The dense‐core vesicle maturation protein CCCP‐1 binds RAB‐2 and membranes through its C‐terminal domain

Dense‐core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled‐coil protein CCCP‐1 was identified as a component of the DCV biogenesis pathway in the nematode Caenorhabditis elegans. CCCP‐1 binds the small GTPase RAB‐2 and colocalizes with it at the trans‐Golgi. Here, we report a structure‐function analysis of CCCP‐1 to identify domains of the protein important for its localization, binding to RAB‐2, and function in DCV biogenesis. We find that the CCCP‐1 C‐terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP‐1 localization and for binding to RAB‐2, and is required for the function of CCCP‐1 in DCV biogenesis. In addition, CCCP‐1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid‐binding motif. We conclude that CCCP‐1 is a coiled‐coil protein that binds an activated Rab and localizes to the Golgi via its C‐terminus, properties similar to members of the golgin family of proteins. CCCP‐1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.      

Expression of an HCV core antigen coding gene in tobacco (N. tabacum L.)

Hepatitis C virus (HCV) is the major agent causing chronic liver disease. The core gene is the most conserved sequence in the HCV genome and proved immunoreactive when expressed in bacteria and antigenic in humans. In order to test the ability of plants to express the core gene for the production of core antigen, transgenic tobacco plants carrying the core gene were generated. The core protein was stably synthesized in T(0) and T(1) generations and was found to be immunoreactive, not only with anti-core polyclonal and monoclonal antibodies, but also was able to recognize the HCV virus in infected human serum. The prospects of producing a plant based vaccine and/or a food vaccine for this important virus are discussed.     

The internal sequence of the peptide-substrate determines its N-terminus trimming by ERAP1

Background

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini.

Methodology/Principal Findings

In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme''s substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme''s active site. This model can readily account for the strong preference for positively charged side chains.

Conclusions/Significance

To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide''s length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.     

Purification from normal human plasma and biochemical characterization of a ribonuclease specific for poly(C) and poly(U)

A new specific ribonuclease from normal human plasma has been purified to homogeneity, following a five-step purification protocol that included DEAE-Sepharose, CM-Sepharose, and Heparin-Sepharose chromatographies. The purified enzyme was found to be glycosylated and appeared as a single 25-kDa band on a SDS polyacrylamide gel. This RNase is poly(C) preferential, degrading poly(U) at a lower rate. Activity of this RNase toward cleavage of native substrates such as ribosomal RNA was also detected. The human plasma ribonuclease is a thermolabile molecule, exhibiting maximum activity at pH 6.5. Comparison between other known plasma RNases and the human plasma ribonuclease described here indicated a variety of differences in their biochemical and catalytic properties.     

JenPep: a database of quantitative functional peptide data for immunology

MOTIVATION: The compilation of quantitative binding data underlies attempts to derive tools for the accurate prediction of epitopes in cellular immunology and is part of our concerted goal to develop practical computational vaccinology. RESULTS: JenPep is a family of relational databases supporting the growing community of immunoinformaticians. It contains quantitative data on peptide binding to Major Histocompatibility Complexes (MHCs) and to Transmembrane Peptide Transporter (TAP), as well as an annotated list of T-cell epitopes. AVAILABILITY: The database is available via the Internet. An HTML interface allowing searching of the database can be found at the following address: http://www.jenner.ac.uk/JenPep.     

Physicochemical explanation of peptide binding to HLA-A*0201 major histocompatibility complex: a three-dimensional quantitative structure-activity relationship study

A three-dimensional quantitative structure-activity relationship method for the prediction of peptide binding affinities to the MHC class I molecule HLA-A*0201 was developed by applying the CoMSIA technique on a set of 266 peptides. To increase the self consistency of the initial CoMSIA model, the poorly predicted peptides were excluded from the training set in a stepwise manner and then included in the study as a test set. The final model, based on 236 peptides and considering the steric, electrostatic, hydrophobic, hydrogen bond donor, and hydrogen bond acceptor fields, had q2 = 0.683 and r2 = 0.891. The stability of this model was proven by cross-validations in two and five groups and by a bootstrap analysis of the non-cross-validated model. The residuals between the experimental pIC50 (-logIC50) values and those calculated by "leave-one-out" cross-validation were analyzed. According to the best model, 63.2% of the peptides were predicted with /residuals/ < or = 0.5 log unit; 29.3% with 1.0 < or = /residuals/ < 0.5; and 7.5% with /residuals/ > 1.0 log unit. The mean /residual/ value was 0.489. The coefficient contour maps identify the physicochemical property requirements at each position in the peptide molecule and suggest amino acid sequences for high-affinity binding to the HLA-A*0201 molecule.     

Quantitative structure-activity relationships and the prediction of MHC supermotifs

The underlying assumption in quantitative structure-activity relationship (QSAR) methodology is that related chemical structures exhibit related biological activities. We review here two QSAR methods in terms of their applicability for human MHC supermotif definition. Supermotifs are motifs that characterise binding to more than one allele. Supermotif definition is the initial in silico step of epitope-based vaccine design. The first QSAR method we review here--the additive method--is based on the assumption that the binding affinity of a peptide depends on contributions from both amino acids and the interactions between them. The second method is a 3D-QSAR method: comparative molecular similarity indices analysis (CoMSIA). Both methods were applied to 771 peptides binding to 9 HLA alleles. Five of the alleles (A*0201, A*0202, A*0203, A*0206 and A*6802) belong to the HLA-A2 superfamily and the other four (A*0301, A*1101, A*3101 and A*6801) to the HLA-A3 superfamily. For each superfamily, supermotifs defined by the two QSAR methods agree closely and are supported by many experimental data.     

A comparative molecular similarity indices (CoMSIA) study of peptide binding to the HLA-A3 superfamily

Epitope identification is the basis of modern vaccine design. The present paper studied the supermotif of the HLA-A3 superfamily, using comparative molecular similarity indices analysis (CoMSIA). Four alleles with high phenotype frequencies were used: A*1101, A*0301, A*3101 and A*6801. Five physicochemical properties-steric bulk, electrostatic potential, local hydrophobicity, hydrogen-bond donor and acceptor abilities-were considered and 'all fields' models were produced for each of the alleles. The models have a moderate level of predictivity and there is a good correlation between the data. A revised HLA-A3 supermotif was defined based on the comparison of favoured and disfavoured properties for each position of the MHC bound peptide. The present study demonstrated that CoMSIA is an effective tool for studying peptide-MHC interactions.     

New expanded bed adsorbents for the recovery of DNA

A 20–40 m pellicular high density (3.7 g cm^–3) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange adsorbents (6 mg ml^–1, c.f. 50 g ml^–1 at 10% breakthrough), due to a combination of small particle and fuzzy surface architecture created through the coupling of polyethylene imine chains.     

CD2AP/CIN85 balance determines receptor tyrosine kinase signaling response in podocytes

Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP(-/-)) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP(-/-) mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP(-/-) podocytes in vitro. Stimulation of CD2AP(-/-) podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP(-/-) mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.