VlsE, the variable surface antigen of Borrelia burgdorferi, contains an immunodominant conserved region named IR(6). In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C(6)) with the IR(6) sequence was explored. Sensitivity was assessed with serum samples (n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C(6) peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects.

A set of heavy-metal-complexing peptides was isolated from plants and plant suspension cultures. The structure of these peptides was established as (gamma-glutamic acid-cysteine)n-glycine (n = 2-11) [(gamma-Glu-Cys)n-Gly]. These peptides appear upon induction of plants with metals of the transition and main groups (Ib-Va, Z = 29-83) of the periodic table of elements. These peptides, called phytochelatins (PC), are induced in all autotrophic plants so far analyzed, as well as in select fungi. Some species of the order Fabales and the family Poaceae synthesize aberrant PC that contain, at their C-terminal end, either beta-alanine, serine or glutamic acid. For this group of peptides the name iso-PC is proposed. The biosynthesis of PC proceeds by metal activation of a constitutive enzyme that uses glutathione (GSH) as a substrate; this enzyme is a gamma-glutamylcysteine dipeptidyl transpeptidase which was given the trivial name PC synthase. It catalyzes the following reaction: gamma-Glu-Cys-Gly + (gamma-Glu-Cys)n-Gly->>(gamma-Glu-Cys)n+1-Gly + Gly. The plant vacuole is the transient storage compartment for these peptides. They probably dissociate, and the metal-free peptide is subsequently degraded. Sequestration of heavy metals by PC confers protection for heavy-metal-sensitive enzymes. The isolation of a Cd(2+)-sensitive cadl mutant of Arabidopsis thaliana, that is deficient in PC synthase, demonstrates conclusively the importance of PC for heavy metal tolerance. In spite of the fact that nucleic acid sequences and proteins are found in higher plants that have distant homology to animal metallothioneins, there is absolutely no experimental evidence that these "plant metallothioneins' are involved in the detoxification of heavy metals. PC synthase will be an interesting target for biotechnological modification of heavy metal tolerance in higher plants.

Basic peptides such as human immunodeficiency virus type 1 (HIV-1) Tat-(48-60) and Drosophila Antennapedia-(43-58) have been reported to have a membrane permeability and a carrier function for intracellular protein delivery. We have shown that not only Tat-(48-60) but many arginine-rich peptides, including HIV-1 Rev-(34-50) and octaarginine (Arg(8)), efficiently translocated through the cell membranes and worked as protein carriers (Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001) J. Biol. Chem. 276, 5836-5840). Quantification and time course analyses of the cellular uptake of the above peptides by mouse macrophage RAW264.7, human cervical carcinoma HeLa, and simian kidney COS-7 cells revealed that Rev-(34-50) and Arg(8) had a comparable translocation efficiency to Tat-(48-60). Internalization of Tat-(48-60) and Rev-(34-50) was saturable and inhibited by the excess addition of the other peptide. Typical endocytosis and metabolic inhibitors had little effect on the internalization. The uptake of these peptides was significantly inhibited in the presence of heparan sulfate or chondroitin sulfates A, B, and C. Treatment of the cells with the anti-heparan sulfate antibody or heparinase III also lowered the translocation of these peptides. These results strongly suggest that the arginine-rich basic peptides share a certain part of the internalization pathway.

BACKGROUND

In patients with acute coronary syndromes (ACS), troponin I (TnI), C-reactive protein (CRP), and B-type natriuretic peptide (BNP) each predict adverse cardiac events. Little is known, however, about the utility of these biomarkers in combination.

RESULTS

Baseline measurements of TnI, CRP, and BNP were performed in 450 patients in OPUS-TIMI 16. Elevations in TnI, CRP, and BNP each were independent predictors of the composite of death, myocardial infarction (MI), or congestive heart failure (CHF). When patients were categorized on the basis of the number of elevated biomarkers at presentation, there was a near doubling of the mortality risk for each additional biomarker that was elevated (P=0.01). Similar relationships existed for the endpoints of MI, CHF, and the composite, both at 30 days and through 10 months. In a validation cohort of 1635 patients in TACTICS-TIMI 18, the number of elevated biomarkers remained a significant predictor of the composite endpoint after adjustment for known clinical predictors: patients with one, two, and three elevated biomarkers had a 2.1- (P=0.006), 3.1- (P<0.001), and 3.7- (P=0.001) fold increase in the risk of death, MI, or CHF by 6 months.

CONCLUSIONS

Troponin, CRP, and BNP each provide unique prognostic information in patients with ACS. A simple multimarker strategy that categorizes patients based on the number of elevated biomarkers at presentation allows risk stratification over a broad range of short- and long-term major cardiac events.

Previous studies indicated that CAPS (calcium-dependent activator protein for secretion) functions as an essential component for the Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells. However, recent mouse knock-out studies suggested an alternative role in the vesicular uptake or storage of catecholamines. To genetically assess the functional role of CAPS, we characterized the sole Caenorhabditis elegans CAPS ortholog UNC-31 (uncoordinated family member) and determined its role in dense-core vesicle-mediated peptide secretion and in synaptic vesicle recycling. Novel assays for dense-core vesicle exocytosis were developed by expressing a prepro-atrial natriuretic factor-green fluorescent protein fusion protein in C. elegans. unc-31 mutants exhibited reduced peptide release in vivo and lacked evoked peptide release in cultured neurons. In contrast, cultured neurons from unc-31 mutants exhibited normal stimulated synaptic vesicle recycling measured by FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide] dye uptake. Conversely, UNC-13, which exhibits sequence homology to CAPS/UNC-31, was found to be essential for synaptic vesicle but not dense-core vesicle exocytosis. These findings indicate that CAPS/UNC-31 function is not restricted to catecholaminergic vesicles but is generally required for and specific to dense-core vesicle exocytosis. Our results suggest that CAPS/UNC-31 and UNC-13 serve parallel and dedicated roles in dense-core vesicle and synaptic vesicle exocytosis, respectively, in the C. elegans nervous system.

OBJECTIVE

Type 1 diabetes is a condition in which pancreatic islets are destroyed by self-reactive T cells. The process is facilitated by deficits in the number and suppressive activity of regulatory T cells (Tregs). Here, we show for the first time that the infusion of autologous Tregs prolongs remission in recently diagnosed type 1 diabetes in children.

METHODS

We have administered Tregs in 10 type 1 diabetic children (aged 8-16 years) within 2 months since diagnosis. In total, 4 patients received 10 × 10(6) Tregs/kg body wt, and the remaining 6 patients received 20 × 10(6) Tregs/kg body wt. The preparation consisted of sorted autologous CD3(+)CD4(+)CD25(high)CD127(-) Tregs expanded under good manufacturing practice conditions.

RESULTS

No toxicity of the therapy was noted. A significant increase in the percentage of Tregs in the peripheral blood has been observed since the day of infusion. These patients were followed along with matched type 1 diabetic patients not treated with Tregs. Half a year after type 1 diabetes onset (4-5 months after Tregs infusion), 8 patients treated with Tregs still required <0.5 UI/kg body wt of insulin daily, with 2 patients out of insulin completely, whereas the remission was over in the nontreated group. In addition, plasma C-peptide levels were significantly higher in the treated group as compared with those not treated.

CONCLUSIONS

This study shows that the administration of Tregs is safe and tolerable in children with recent-onset type 1 diabetes.

Epitopes presented by major histocompatibility complex (MHC) class I molecules are selected by a multi-step process. Here we present the first computational prediction of this process based on in vitro experiments characterizing proteasomal cleavage, transport by the transporter associated with antigen processing (TAP) and MHC class I binding. Our novel prediction method for proteasomal cleavages outperforms existing methods when tested on in vitro cleavage data. The analysis of our predictions for a new dataset consisting of 390 endogenously processed MHC class I ligands from cells with known proteasome composition shows that the immunological advantage of switching from constitutive to immunoproteasomes is mainly to suppress the creation of peptides in the cytosol that TAP cannot transport. Furthermore, we show that proteasomes are unlikely to generate MHC class I ligands with a C-terminal lysine residue, suggesting processing of these ligands by a different protease that may be tripeptidyl-peptidase II (TPPII).

CONCLUSIONS

ProPred1 is an on-line web tool for the prediction of peptide binding to MHC class-I alleles. This is a matrix-based method that allows the prediction of MHC binding sites in an antigenic sequence for 47 MHC class-I alleles. The server represents MHC binding regions within an antigenic sequence in user-friendly formats. These formats assist user in the identification of promiscuous MHC binders in an antigen sequence that can bind to large number of alleles. ProPred1 also allows the prediction of the standard proteasome and immunoproteasome cleavage sites in an antigenic sequence. This server allows identification of MHC binders, who have the cleavage site at the C terminus. The simultaneous prediction of MHC binders and proteasome cleavage sites in an antigenic sequence leads to the identification of potential T-cell epitopes.

BACKGROUND

Server is available at http://www.imtech.res.in/raghava/propred1/. Mirror site of this server is available at http://bioinformatics.uams.edu/mirror/propred1/ SUPPLEMENTARY INFORMATION: Matrices and document on server are available at http://www.imtech.res.in/raghava/propred1/page2.html

The genomic RNA of pestiviruses is translated into a large polyprotein that is cleaved into a number of proteins. The structural proteins are N terminal in this polyprotein and include three glycoproteins called E0, E1, and E2 on the basis of the order in which they appear in the polyprotein. Using pulse-chase experiments, we show that a pestiviral glycoprotein precursor, E012, is formed that is processed into E0, E1, and E2 in an ordered fashion. Processing is initiated by a nascent cleavage between the capsid and the translocated E012 followed by cleavage at the C terminus of E2. E012 is then rapidly cleaved to form E01 and E2. After E2 is released from the precursor, E01 is processed into E0 and E1. To identify the sites of cleavage, the N termini of the glycoproteins of the pestivirus classical swine fever virus (formerly termed hog cholera virus) were sequenced after expression in the vaccinia virus system. The N termini are Glu-268 for E0 (gp44/48), Leu-495 for E1 (gp33) and Arg-690 for E2 (gp55). The sequences around the cleavage sites capsid/E0 and E1/E2 conform to the rules known for cellular signal proteases, as does the sequence at the presumed C terminus of E2. The sequence upstream of the E0/E1 cleavage site also shows sequence characteristics of signalase processing sites but lacks the typical hydrophobic signal peptide; this cleavage site has characteristics in common with a site in flaviviruses that is also cleaved in a delayed fashion. The absence of any membrane-spanning region results in the shedding of E0 by infected cells, and E0 can be detected in the virus-free supernatant. Comparison of the sequences around the cleavage sites of pestiviruses suggests a general processing scheme for the structural glycoproteins. Comparison of the pesti- and flaviviral structural glycoproteins suggests analogies between E012 and prM-E.

We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted 13C-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are closely linked to degenerative diseases such as Alzheimer's disease, Parkinson's, neuronal death including ischemic and hemorrhagic stroke, acute and chronic degenerative cardiac myocyte death, and cancer. As a byproduct of oxidative phosphorylation, a steady stream of reactive species emerge from our cellular energy plants, the mitochondria. ROS and RNS potentially cause damage to all cellular components. Structure alteration, biomolecule fragmentation, and oxidation of side chains are trade-offs of cellular energy production. ROS and RNS escape results in the activation of cytosolic stress pathways, DNA damage, and the upregulation of JNK, p38, and p53. Incomplete scavenging of ROS and RNS particularly affects the mitochondrial lipid cardiolipin (CL), triggers the release of mitochondrial cytochrome c, and activates the intrinsic death pathway. Due to the active redox environment and the excess of NADH and ATP at the inner mitochondrial membrane, a broad range of agents including electron acceptors, electron donors, and hydride acceptors can be used to influence the biochemical pathways. The key to therapeutic value is to enrich selective redox modulators at the target sites. Our approach is based on conjugating nitroxides to segments of natural products with relatively high affinity for mitochondrial membranes. For example, a modified gramicidin S segment was successfully used for this purpose and proven to be effective in preventing superoxide production in cells and CL oxidation in mitochondria and in protecting cells against a range of pro-apoptotic triggers such as actinomycin D, radiation, and staurosporine. More importantly, these mitochondria-targeted nitroxide/gramicidin conjugates were able to protect against apoptosis in vivo by preventing CL oxidation induced by intestinal hemorrhagic shock. Optimization of nitroxide carriers could lead to a new generation of effective antiapoptotic agents acting at an early mitochondrial stage. Alternative chemistry-based approaches to targeting mitochondria include the use of proteins and peptides, as well as the attachment of payloads to lipophilic cationic compounds, sulfonylureas, anthracyclines, and other agents with proven or hypothetical affinities for mitochondria. Manganese superoxide dismutase (MnSOD), SS tetrapeptides with 2',6'-dimethyltyrosine (Dmt) residues, rhodamine, triphenylphosphonium salts, nonopioid analgesics, adriamycin, and diverse electron-rich aromatics and stilbenes were used to influence mitochondrial biochemistry and the biology of aging. Some general structural principles for effective therapeutic agents are now emerging. Among these are the presence of basic or positively charged functional groups, hydrophobic substructures, and, most promising for future selective strategies, classes of compounds that are actively shuttled into mitochondria, bind to mitochondria-specific proteins, or show preferential affinity to mitochondria-specific lipids.

Soluble oligomers of Abeta42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP(C), on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128-1132). Here we characterized the interaction between synthetic Abeta42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met(129) and Val(129) polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with Abeta oligomers. It was also found that in addition to the previously identified approximately 95-110 segment, the second region of critical importance for the interaction with Abeta42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23-27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for Abeta42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well.

The colored complex formed between Cu+ and bicinchoninic acid is the basis of the bicinchoninic acid protein assay (P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B.J. Olson, and D.C. Klenk (1985) Anal. Biochem. 150, 76-85). Studies show that cysteine, tryptophan, tyrosine, and the peptide bond are capable of reducing Cu2+ to Cu+. Electrochemical studies and the magnitude of the color changes observed when the reaction is carried out at 37 degrees C indicate that tryptophan, tyrosine, and the peptide bond are not completely oxidized at this temperature. When the reaction temperature is increased to 60 degrees C, significantly more color formation is observed for these three groups. Studies with di-, tri-, and tetrapeptides and with proteins indicate that the extent of color formation is not the sum of the contributions of the individual color producing functional groups. Compounds with functional groups similar to those of cysteine, cystine, tyrosine, or tryptophan are shown to react with the bicinchoninic acid reagent. The color formed by these compounds in the presence of bovine serum albumin cannot be compensated for by using a reagent blank containing an identical concentration of the interfering compound.

BACKGROUND

The nematode Caenorhabditis elegans has emerged as an important model for studies of the regulation of fat storage. C. elegans feed on bacteria, and various strains of E. coli are commonly used in research settings. However, it is not known whether particular bacterial diets affect fat storage and metabolism.

RESULTS

Fat staining of fixed nematodes, as well as biochemical analysis of lipid classes, revealed considerable differences in fat stores in C. elegans growing on four different E. coli strains. Fatty acid composition and carbohydrate levels differ in the E. coli strains examined in these studies, however these nutrient differences did not appear to have a causative effect on fat storage levels in worms. Analysis of C. elegans strains carrying mutations disrupting neuroendocrine and other fat-regulatory pathways demonstrated that the intensity of Nile Red staining of live worms does not correlate well with biochemical methods of fat quantification. Several neuroendocrine pathway mutants and eating defective mutants show higher or lower fat storage levels than wild type, however, these mutants still show differences in fat stores when grown on different bacterial strains. Of all the mutants tested, only pept-1 mutants, which lack a functional intestinal peptide transporter, fail to show differential fat stores. Furthermore, fatty acid analysis of triacylglycerol stores reveals an inverse correlation between total fat stores and the levels of 15-methylpalmitic acid, derived from leucine catabolism.

CONCLUSIONS

These studies demonstrate that nutritional cues perceived in the intestine regulate fat storage levels independently of neuroendocrine cues. The involvement of peptide transport and the accumulation of a fatty acid product derived from an amino acid suggest that specific peptides or amino acids may provide nutritional signals regulating fat metabolism and fat storage levels.

Octopamine (OA), a biogenic monoamine structurally related to noradrenaline, acts as a neurohormone, a neuromodulator and a neurotransmitter in invertebrates. It is present in relatively high concentrations in neuronal as well as in non-neuronal tissues of most invertebrate species studied. It functions as a model for the study of modulation in general. OA modulates almost every physiological process in invertebrates studied so far. Among the targets are peripheral organs, sense organs, and processes within the central nervous system. The known actions of OA in the central nervous system include desensitization of sensory inputs, influence on learning and memory, or regulation of the 'mood' of the animal. Together with tyramine, OA it is the only neuroactive non-peptide transmitter whose physiological role is restricted to invertebrates. This focussed the interest on the corresponding OA receptors. They are believed to be good targets for highly specific insecticides as they are not found in vertebrates. All octopamine receptors belong to the family of G-protein coupled receptors. Four of them could be distinguished using pharmacological tools. They show different coupling to second messenger systems including activation and inhibition of adenylyl cyclase, activation of phospholipase C and coupling to a chloride channel. Recently, octopamine receptors from molluscs and insects have been cloned. Further studies of all aspects of octopaminergic neurotransmission should give deeper insights into modulation of peripheral and sense organs and within the central nervous system in general.

Wheat germ RNA polymerase II was used to raise monoclonal antibodies (mAbs) that cross-react with the largest subunit of calf thymus RNA polymerase II. Most of these mAbs were of the IgM isotype and were shown to react with a synthetic peptide containing the consensus sequence for the C-terminal heptapeptide repeat that has been found on the largest subunit of RNA polymerase II from a variety of eukaryotic organisms. A representative mAb (3WG2) was tested for its effect on transcription in both in vitro and in vivo systems. Antibody 3WG2 did not affect the transcription (elongation) of wheat germ RNA polymerase II on denatured calf thymus DNA. When HeLa cell nuclear extracts were preincubated with the mAb, run-off transcription from a promoter that contains a TATA box (the adenovirus-2 major late promoter) and from a promoter that does not contain a TATA box (the murine dihydrofolate reductase gene promoter = dhfr) was inhibited. Transcription from these promoters was also inhibited by the synthetic peptide containing the consensus sequence when it was conjugated to bovine serum albumin. HeLa cell nuclear extract in which the endogenous RNA polymerase II had been inhibited by the specific mAb was used to examine the ability of added mammalian RNA polymerase II that lacks the C-terminal domain to accurately transcribe specific genes. When calf thymus RNA polymerase II that lacked the C-terminal domain was added back to the inhibited extract, a discrete transcript that was initiated correctly was obtained with the adenovirus-2 major late promoter; however, no discrete transcript was observed from the mouse dhfr gene promoter. When injected into Xenopus laevis oocytes, antibody 3WG2 inhibited transcription of the human histone H2b gene (contains a TATA box) and the human U1 small nuclear RNA gene (does not contain a TATA box), but did not inhibit transcription from RNA polymerase I or RNA polymerase III promoters. These results indicate that the C-terminal heptapeptide repeat plays a critical role in promoter-directed transcription, although enzyme that lacks this domain can initiate from some promoters in vitro.

Human herpesvirus 8 (HHV-8) is implicated in the pathogenesis of Kaposi's sarcoma. HHV-8 envelope glycoprotein B (gB) possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, by antibodies against alpha3 and beta1 integrins, and by soluble alpha3beta1 integrin (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Anti-gB antibodies immunoprecipitated the virus alpha3 and beta1 complexes, and virus-binding studies suggest a role for alpha3beta1 in HHV-8 entry. HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK), implicating a role for integrin and the associated signaling pathways in HHV-8 entry into the target cells. Immediately after infection, target cells exhibited morphological changes and cytoskeletal rearrangements, suggesting the induction of signal pathways. As early as 5 min postinfection, HHV-8 activated the MEK-ERK1/2 pathway. The focal adhesion components phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C-zeta (PKC-zeta) were recruited as upstream mediators of the HHV-8-induced ERK pathway. Anti-HHV-8 gB-neutralizing antibodies and soluble alpha3beta1 integrin inhibited the virus-induced signaling pathways. Early kinetics of the cellular signaling pathway and its activation by UV-inactivated HHV-8 suggest a role for virus binding and/or entry but not viral gene expression in this induction. Studies with human alpha3 integrin-transfected Chinese hamster ovary cells and FAK-negative mouse DU3 cells suggest that the alpha3beta1 integrin and FAK play roles in the HHV-8 mediated signal induction. Inhibitors specific for PI 3-kinase, PKC-zeta, MEK, and ERK significantly reduced the virus infectivity without affecting virus binding to the target cells. Examination of viral DNA entry suggests a role for PI 3-kinase in HHV-8 entry into the target cells and a role for PKC-zeta, MEK, and ERK at a post-viral entry stage of infection. These findings implicate a critical role for integrin-associated mitogenic signaling in HHV-8's infection of target cells and suggest that, by orchestrating the signal cascade, HHV-8 may create an appropriate intracellular environment to facilitate the infection.

Immunochemical studies have suggested a tight association of syntaxin with N-type calcium channels. Syntaxin specifically interacts with the fusion proteins containing the cytoplasmic loop (LII-III) between homologous repeats II and III of the alpha 1 subunit of the class B N-type calcium channel (alpha 1B) from rat brain, but not with those of the class A Q-type (alpha 1A) or the class S L-type (alpha 1S) calcium channels. This interaction is mediated by an 87 amino acid sequence (773-859) containing two overlapping predicted helix-loop-helix domains. The 87 amino acid peptide can specifically block binding of native N-type calcium channels to syntaxin, indicating that this binding site is required for stable interaction of these two proteins. Interaction takes place with the C-terminal one-third of syntaxin (residues 181-288), which is thought to be anchored in the presynaptic plasma membrane. Our results suggest a direct interaction between the cytoplasmic domains of these two presynaptic membrane proteins that could have an important role in the targeting and docking of synaptic vesicles near N-type calcium channels, enabling tight structural and functional association of calcium entry sites and neurotransmitter release sites.

When Swiss 3T3 cells are treated with Insulin-like Growth Factor I, a rapid decrease in the mass of polyphosphoinositol lipids (phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate) occurs within the nuclei, with a concomitant increase in nuclear diacylglycerol and translocation of protein kinase C to the nuclear region. This is in contrast to the effects of the regulatory peptide, bombesin, which causes similar inositol lipid changes in the plasma membrane, has no effect on nuclear inositide levels and causes a translocation of protein kinase C to post-nuclear membranes. These results suggest the existence of a discrete nuclear polyphosphoinositide signalling system entirely distinct from the well-known plasma membrane-located system, which is under regulatory control by cell surface-located receptors.

Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H(+)-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.

Proteolytic processing of the amyloid precursor protein (APP) by beta-secretase, beta-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid beta (Abeta) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP(C)), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, remains enigmatic. Because both APP and PrP(C) are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP(C) in the proteolytic processing of APP. Cellular overexpression of PrP(C) inhibited the beta-secretase cleavage of APP and reduced Abeta formation. Conversely, depletion of PrP(C) in mouse N2a cells by siRNA led to an increase in Abeta peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Abeta levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the beta-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP(C) on the beta-secretase cleavage of APP required the localization of PrP(C) to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP(C) via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Abeta is regulated by PrP(C) and may have implications for both Alzheimer's and prion diseases.

Bones and teeth are biocomposites that require controlled mineral deposition during their self-assembly to form tissues with unique mechanical properties. Acidic extracellular matrix proteins play a pivotal role during biomineral formation. However, the mechanisms of protein-mediated mineral initiation are far from understood. Here we report that dentin matrix protein 1 (DMP1), an acidic protein, can nucleate the formation of hydroxyapatite in vitro in a multistep process that begins by DMP1 binding calcium ions and initiating mineral deposition. The nucleated amorphous calcium phosphate precipitates ripen and nanocrystals form. Subsequently, these expand and coalesce into microscale crystals elongated in the c-axis direction. Characterization of the functional domains in DMP1 demonstrated that intermolecular assembly of acidic clusters into a beta-sheet template was essential for the observed mineral nucleation. Protein-mediated initiation of nanocrystals, as discussed here, might provide a new methodology for constructing nanoscale composites by self-assembly of polypeptides with tailor-made peptide sequences.

Many proteins contain targeting signals within their sequences that specify their delivery to particular organelles. The peroxisomal targeting signal-1 (PTS1) is a C-terminal tripeptide that is sufficient to direct proteins into peroxisomes. The PTS1 sequence closely approximates Ser-Lys-Leu-COO-. PEX5, the receptor for PTS1, interacts with the signal via a series of tetratricopeptide repeats (TPRs) within its C-terminal half. Here we report the crystal structure of a fragment of human PEX5 that includes all seven predicted TPR motifs in complex with a pentapeptide containing a PTS1 sequence. Two clusters of three TPRs almost completely surround the peptide, while a hinge region, previously identified as TPR4, forms a distinct structure that enables the two sets of TPRs to form a single binding site. This structure reveals the molecular basis for PTS1 recognition and demonstrates a novel mode of TPR-peptide interaction.

Accumulation of globin mRNAs during erythroid differentiation is dependent on their extraordinary stability. The longevity of human alpha-globin mRNA is associated with a ribonucleoprotein complex (alpha-complex) formed on the 3' untranslated region (3'UTR). One or more of the proteins within this alpha-complex contain strong polycytosine [poly(C)] binding (alpha PCB) activity. In the present report we purify alpha PCB activity from human erythroid K562 cells. Although not able to bind the alpha-globin 3'UTR directly, alpha PCB activity is sufficient to complement alpha-complex formation in a cytosolic extract depleted of poly(C) binding activity. Peptide microsequencing demonstrates that alpha PCB activity contains two structurally related poly(C) binding proteins. These two proteins, alpha-complex protein (alpha CP)-1 and -2, have an overall structural identity of 80% and contain three repeats of the K homology (KH) domain which is found in a subset of RNA binding proteins. Epitope-tagged recombinant alpha CP-1 and alpha CP-2 expressed in cells are each incorporated into the alpha-complex. We conclude that alpha CP-1 and alpha CP-2, members of the KH domain RNA binding protein family, are involved in formation of a sequence-specific alpha-globin mRNP complex associated with alpha-globin mRNA stability. As such this represents the first example of a specific function for this class of proteins and suggests potential roles for other members of this protein family.