino acid cluster in their juxta-membrane cytoplasmic domain.

Glycosaminoglycan‐protein interactions: definition of consensus sites in glycosaminoglycan binding proteins

Abstract: Although interactions of proteins with glycosaminoglycans (GAGs), such as heparin and heparan sulphate, are of great biological importance, structural requirements for protein-GAG binding have not been well-characterised. Ionic interactions are important in promoting protein-GAG binding. Polyelectrolyte theory suggests that much of the free energy of binding comes from entropically favourable release of cations from GAG chains. Despite their identical charges, arginine residues bind more tightly to GAGs than lysine residues. The spacing of these residues may determine protein-GAG affinity and specificity. Consensus sequences such as XBBBXXBX, XBBXBX and a critical 20 A spacing of basic residues are found in some protein sites that bind GAG. A new consensus sequence TXXBXXTBXXXTBB is described, where turns bring basic interacting amino acid residues into proximity. Clearly, protein-GAG interactions play a prominent role in cell-cell interaction and cell growth. Pathogens including virus particles might target GAG-binding sites in envelope proteins leading to infection.

Human beta-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung

Abstract: Previous studies have implicated the novel peptide antibiotic human beta-defensin 1 (hBD-1) in the pathogenesis of cystic fibrosis. We describe in this report the isolation and characterization of the second member of this defensin family, human beta-defensin 2 (hBD-2). A cDNA for hBD-2 was identified by homology to hBD-1. hBD-2 is expressed diffusely throughout epithelia of many organs, including the lung, where it is found in the surface epithelia and serous cells of the submucosal glands. A specific antibody made of recombinant peptide detected hBD-2 in airway surface fluid of human lung. The fully processed peptide has broad antibacterial activity against many organisms, which is salt sensitive and synergistic with lysozyme and lactoferrin. These data suggest the existence of a family of beta-defensin molecules on mucosal surfaces that in the aggregate contributes to normal host defense.

A newly identified N‐terminal amino acid sequence of human eIF4G binds poly(A)‐binding protein and functions in poly(A)‐dependent translation

Abstract: Most eukaryotic mRNAs possess a 5' cap and a 3' poly(A) tail, both of which are required for efficient translation. In yeast and plants, binding of eIF4G to poly(A)-binding protein (PABP) was implicated in poly(A)-dependent translation. In mammals, however, there has been no evidence that eIF4G binds PABP. Using 5' rapid amplification of cDNA, we have extended the known human eIF4GI open reading frame from the N-terminus by 156 amino acids. Co-immunoprecipitation experiments showed that the extended eIF4GI binds PABP, while the N-terminally truncated original eIF4GI cannot. Deletion analysis identified a 29 amino acid sequence in the new N-terminal region as the PABP-binding site. The 29 amino acid stretch is almost identical in eIF4GI and eIF4GII, and the full-length eIF4GII also binds PABP. As previously shown for yeast, human eIF4G binds to a fragment composed of RRM1 and RRM2 of PABP. In an in vitro translation system, an N-terminal fragment which includes the PABP-binding site inhibits poly(A)-dependent translation, but has no effect on translation of a deadenylated mRNA. These results indicate that, in addition to a recently identified mammalian PABP-binding protein, PAIP-1, eIF4G binds PABP and probably functions in poly(A)-dependent translation in mammalian cells.

Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha.

Abstract: Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS (bHLH-PAS) superfamily. In our efforts to identify new bHLH-PAS proteins, we cloned a cDNA encoding a novel alpha-class hypoxia inducible factor, HIF3alpha. The HIF3alpha open reading frame encodes a 662-amino acid protein with a predicted molecular weight of 73 kDa and is expressed in adult thymus, lung, brain, heart, and kidney. The N-terminal bHLH-PAS domain of this protein shares amino acid sequence identity with that of HIF1alpha and HIF2alpha (57% and 53% identity, respectively). The C-terminus of HIF3alpha contains a 36-amino acid sequence that shares 61% identity with the hypoxia responsive domain-1 (HRD1) of HIF1alpha. In transient transfections, this domain confers hypoxia responsiveness when linked to a heterologous transactivation domain. In vitro studies reveal that HIF3alpha dimerizes with a prototype beta-class subunit, ARNT, and that the resultant heterodimer recognizes the hypoxia responsive element (HRE) core sequence, TACGTG. Transient transfection experiments demonstrate that the HIF3alpha-ARNT interaction can occur in vivo, and that the activity of HIF3alpha is upregulated in response to cobalt chloride or low oxygen tension.

A structural explanation for the recognition of tyrosine-based endocytotic signals.

Abstract: Many cell surface proteins are marked for endocytosis by a cytoplasmic sequence motif, tyrosine-X-X-(hydrophobic residue), that is recognized by the μ2 subunit of AP2 adaptors. Crystal structures of the internalization signal binding domain of μ2 complexed with the internalization signal peptides of epidermal growth factor receptor and the trans-Golgi network protein TGN38 have been determined at 2.7 angstrom resolution. The signal peptides adopted an extended conformation rather than the expected tight turn. Specificity was conferred by hydrophobic pockets that bind the tyrosine and leucine in the peptide. In the crystal, the protein forms dimers that could increase the strength and specificity of binding to dimeric receptors.

Method for the production of non-immunogenic proteins

Abstract: Protein, or parts of proteins, may be rendered non-immunogenic, or less immunogenic, to a given species by identifying in their amino acid sequences one or more potential epitopes for T-cells of the given species and modifying the amino acid sequence to eliminate at least one of the T-cell epitopes. This eliminates or reduces the immunogenicity of the protein when exposed to the immune system of the given species. Monoclonal antibodies and other immunoglobulin-like molecules can particularly benefit from being de-immunised in this way: for example, mouse-derived immunoglobulins can be de-immunised for human therapeutic use.

The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

Abstract: The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.

Solution structure of amyloid beta-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?

Abstract: The three-dimensional solution structure of the 40 residue amyloid beta-peptide, Abeta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles. In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of Abeta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6. 0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for Abeta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of Abeta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease.

Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex.

Abstract: Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility.

Abstract: The specificities of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases which link the carbohydrate GalNAc to the side-chain of certain serine and threonine residues in mucin type glycoproteins, are presently unknown. The specificity seems to be modulated by sequence context, secondary structure and surface accessibility. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Non-clustered sites had a sequence context different from that of clustered sites. Charged residues were disfavoured at position – 1 and +3. A jury of artificial neural networks was trained to recognize the sequence context and surface accessibility of 299 known and verified mucin type O-glycosylation sites extracted from O-GLYCBASE. The cross-validated NetOglyc network system correctly found 83% of the glycosylated and 90% of the non-glycosylated serine and threonine residues in independent test sets, thus proving more accurate than matrix statistics and vector projection methods. Predictions of O-glycosylation sites in the envelope glycoprotein gp120 from the primate lentiviruses HIV-1, HIV-2 and SIV are presented. The most conserved O-glycosylation signals in these evolutionary-related glycoproteins were found in their first hypervariable loop, V1. However, the strain variation for HIV-1 gp120 was significant. A computer server, available through WWW or E-mail, has been developed for prediction of mucin type O-glycosylation sites in proteins based on the amino acid sequence. The server addresses are http://www.cbs.dtu.dk/services/NetOGlyc/ and netOglyc@cbs.dtu.dk.

HLA-E Surface Expression Depends on Binding of TAP-Dependent Peptides Derived from Certain HLA Class I Signal Sequences

Abstract: Previous studies showed that HLA-E was expressed in lymphoblastoid cell line (LCL) 721.221 cells, but surface expression was lacking. To determine the signals controlling surface expression, we constructed a series of hybrid genes using complementary portions derived from the HLA-E and HLA-A2 genes. In this manner, a hybrid of HLA-E was identified, designated AEH, which differed from HLA-E by having the HLA-A2 signal sequence substituting for the HLA-E leader peptide. Transfection of LCL 721.221 cells with AEH induced HLA-E surface expression. Analysis of peptides bound to HLA-E revealed that a nonamer peptide derived from the A2 signal sequence was the predominant peptide bound. LCL 721.221 cells transfected with certain class I genes, including HLA-G, were also sufficient to promote peptide binding and HLA-E surface expression without increasing the level of HLA-E heavy chain synthesis. Peptides bound to HLA-E consisted of nine amino acids, with methionine at position 2 and leucine in the carboxyl-terminal position, and were nearly identical to the leader sequence-derived peptide previously shown to be a predominant peptide bound to the murine Qa-1 Ag. Signal peptides derived from certain HLA-B proteins with threonine in position 2 only marginally up-regulated HLA-E surface expression in .221 cells. An examination of HLA-E peptide binding in the TAP negative cell line .134 indicated that peptide binding to HLA-E was dependent on a functional TAP heterodimer regardless of whether peptide was available in cis, as in the AEH construct, or in trans, as in the class I transfectants of .221 cells.

The dwf4 gene of arabidopsis encodes a cytochrome p450 that mediates multiple 22alpha -hydroxylation steps in brassinosteroid biosynthesis

Abstract: dwarf4 (dwf4) mutants of Arabidopsis display a dwarfed phenotype due to a lack of cell elongation. Dwarfism could be rescued by the application of brassinolide, suggesting that DWF4 plays a role in brassinosteroid (BR) biosynthesis. The DWF4 locus is defined by four mutant alleles. One of these is the result of a T-DNA insertion. Plant DNA flanking the insertion site was cloned and used as a probe to isolate the entire DWF4 gene. Sequence analysis revealed that DWF4 encodes a cytochrome P450 monooxygenase with 43% identity to the putative Arabidopsis steroid hydroxylating enzyme CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM. Sequence analysis of two other mutant alleles revealed deletions or a premature stop codon, confirming that DWF4 had been cloned. This sequence similarity suggests that DWF4 functions in specific hydroxylation steps during BR biosynthesis. In fact, feeding studies utilizing BR intermediates showed that only 22alpha-hydroxylated BRs rescued the dwf4 phenotype, confirming that DWF4 acts as a 22alpha-hydroxylase.